Dissertations / Theses on the topic 'Detection PCR real-time'

Babesia rossi is the most virulent Babesia species in dogs occurring in South Africa and is associated with severe clinical manifestation and mortalities. Babesia rossi is highly pathogenic and reacting dogs requires treatment to prevent mortalities. Mild, uncomplicated forms of the disease are effectively treated with antibabesial drugs. Complicated forms of the disease are difficult to treat with high mortality rate.This results in the disease being of economic importance in South Africa. There is a lack of information regarding the relationship between parasite genotype and disease phenotype. The broad objective of this study is to detect B. rossi genotypes using real-time PCR. This test is a method that can be used to monitor amplicon formation throughout the PCR reaction and to estimate the initial concentration of target DNA in samples. Polymerase chain reaction, sequencing and phylogenetic analysis were used to determine the genotype detection of Babesia rossi isolated from infected dogs in South Africa. The correlation between the parasite genotype and disease phenotype was also investigated. Correlation between B. rossi Erythrocyte Membrane Antigen (BrEMA1) genotypes and age of dogs was studied in 44 cases. A total of 101 blood samples were tested using the reverse line blot (RLB) assay. Ninety six percent hybridized to the Babesia rossi species-specific probe. Our findings demonstrate that the most encountered (BrEMA1) genotype is genotype29, followed by genotype28 and genotype19, with genotype29 associated with most of the severe clinical signs diagnosed compared to genotype19. The number of cases caused by genotype19 was low, constituting 22% of the cases. This is comparable with the 2009 report where genotype19 appeared highly prevalent and virulent, whereas the prevalence of genotype28/29 appeared moderate. In this dissertation, we present the first report on the detection of an amplification product of BrEMA1 genotypes using real-time PCR. Samples which were below the detectable limit of conventional PCR and could not be sequenced probably due to low parasitaemia were also used and real-time PCR provided the ability to detect B. rossi positive animals. This was able to detect 10 BrEMA1 genotypes. However, it was not reliable enough in differentiating between various BrEMA1 genotypes. When evaluating the relationship between BrEMA1 genotypes, clinical manifestation and age of the dogs, collapse was found to be a poor prognostic sign in dogs with babesiosis. Genotype29 was associated with most of the collapsed cases and with high number of the dogs that died. Although B. rossi can infect dogs of all ages, young dogs showed to be more susceptible to canine babesiosis than older dogs. This is in agreement with the survey carried out from the Onderstepoort Veterinary Academic Hospital (OVAH) in 1994. Since B. rossi is the most pathogenic species of the large babesias of dogs, the ability to manage the disease is dependent on rapid detection of the organism. Real-time PCR test is indeed a quicker method to confirm diagnoses of B. rossi infected dogs. It can detect B. rossi infection at a low DNA concentration (0.185 ng/μl) which provides a major advantage in detecting B. rossi infection in field blood samples.
Dissertation (MSc)--University of Pretoria, 2011.
Veterinary Tropical Diseases
MSc
Unrestricted

Directive EC 2003/89/EC of the European Parliament and of the Council states that certain

ingredients and products derived there of known to cause allergen reactions must always be

declared. Furthermore labelling is mandatory irrespective of the amount included. The National

Food Administration therefore needs methods for monitoring the presence of allergens in food.

Methods already exist for most of the allergens on the EU-list, but an operational method for

celery (Apium graveolens) is missing.

A specific DNA-method was developed, based on TaqMan Real-Time PCR with the celery

mannitol dehydrogenase gene as target sequence. The analysis was started with homogenisation

of the sample followed by extraction of DNA. The Real-Time PCR method was shown to be

specific for celery, producing a 113 bp fragment with two celery varieties and negative results

with other closely selected species commonly present together with celery in food products (12

samples). The detection limit was 2-20 pg DNA, which corresponds to 1-7 haploid genome

copies. When evaluated with model samples of celery in meat, a detection limit of less than

0,01 % was determined. When used to analyse food products from the market, six out of seven

products declared to contain celery were correctly identified as positive.

As the area of nucleic acid based technologies develops, so will our understanding of how structural variations in DNA and RNA pathogens are associated with disease. The overall goal of this thesis is the development of broadly targeted measurement techniques for variable viral RNA by Real-Time PCR (here referred to as quantitative reverse transcriptase PCR, QRT-PCR). In papers I & II, broadly targeted and specific QRT-PCRs were used to study expression of endogenous and exogenous betaretrovirus sequences in human tissues. Results from human tissues demonstrated endogenous betaretrovirus expression in a tissue-specific manner, highest in reproductive tissues. Despite the high sensitivity, no exogenous betaretrovirus was found in human breast cancer samples. The limits of primer and probe degeneracy for detection of a diverse set of retroviral sequences was evaluated. These methods are useful for further investigations on the pathophysiological contribution(s) of endogenous betaretrovirus and to investigate whether an exogenous betaretrovirus is involved in human breast cancer. In papers III & IV, we developed and applied broadly targeted one-step QRT-PCRs for influenza viruses and coronaviruses. In addition to the generic primers, two novel probe design strategies were used in order to be able to broadly amplify these diverse sets of viruses: A triplex system for simultaneous detection and quantification of influenza A, B and C (3QRT-PCR and further developed 3QRT-PCR-MegB; where MegB stands for MegaBeacon) based on TaqMan® and MegB probes, and a pan-CoV QRT-PCR, based on three TaqMan® probes i.e., degeneracy was distributed on three probes. Probe fault tolerance was thus increased in two ways, either with short probes with/without locked nucleic acid (LNA) nucleotides concentrated to conserved stretches, or with long probes (MegB), compensating mismatching positions with many matching ones. Clinical samples, negative by antigen detection with immunofluorescence (IFA), were influenza A positive with 3QPCR-MegB. Avian pooled samples, negative with an earlier pan-CoV QPCR, came out positive with the triple-probe system. Assay evaluation with clinical samples and reference strains revealed good clinical diagnostic potential. Thus, the thesis describes several strategies to counteract sequence variation of RNA viruses and describes a set of broadly targeted QRT-PCRs useful for scientific screening or diagnostics of betaretroviruses and respiratory viruses.

Thesis (M.S.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Sep. 16, 2008). "... in partial fulfillment of the requirements for the degree of Master of Science in the Curriculum of Genetics and Molecular Biology." Discipline: Genetics and Molecular Biology; Department/School: Medicine.

Respiratory infection is a major disease burden worldwide. Statistical reports revealed it is one of the main causes of mortality and morbidity especially in young children. Influenza infection is one of the predominant cause associate with respiratory infection. Traditionally, studies have been emphasized on the detection of influenza A and B virus owing to their significance in clinical and economic impact. Attention of influenza C virus is rarely recognized due to its difficulty in isolation. However, recently, increasing reports have been illustrated the co-circulation of influenza C virus globally. Serological studies also suggested majority of people worldwide acquired influenza C virus infection in their early childhood or adolescent stage, yet information regarding influenza C virus is still inadequate. Epidemiological and clinical impact of influenza C virus in pediatric patients in Hong Kong was examined by the approach of real-time PCR. From November 2007 to April 2011, a total of 1, 037 specimens were obtained from pediatric patients exhibited apparent respiratory tract illness in Hong Kong. Eleven strains of influenza C virus were detected by real-time PCR approach. All patients with influenza C virus infection were below 5 years of age with the youngest age of 11 months. The ratio of infection in male to female was approximately one to one. High grade fever appeared to be the most frequent clinical manifestations (10/11) of influenza C virus infection. Upper respiratory tract infection was also occasionally observed. The clinical presentation of influenza C virus was similar to its influenza counterpart. Phylogenetic analysis of influenza C virus was examined in 6/11 of the isolates to determine the lineages of co-circulating influenza C viruses in Hong Kong. Nucleotide sequencing was performed with primer targeting the hemagglutinin-esterase (HE) gene. Result revealed that most of the detected influenza C virus associate with the C/Sao Paulo/378/82 related lineage. Results from this study revealed the positive rate of influenza C was comparable to influenza B and resultant respiratory symptoms could be severe in pediatric patients It is suggested to consider the inclusion of influenza C virus detection in routine diagnostic panel and real-time PCR could be a desirable detection platform account for its sensitivity and rapidity.
published_or_final_version
Microbiology
Master
Master of Medical Sciences

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2004.
Includes bibliographical references (leaf 21).
Tissue print technology allows for the transfer of cellular material from tissue onto a nitrocellulose film for immunocytochemical assays. The MIT BioInstrumentation Laboratory is currently developing a novel cancer marker imaging system for detection of cancerous tissue, which will be useful for discerning tumor margins. This research will advance the recent application of tissue print technology in bio-medicine by combining it with imaging and real time polymerase chain reaction (PCR) amplification and detection. A major objective in the design of this instrumentation is to develop the capacity to evaluate much larger areas of tissue. An approach to fulfilling this objective is the creation of a gasket that can seal individual wells of a nitrocellulose array. A gasket was created by laser cutting an assembly of molded silicone rubber and a double-sided tape (silicone-acrylic). Experiments showed when the gasket was adhered to a glass slide and subjected to the PCR, there was no leakage. FAST Slides, nitrocellulose slides provided by Grace Bio-Labs, are cut with a laser to generate the nitrocellulose arrays.
by Helena Nien-Hwa Chia.
S.B.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination.
AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

Pseudomonas aeruginosa (PA) infection is the main cause of mortality in Cystic Fibrosis (CF) patients. Rapid and sensitive PA detection is pivotal to improve the prognosis. This thesis includes two parts. The first focuses on the development of a Real-time PCR protocol to detect dormant- non-culturable PA in sputum samples from CF patients (CFPA). 12 culture-positive (CP) and 29 culture-negative (CN) samples were analyzed by a qPCR targeting ecfX, 24% resulted positive (P), with bacterial counts (102 - 106 cells/ml) also higher than those obtained for CP samples. A significant association of culture-negative/PCR-positive samples from chronic patients with symptoms relapse (P=0.018) and following culture-positive samples (P=0.034) was found. These findings stress the value of qPCR approaches in monitoring P. aeruginosa colonization and foreseeing symptom recurrence in CF patients. In second part we investigated the epidemiological relatedness of CFPA. 77 CFPA and 19 PA from non-CF patients (NCFPA) were analysed by pulsed field gel electrophoresis (PFGE) profile, antibiotic resistance (AR) and mucous production. Population structure analysis showed an overall polyclonality, more evident among CF strains. 100% similarity was only found within each of 10 CFPA pairs from the same patient, and 95% between 2 CFPA isolates from different patients who never met each other in hospital, suggesting a common source of infection rather than cross-contamination. Among NCFPA four isolates from as many inpatients and two from as many outpatients showed the same pulsotype (H and M, respectively). AR was more common among CFPA and nonmucoid phenotype. In particular, a significant (P=0.016) association was found between gentamycin-resistance CFPA and nonmucoid phenotype. AR patterns were however highly variable, also among PA belonging to the same pulsotype; suggesting the lack of involvement of multiple antibiotic resistance (MAR) in cross-colonization.

Genetically modified organisms are entering the human diet in all over the world. In order to have transparency in the foods that are being consumed, there is a need to trace the genetically modified organisms (GMOs) in the market and consequently this need brings the necessity of analytical methods that are capable of detecting, identifying and quantifying the transgenic events. These analytical methods also form the basis of the labeling regulations that are tried to be formed regarding GMOs. The main aim of this study is to develop and apply the detection methods for the two of the tomato events, delayed ripening and insect resistant. Currently the only validated detection methods are mainly for the corn, soybean, and cotton. There is no validated detection method for tomato. Tomato is one of the most consumed food products in Turkey and it is also among the controversial organisms in terms of genetic modifications and labeling, therefore the analysis of the genetic modifications in tomato is crucial. In this study, DNA-based detection is performed, with PCR being the chosen method of study. In order to detect the GMO-derived DNA, the method of analysis includes the following studies: species-specific, screening, gene-specific, construct-specific and inverse PCR. In addition, the quantification method is developed using the real-time PCR. In order to develop the procedure of identification method, the reference samples are used and the unknown varieties that are to be analyzed using this method are expected to have similarities with the authorized transgenic events.

Molecular diagnostics offer quick access to information for healthcare decision-making towards personalized therapeutics, but complicated procedures requiring extensive labor and infrastructure restrict their use. Droplet-based technologies can expand the accessibility of molecular diagnostics by miniaturizing devices, shortening sample-to-answer times, decreasing costs and increasing throughput. Methods for droplet manipulation are central to the automation of molecular diagnostics protocols. The innovative method, wire-guided droplet manipulation (WDM), is the actuation of liquid droplets in a hydrophobic milieu with a wire, or needle, guide. In this work, WDM is demonstrated for the automation of the polymerase chain reaction (PCR) on reprogrammable platforms for the diagnosis of cardiovascular infections. WDM is used to minimize thermal resistance by convective heat transfer for PCR amplification at a maximum speed of 8.67 s/cycle. The oil-water interfacial boundary is shown to passively partition molecular contaminants from sample matrices, including blood and heart valve tissue. Molecular self-assembly at the oil-water interface is used to increase PCR efficiency with blood in situ and is used as an innovative sensing modality for real-time monitoring of PCR amplification. Temperature feedback controlled droplet actuation is achieved by using a thermocouple loop as a functionalized wire-guide. Our novel methodology for real-time PCR, droplet-on-thermocouple silhouette real-time PCR (DOTS qPCR), utilizes interfacial effects to achieve droplet actuation, relief from PCR inhibitors and amplification sensing, for a sample-to-answer time as short as 3 min 30 s. DOTS qPCR addresses three major issues for rapid PCR—sample preparation, rapid thermocycling and sensitive real-time detection—on an inexpensive, disposable device with smartphone-based detection. In contrast, commercially available real-time PCR systems rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection we anticipate extending this technology towards trending biological research applications such as single cell, single nucleus, and single DNA molecule analyses, especially in droplet microfluidic platforms.

A new European labeling directive (2003/89/EC) states that certain foods and products derived thereof must always be declared. Among the tree nuts specified is Macadamia nut (Macadamia spp.). During the last few years, cases of IgE-allergic reactions, even severe anaphylaxes, have been reported. Reliable methods for the detection of this nut are needed.

Protein from Macadamia nuts was isolated. Polyacrylamide gel electrophoresis in SDS revealed two main protein bands of about 20 and 50kDa. These protein bands were cut and extracted from the gel and rabbits were immunized with each protein.

Immunoblotting showed dominant reactivity with the respective antigens. The antisera were further tested for specificity in immunodiffusion and in rocket immunoelectrophoresis.

In addition, a specific DNA-method was developed, based on Real-Time PCR using Macadamia vicilin as target sequence. Two different primer pairs were tested. Specificity was tested against potentially related nuts. Optimisation of primer and probe concentrations was performed. The limit of detection was 2-4 pg DNA, corresponding to a macadamia nut concentration of 50 to 100 μg per g. In a background of soybean DNA, down to 0,01 % macadamia DNA could be detected.

Influenza is a widespread disease occurring in seasonal epidemics, and each year is responsible for up to 500,000 deaths worldwide. Influenza can develop into strains which cause severe symptoms and high mortality rates, and could potentially reach pandemic status if the virus’ properties allow easy transmission. Influenza is transmissible via contact with the virus, either directly (infected people) or indirectly (contaminated objects); via reception of large droplets over short distances (one metre or less); or through inhalation of aerosols containing the virus expelled by infected individuals during respiratory activities, that can remain suspended in the air and travel distances of more than one metre (the aerosol route). Aerosol transmission of viruses involves three stages: production of the droplets containing viruses; transport of the droplets and ability of a virus to remain intact and infectious; and reception of the droplets (via inhalation). Our understanding of the transmission of influenza viruses via the aerosol route is poor, and thus our ability to prevent a widespread outbreak is limited. This study explored the fate of viruses in droplets by investigating the effects of some physical factors on the recovery of both a bacteriophage model and influenza virus. Experiments simulating respiratory droplets were carried out using different types of droplets, generated from a commonly used water-like matrix, and also from an ‘artificial mucous’ matrix which was used to more closely resemble respiratory fluids. To detect viruses in droplets, we used the traditional plaque assay techniques, and also a sensitive, quantitative PCR assay specifically developed for this study. Our results showed that the artificial mucous suspension enhanced the recovery of infectious bacteriophage. We were able to report detection limits of infectious bacteriophage (no bacteriophage was detected by the plaque assay when aerosolised from a suspension of 103 PFU/mL, for three of the four droplet types tested), and that bacteriophage could remain infectious in suspended droplets for up to 20 minutes. We also showed that the nested real-time PCR assay was able to detect the presence of bacteriophage RNA where the plaque assay could not detect any intact particles. Finally, when applying knowledge from the bacteriophage experiments, we reported the quantitative recoveries of influenza viruses in droplets, which were more consistent and stable than we had anticipated. Influenza viruses can be detected up to 20 minutes (after aerosolisation) in suspended aerosols and possibly beyond. It also was detectable from nebulising suspensions with relatively low concentrations of viruses.

Campylobacter organisms are the most commonly reported bacterial causes of foodborne infection in the world, with Campylobacter jejuni and Campylobacter coli responsible for over 99% of reported infections. Traditionally, Campylobacter species detection is an arduous process, requiring a special incubation environment as well as specific growth media for an extended growth period. The development of a rapid and reliable diagnostic tool for the detection of Campylobacter species would be a valuable aid to the medical diagnostic decision process, especially to rule out Campylobacter infection during the enteric pre-surgical time period. Improved patient outcomes would result if this rapid assay could reduce the number of enteric surgeries. Assays performed during this dissertation project have demonstrated that both SYBR® green and hydrolysis probe assays targeting an 84 nucleotide portion of cadF, a fibronectin-binding gene of Campylobacter jejuni and Campylobacter coli, were able to detect from 101 to 108 copies of organism from stool specimens, did not detect nonspecific targets, and exhibited a coefficient of variation (CV) of 1.1% or less. Analytical validation of sensitivity, specificity and precision, successfully performed in these studies, warrants additional clinical validation of these assays.

In recent years, foods produced by genetic engineering technology have been on the world food market. The biosafety aspects, regulations, and labelling of these foods are still contentious issues in most countries. It is necessary to have approval for the use of GMOs in the production of food. Thus, detection and quantification of GMOs play crucial role for developing regulations on GM foods. In this study, raw and processed maize samples were analysed for genetic modification using a DNA based detection method, the Polymerase Chain Reaction. Ten raw food and 18 processed maize food including maize flour, starch, corn flakes, maize chips were collected from different markets located in different places in Turkey. The samples were examined for the presence of genetic elements located in the majority of transgenic crops such as NOS terminator, CaMV 35S promoter, kanamycin resistance (KanR) gene, using conventional PCR with oligonucleotide sets targeting to novel genes. Furthermore screening was conducted via Real-Time PCR assay for NOS terminator and 35S promoter. For confirming the presence of Bt11 maize lines event specific primers were utilised. Quantification of Bt11 maize lines were performed via Real-Time PCR. The result indicates that foreign genetic elements were found in all analysed raw material. In six out of 10 raw material, presence of Bt11 gene were identified. GMO detection was also possible for maize flour and starch, however in processed material as corn starch, corn flakes, corn chips and pop corn, transgenes were not detected.

The transmission of human pathogens by faecally contaminated fruit and vegetables is well established, but the burden of disease caused by foodborne pathogens is unknown. Fresh produce can be contaminated through the use of polluted irrigation water or by the handling of the produce by infected individuals either pre- or post harvest. There is very little known regarding the extent of viral contamination of irrigation water and fresh produce in South Africa. Noroviruses (NoV) and hepatitis A virus (HAV) are recognized as leading causes of foodborne viral disease. These viruses are transmitted predominantly via the faecal–oral route, primarily person-to-person by direct contact with an infected person, or indirectly by ingestion of contaminated food and water. The detection of enteric viruses in food or water is problematical and complex as many foodborne viruses, including HAV and NoV, cannot be readily isolated in cell culture. The aim of this investigation was to develop and optimise simple and efficient methods for the concentration and detection of NoV GII and HAV in irrigation water and fresh produce. These methods would then be applied to field samples of irrigation water and fresh produce to try and establish a link between viral contamination detected in irrigation water and that on associated irrigated fresh produce. The efficiency of different commercial real-time reverse transcriptase-polymerase chain reaction amplification kits for the realtime detection of HAV, NoV GI and NoV GII was assessed, and standard curves for the quantitative detection of these viruses were constructed using the most appropriate kit. Using two types of fresh produce, three different elution buffers, each at two pHs, with two different elution times were compared to establish which buffer was the most efficient for the extraction of viruses from the fresh produce. The tris-glycine beef extract buffer (pH 9.5) with an elution time of 20 minutes most efficient for the extraction of the selected enteric viruses from fresh produce. From April 2008 to November 2009, 86 irrigation water and 72 fresh produce samples were collected from commercial and subsistence farms, street vendors and commercial outlets. All the irrigation water and fresh produce samples were analysed for HAV, NoV GI and NoV GII. Overall, 16.3 % (13/86) and 12.5 % (9/72) of irrigation water and fresh produce samples tested positive for one or more human pathogenic viruses, namely NoV GII and HAV, respectively. Nucleotide sequence and phylogenetic analysis of the HAV and NoV GII strains identified clinically relevant viruses in the irrigation water and on the fresh produce. A direct link between contaminated irrigation water and contamination of fresh produce could not be established, but irrigation water was identified as a possible source of contamination of the fresh produce. The results also suggested that food handlers contributed significantly to the viral contamination of the fresh produce. This study highlights the potential health risk posed by fresh produce to consumers in South Africa and highlights the need for further in depth studies to quantify the risk to consumers. This study represents new data on the occurrence of enteric viruses in food and water in South Africa and is crucial for the development of effective intervention and control strategies for food safety in South Africa. Copyright
Dissertation (MSc)--University of Pretoria, 2011.
Medical Virology
unrestricted

Master of Science
Department of Diagnostic Medicine/Pathobiology
T. G. Nagaraja
Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.

no
Yellow leaf syndrome (YLS) of sugarcane has been associated with Sugarcane yellow leaf virus (ScYLV) and has been reported from most sugarcane growing countries around the world. As sugarcane is vegetatively propagated, it is important to use effective and sensitive detection methods to screen new propagating material. Virus detection in symptomatic tissue is currently achieved using enzyme linked immunosorbent assay (ELISA), tissue blot immunoassay (TBIA) or a conventional RT-PCR based assay. This paper reports the development of an improved assay based on multiplex real-time fluorescent RT-PCR. The new assay is 100-fold more sensitive than conventional RT-PCR, and incorporates a novel `RNA specific¿ internal positive control (based around the intron of the caffeic acid 3-o-methyltransferase gene) to guard against false negative results. The paper also describes the comparison of eight RNA extraction methods for sugarcane tissue giving a number of alternatives for different laboratory situations. The sensitivity of this assay has allowed the detection of ScYLV in many samples that were thought to be healthy following conventional testing (RT-PCR, ELISA or TBIA). The detection of ScYLV using this TaqMan assay can be applied to the production of ScYLV-free plants and prevents its spread through the propagation material.

Introduction: Mycoplasma pneumoniae (M. pneumoniae) has been a major cause of community-acquired pneumonia (CAP), accounting for about 10-30% of the cases. Previously, a local study revealed that more than 60% of clinical isolates of M. pneumoniae exerted A2063G mutation, which confers a high level of macrolide drug resistance and results in treatment failure. While A2063G is the only mutation identified locally, a rapid diagnostic assay for detection of this single point mutation is urgently needed for switching the drug of choice. Aims: This study aims to develop a rapid PCR assay for detection of A2063G mutation of M. pneumoniae isolates for our locality, to compare with other commercially available assays and to further confirm the prevalence of A2063G mutation in macrolide-resistance M. pneumoniae (MRMP) in Hong Kong. Methods: A total of 110 respiratory tract samples were collected from 102 patients in Hong Kong Sanatorium and Hospital during April 2013 to April 2014. They were analyzed by an in-house hybridization-probe real-time PCR assay coupled with melting curve analysis to detect the presence of M. pneumoniae and the target A2063G point mutation. Results were compared with a commercial real-time PCR assay and the A2063G point mutation was further confirmed by 23S rRNA gene sequencing. The limit of detection (LOD), mutation threshold determination and cross reactivity of the in-house assay were also evaluated. Results: Over 40% (47/110) of the respiratory tract samples were tested positive for M. pneumoniae by the in-house assay and 36.2% (17/47) of the positive samples exerted A2063G mutation. The limit of detection was 500 copies/ml as evaluated using external quality control samples. Twenty well-characterized clinical isolates of M. pneumoniae were used to evaluate the A2063G mutation threshold. The mutation threshold for A2063G mutant detection was above 60%. This assay did not show any cross-reactivity with common clinical isolates from the respiratory tract samples. Conclusion: In this study, an in-house real-time PCR assay was evaluated and demonstrated its great potential as a rapid clinical diagnostic tool. The assay was highly sensitive and specific in detecting M. pneumoniae and its A2063G mutation from clinical samples in Hong Kong. The results were almost concordant to the current routine testing, with the advantage of lower cost and shorter turnaround time for rapid detection.
published_or_final_version
Microbiology
Master
Master of Medical Sciences

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
A hansenÃase à uma doenÃa infecciosa crÃnica e granulomatosa responsÃvel por afetar a pele e os nervos perifÃricos, sendo ocasionada pelo Mycobacterium leprae, um agente intracelular obrigatÃrio incapaz de ser cultivado em meios de cultura axÃnicos. No ano de 2012, foram detectados 232.857 casos novos no mundo, sendo desses 33.303 (14, 30%) detectados somente no Brasil. O Estado do Cearà diagnosticou 2.066 casos novos sà no ano de 2012, com um coeficiente detecÃÃo geral de 24,0/100.000 habitantes. A transmissÃo da doenÃa està relacionada com eliminaÃÃo dos bacilos provenientes de pacientes multibacilares atravÃs do trato respiratÃrio superior e, como a bactÃria fica em suspensÃo no ar, ocorre a posterior contaminaÃÃo de outra pessoa. A existÃncia de casos clÃnicos da doenÃa nos quais os pacientes nÃo tiveram nenhum contato anterior com portadores de hansenÃase e, ainda, Ãreas com casos prÃximos de fontes de Ãgua sugere infecÃÃo do ser humano com fontes ambientais. Por isso, atualmente, pesquisa-se a possÃvel interferÃncia de fatores ambientais e animais silvestres na transmissÃo da hansenÃase, como solo, Ãgua, vegetaÃÃo e tatus. O ambiente funcionaria como uma espÃcie de reservatÃrio, permitindo que o bacilo permaneÃa infeccioso apÃs longos perÃodos fora do corpo humano. Este estudo tem como objetivo principal detectar e quantificar bacilos de M. leprae por qPCR em amostras de Ãguas ambientais provenientes de municÃpios cearenses. Foram coletadas cinco rÃplicas de cada um dos 30 reservatÃrios selecionados, totalizando 149 amostras. O DNA total foi extraÃdo atravÃs de kit especÃfico para amostras ambientais de acordo com as recomendaÃÃes do fabricante. Posteriormente, foi realizado a amplificaÃÃo do gene 16S rRNA de M. leprae atravÃs de qPCR com o uso do kit SYBR Green PCR Master Mix. Utilizou-se uma curva padrÃo com concentraÃÃes conhecidas de plasmÃdeo pIDTBlue 16SrRNAMlep para quantificar o DNA presente nas amostras ambientais. As amostras ambientais do municÃpio de Juazeiro do Norte e as amostras clÃnicas provenientes de pacientes atendidos no CDREM foram genotipadas e subtipadas por PCR-RFLP. O DNA de M. leprae foi detectado em todos os municÃpios estudados. Do total de 149 amostras de Ãgua analisadas, 81 (54,4%) foram positivas para a pesquisa de DNA. O nÃmero de cÃpias de M. leprae se manteve no intervalo de 1,42 x 10-1 a 1,44 x 10+2 . A maioria das amostras clÃnicas apresentou genÃtipo 4 (64%) enquanto que 100% das amostras de Juazeiro do Norte foram SNP 4. Com relaÃÃo a subtipagem, o SNP 4-N foi o mais presente dentre as amostras analisadas. Este estudo indica a existÃncia de DNA de M. leprae nas amostras de Ãguas ambientais, mostrando fundamentalmente a presenÃa de bacilos nas Ãguas analisadas. TambÃm relata que a maioria das cepas de M. leprae das fontes ambientais estudadas à do mesmo subtipo das isoladas do homem. Dessa forma, sÃo necessÃrios mais estudos a fim de ampliar o conhecimento da influÃncia da Ãgua na transmissÃo da hansenÃase.

Reliable methods to analyze food for the presence of almond are important – not only for those allergic to almond, but also for monitoring the compliance with labelling regulations (EG directive 2003/89). Until now the Swedish National Food Administration has used methods like rocket immunoelectrophoresis and real-time PCR to detect almond in food. These methods are, however, not sensitive enough for protecting the most sensitive individuals. Therefore, the performance of a commercial ELISA kit was tested with regard to specificity/cross reactivity and limit of detection for almond both in solution and in different matrixes.

The limit of quantitation was at least 3,1 ppm (mg/kg) in solution and similar concentrations were measured in bisquits and chocolate. The ELISA method was about 100-fold more sensitive than rocket immunoelectrophoresis and PCR.

The specificity of the test kit was evaluated against a number of different nuts and seeds. No important cross reactivity was found. The antibodies against almond used in the kit can not differentiate between almond and apricot kernel. For such purposes the PCR method could be used.

Chlamydia and gonorrhea are major causes of sexually transmitted infections (STI) in adolescents worldwide. The infections are caused by Chlamydia trachomatis or Neisseria gonorrhoeae, bacteria with clinical manifestations such as urethritis, prostatitis and epididymitis among men, and urethritis, cervicitis and upper genital tract infection (i.e. pelvic inflammatory disease) among women. However, in many cases of genital tract infection, the etiology remains uncertain. In light of this, Mycoplasma genitalium was somewhat accidentally isolated in 1980 after prolonged incubation of urogenital specimens from men with non-gonococcal urethritis. Following the initial isolation in 1980, repeated attempts have been made to recover the extremely fastidious organism from clinical samples by culture techniques, but isolates have been rare and difficult to obtain. With the development of PCR methods in the early 1990s, detection of M. genitalium infection became more feasible. The aim in paper I was to compare three different PCR assays (conventional and real-time 16S rRNA gene PCR as well as real-time Mycoplasma genitalium adhesin protein (MgPa) gene PCR) for detection of M. genitalium. The study also determined the prevalence of M. genitalium. Clinical specimens collected from STI attendees, 381 men and 298 women, were used to determine the prevalence of M. genitalium and 213 of these specimens were used in the PCR comparative study. The prevalence of M. genitalium infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %) respectively. In the PCR comparative study, M. genitalium DNA were detected in 61/76 (80.3 %) of true-positive specimen by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Hence, real-time MgPa gene PCR is well suited for clinical diagnosis of M. genitalium in urogenital specimens from men and women. The aim in paper II was to determine whether a patients’ endocervical swab specimen can be transported in first void urine (FVU) as combined specimens in detection of Mycoplasma genitalium by real-time PCR. The study also compared two different DNA extraction methods (manual Chelex DNA extraction and automated BioRobot M48 DNA extraction) for observation of possible PCR inhibition. Clinical specimens collected from 329 women attending a STI clinic were used in the study. A total of 100 endocervical swab specimens transported in FVU was used in the PCR inhibition analysis. M. genitalium was detected in 25/329 (7.6 %) women. Endocervical swab specimens transported in FVU demonstrate higher sensitivity compared to both FVU alone and specimens transported in 2-SP medium detecting 24/25 (96 %), 22/25 (88 %) and 17/25 (68 %) of M. genitalium positive women, respectively. Automated BioRobot M48 DNA extraction was shown to be superior to manual Chelex extraction leaving no PCR inhibition and slightly higher DNA yield and/or better sensitivity. The results from these two studies are important knowledge in establishing the future diagnostic level of this STI in our county and also nationally.

Odontoglossum ringspot virus (ORSV) is one of the most prevalent orchid viruses that infects greenhouse-grown orchids worldwide. In order to prevent the spread of viruses in greenhouses and to cultivate clones from virus-free mother plants, it is necessary to develop a more sensitive technique for the detection of viruses in orchids. SYBR green real-time RT-PCR is a highly sensitive technique that can specifically detect ORSV in orchid tissue. By harvesting tissue at the inoculation site and at specific distances from the inoculation site at different times past inoculation, this technique can also be used to study the rate of spread of ORSV in orchids. Orchid clones were inoculated with ORSV and other clones were mock-inoculated with molecular grade water. Leaf tissue was harvested from the ORSV-inoculated and mock-inoculated clones at the site of inoculation and at specific distances from this site at 16 h, 24 h, and 72 h past inoculation. Total RNA was extracted from the harvested tissue. Competitive RTPCR was going to be used for the quantification and detection of ORSV in the samples, but attempts at cloning an ORSV fragment into a vector in order to form a competitive standard were unsuccessful. Instead, a highly sensitive qualitative approach called SYBR green real-time RT-PCR was used for the detection of ORSV. ORSV was detected in all virus-inoculated orchids, except for one. Therefore, all of the ORSV inoculated plants except for one were infected with the virus. Unexpectedly, ORSV was also detected in all of the mock-inoculated orchids. Most likely the orchids were previously infected with ORSV, but the viral titer was too low to be detected by commercial techniques. However, there is a small possibility that the orchids were contaminated during experimentation, despite careful technique. The rate of spread of the virus could not be studied because the mock-inoculated samples also contained the virus. Although viral amplification was demonstrated in the mock-inoculated plants, SYBR green real-time RT-PCR is still a sensitive and consistent method for ORSV detection in orchids. With additional controls, this method could prove to be the ideal method for reliable detection of ORSV in commercially-grown orchids.
Department of Biology

This project was designed to develop a method for the collection of environmental samples during prolonged Norovirus (NoV) outbreak investigations, and to develop real-time RT-PCR assays to analyze environmental samples for GI and GII noroviruses. The collection and processing of environmental samples could provide epidemiological data to facilitate investigations of prolonged NoV outbreaks and could guide public health NoV intervention strategies. Real-time RT-PCR assays for the detection of GI and GII NoVs were developed by adapting the State Hygienic Laboratory clinical GI and GII assays to the AB 7500 Fast platform. Analysis of the GI assay performance yielded a dilution curve slope = 3.28, R2 = 0.999 and a calculated amplification efficiency of 102%. The GII assay yielded a dilution curve slope = 3.39, R2 = 0.999 and a calculated amplification efficiency of 97%. Amplification efficiencies determine the sensitivity and the limit of detection of real-time RT-PCR assays. Optimum efficiencies range from 95%-105%, with a 100% efficiency indicating exponential amplification of targeted nucleic acid. To develop a method for the collection of environmental samples, multiple swab types were tested to determine their ability to recover NoV from laboratory spiked environmental surfaces. It was determined that foam swabs moistened with viral transport media were most effective in recovering NoV from spiked surfaces. A field test of the environmental sampling method was conducted by sampling environmental surfaces in four restaurants in one Iowa community. NoVs were not detected in the environmental samples. The collection and processing of environmental samples when conducting an investigation of a prolonged NoV outbreak could provide additional information on the epidemiology of NoV transmission and infection.

Introduction: Mycoplasma pneumoniae(M. pneumoniae) causes 10% to 30% of community-acquired pneumonia (CAP). The commonly used first-line antibiotic macrolide (ML) against respiratory tract infection may lead to the increase of ML-resistant M. pneumoniaeinfection. To resolve the problem, a rapid and accurate method for detection of ML-resistant M. pneumoniaeis necessary for treatment adjustment. Aims: The study aims to (1) develop a rapid method for diagnosis of ML-resistance of M. pneumoniaedirectly from clinical specimens; and (2) investigate the prevalence of M. pneumoniaeand ML-resistant M. pneumoniae. Methods: The M. pneumoniaeqPCR results of 689 respiratory tract samples from Queen Mary Hospital collected during April 2010 to May of 2012 were analyzed. Positive nucleic acid from M. pneumoniaeqPCR samples were tested with SimpleProbe real-time PCR coupled to melting curve analysis (SimpleProbe PCR), pyrosequencing and 23S rRNA gene sequencing(23S sequencing) for detection of ML-resistance. Results: A total of 111 samples (16.11%) in 689respiratory tract samples were found M. pneumoniaepositive by qPCR. Of 111, 96 positive nucleic acids were available for this study. Overall, 29 (30.21%, n=96) of ML-resistant M. pneumoniaewere found. 23S sequencing identified 28 mutants (29.17%) and 62 wild–type (64.58%), while 6 (6.25%) of them are failed to be identified. Pyrosequencing identified 28 mutants (29.17%) and 63 wild–type (65.63%), while 5 (5.21%) of them are failed to be identified. The SimpleProbe PCR identified 29 mutants (30.21%) and 65 wild–type (67.71%), while 2 (2.08%) of them are failed to be identified. All ML-resistant M. pneumoniaepositives were found to have A2063G mutation either by 23S sequencing or pyrosequencing. Conclusion: From this study, SimpleProbe PCR is the most sensitive and simple to perform. Therefore, it is highly recommended to be included in the routine testing with positive M. pneumoniaesamples for diagnosis of ML-resistant strain. 23S sequencing or pyrosequencing is recommended to use as a confirmatory test if necessary.
published_or_final_version
Microbiology
Master
Master of Medical Sciences

Influenza is a viral infection that affects global health and economy with its endemic and sometimes pandemic spread. Rapid detection of Influenza viruses enables antiviral use and can bring financial savings. It is also essential for the global surveillance of prevalent Influenza strains. RT-PCR is considered the most specific and sensitive method for detection of Influenza, but Influenza mutates at a high rate and it is therefore crucial that RT-PCR methods are updated regularly. In 2014, Cepheid released their Xpert Flu/RSV XC assay, which can detect Influenza A and B and RSV by multiplex RT-PCR in approximately one hour. The aim of this study was to evaluate this assay at Laboratoriemedicin Västernorrland by using the laboratory’s previous PCR assay for detection of Influenza viruses as reference method. Real-time RT-PCR was used to compare Xpert Flu/RSV XC to the reference method. A dilution series was performed to estimate the methods’ PCR efficiencies and precision was calculated from quadruplicates of a positive control sample. Clinical specimens (n=42) were used to evaluate the diagnostic sensitivity and specificity of Xpert Flu/RSV XC. Objective statistical analysis of PCR data was performed and discussed. The Xpert Flu/RSV XC was equivalent to the reference method and demonstrated high diagnostic sensitivity and specificity. Estimated PCR efficiencies were however low. With the introduction of Xpert Flu/RSV XC to the laboratory follows many potential benefits, primarily in form of a simplified pre analytical procedure and a shortened analysis time. The Xpert Flu/RSV XC assay enables fast diagnosis of Influenza infection.

Francisella tularensis is the etiological agent of tularemia, a zoonotic disease with worldwide prevalence. F. tularensis is a highly pathogenic organism and has been designated as a potential biothreat agent. Currently there are four recognized subspecies of F. tularensis: tularensis (type A), holarctica (type B), mediasiatica, and novicida. In addition, genomic studies have further subdivided type A tularensis into two subclassifications, type A.I and type A.II. These two subclassifications differ in geographic distribution with type A.I appearing mainly in the Eastern United States and type A.II appearing mainly in the Western United States. Because of differences of virulence among the subspecies, it is important to be able to quickly identify each of the subspecies rapidly and accurately. This work describes the development of a multiplex real-time polymerase chain reaction (PCR) assay which was shown to be ~98% successful at identifying the known subspecies of F. tularensis. Furthermore, F. tularensis is thought be a genome in decay (losing genes) because of the relatively large number of pseudogenes present in its genome. We hypothesized that the observed frequency of gene loss/pseudogenes may be an artifact of evolution in response to a changing environment, and that genes involved in virulence should be under strong positive selection. Eleven arbitrarily chosen virulence genes were screened for positive selection along with 10 arbitrarily chosen housekeeping genes. Analyses of selection yielded one housekeeping gene and 7 virulence genes which showed significant evidence of positive selection. Our results suggest that while the loss of functional genes through disuse could be accelerated by negative selection, the genome decay in Francisella could also be the byproduct of adaptive evolution, as evidenced by several of its virulence genes which are undergoing strong, positive selection.

Orientador: Glauco Issamu Miyahara
Coorientador: Daniel Galera Bernabé
Coorientadora: Kellen Cristine Tjioe
Banca: Maria José Hitomi Nagata
Banca: Luciana Estevam Simonato de Oliveira
Resumo: Objetivo: Avaliar a presença do HPV-16 em tecido fresco, saliva e plasma sanguíneo de pacientes com leucoplasia bucal pela real time PCR na região noroeste do estado de São Paulo, Brasil. Pacientes e métodos: Trinta e sete pacientes com diagnóstico de leucoplasia bucal foram incluídos no estudo. Destes, foram obtidos dados sociodemográficos, clinicopatológicos, estilo de vida e amostras de tecido fresco, sangue e saliva que foram armazenados a -80ºC para posterior análise molecular. Os materiais obtidos destes pacientes foram submetidos à detecção do DNA viral pela técnica da real time PCR com sonda específica para o HPV-16. Resultados: Dos 37 pacientes incluídos no estudo, 64,8% eram homens e a idade variou de 25 a 82 anos, com uma média de 58,72 anos. Dezesseis pacientes (43,2%) eram idosos e 43,2%, adultos de meia idade, e apenas 13,6%, adultos jovens. A maioria dos pacientes era fumante (72,9%), sendo que 16,3% eram ex-fumantes e 10,8%, não fumantes. Da mesma forma, a maioria (62,2%) era etilista, 21,6%, ex-etilistas e 16,2%, não-etilistas. Vinte e sete por cento das lesões apresentaram algum grau de displasia epitelial. A detecção do HPV-16 pela PCR em tempo real não foi positiva para nenhuma amostra, resultando em um índice de 0% de detecção. Conclusão: O HPV-16 não foi identificado na população estudada. No entanto, outros subtipos do HPV de baixo e alto risco podem estar associados à ocorrência de leucoplasia bucal nesta população, o que requer novas investigações. Es... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Objective: To evaluate the prevalence of HPV-16 DNA detection in fresh tissue, saliva and blood plasma from patients with oral leukoplakia by the real time PCR in the northwest region of the São Paulo state, Brazil. Patients and methods: Thirty-seven patients diagnosed with oral leukoplakia were included in the study. Sociodemographic, clinicopathologic and lifestyle data, fresh tissue, saliva and blood plasma samples were collected. Biologic material was stored at -80ºC and then submitted to viral DNA detection by the real time PCR technique with a probe specific for HPV-16. Results: Of the 37 patients included in the study, 64.8% were men, and the age ranged from 25 to 82 years, with a mean of 58.72. Sixteen patients (43.2%) were elderly, 43.2% were middle-aged adults, and only 13.6% were young adults. Most patients were smokers (72.9%), 16.3% were former smokers, and 10.8% were non-smokers. Most patients (62.2%) were current drinkers, 21.6% were ex-drinkers and 16.2% were non-drinkers. Twenty seven percent of the lesions presented some degree of dysplasia. HPV-16 detection by real-time PCR was not positive for any sample, resulting in a 0% detection rate. Conclusion: The HPV-16 was not identified in the population studied. However, other low and high-risk HPV subtypes might be associated to the occurrence of oral leukoplakia in this population, which requires further investigations. Broader epidemiological studies are required to clarify the geographic variability in the p... (Complete abstract click electronic access below)
Mestre

Os rotavírus são um dos principais agentes causadores de doenças entéricas em várias espécies animais, com ocorrência generalizada na suinocultura do Brasil. O seu diagnóstico é um componente essencial para estudos epidemiológicos e delimitação de medidas profiláticas visando o controle da doença. Apesar da relevância, não existem testes, tais como PCR em tempo real desenvolvido para detectar a diversidade genética de rotavírus suíno do grupo A (RVA). Este trabalho descreve o desenvolvimento de uma PCR em tempo real com SYBR® Green, para a detecção de rotavírus suíno e os seus resultados comparados com a PCR convencional e ELISA. Foram desenhados primers visando o segmento codificador da proteína NSP5 (137pb) e também foram utilizados primers visando o mRNA do gene mitocondrial bovino NADH5 (191pb) para o controle interno exógeno. Amostras de fezes de suínos de até 60 dias de idade de suínos do estado de São Paulo foram usadas para compor um painel de teste. Foram utilizadas como amostras de referencia o isolado 32/00 (controle positivo de rotavírus suíno do grupo A), concentrado de células MDBK (controle positivo do controle interno exógeno) e água tratada com DEPC (controle negativo). A extração do RNA total foi realizado com Trizol a partir de suspensões fecais contendo MDBK e o cDNA foi sintetizado utilizando primers aleatórios e M-MLV. Para as reações de PCR em tempo real utilizou-se o reagente MaximaTM SYBR Green qPCR Master Mix (Fermentas Life Science). Durante a padronização da PCR convencional, a temperatura de 54°C foi definida como a Tm ótima para a reação. O desempenho do ensaio foi validado em sete amostras positivas inicialmente testadas pelos métodos ELISA e PAGE. O isolado viral RV8209 foi utilizado para determinar o limiar de detecção da PCR em tempo real através de diluições seriadas sendo defino como ponto de corte Ct=33,5 (10-12,28TCID50%). O segmento codificador da NSP5 foi clonado no vetor pTZ57R/T submetido a restrição enzimática e usado como alvo para gerar uma curva padrão, onde obteve-se uma eficiência de 93,39%, slope de -3,49 e R2 de 0,993. A detecção do controle interno exógeno mostrou 82,9% de positividade para PCR convencional e 76,31% para a PCR em tempo real, com correlação significativa (0,718). O ensaio de ELISA para RVA apresentou 10,5% (8/78) de positividade, enquanto que as taxas de detecção da PCR em tempo real e PCR convencional foram de 50% (29/58) e 30,1% (24/63), respectivamente. Foi encontrada uma correlação moderada (0,546) entre PCR convencional e PCR em tempo real; baixa (0,056) entre a PCR convencional e ELISA; ausente (0,0) entre a PCR em tempo real e ELISA. Os resultados obtidos sugerem que a detecção por PCR em tempo real para a detecção do rotavírus suíno do grupo A em amostras fecais possa ser utilizada como diagnostico rápido e eficiente aumentando o repertório dos testes já estabelecidos, de modo a proporcionar uma maior sensibilidade para o diagnóstico clínico e epidemiológico.
Rotavirus is one of the main causative agents of enteric diseases in several animal species with widespread occurrence in Brazilian pig farm. Diagnosis is an essential component for epidemiological studies and delineation of prophylactic measures aiming disease control. Despite the relevance, there are no assays such as real-time PCR developed to detect the genetic diversity of porcine rotavirus from group A (RVA). This work describes the development of SYBR Green real-time PCR assay for the detection of porcine rotavirus and the results were compared with conventional PCR and ELISA. Primers were designed targeting the coding segment of the protein NSP5 (137pb) and also primers targeting the bovine mitochondrial gene mRNA NAD5 (191pb) were used for the exogenous internal control. Fecal samples from pigs up to 60 days of age from São Paulo state were used to compose a panel test. The reference sample was the isolate 32/00 (positive control for porcine rotavirus group A), concentrated MDBK cells (Exogenous Internal Positive Control) and DEPC-treated water (negative control). Total RNA extraction from supernatants of fecal samples containing MDBK cells were carried out with TRIzol reagent and cDNA was synthesized using random primers and M-MLV Reverse Transcriptase. For real-time PCR reactions were used MaximaTM SYBR Green qPCR Master Mix (Fermentas Life Science). For conventional PCR optimization, 54oC was defined as the optimum reaction temperature (Tm). The performance of the assay was validated on seven samples initially tested positive by ELISA and PAGE methods. The limit of detection of the developed real-time-PCR assay was determined using serial dilutions of the isolated RV8209 with Ct=33,5 (10-12,28TCID50%). The NSP5 gene segment was cloned into vector pTZ57R/T submitted to enzymatic restriction and used as template to generate a standard curve which Efficiency of 93.39%, slope of -3.49 and R2 □ 0.993. The Exogenous internal control showed 82.9% positivity for conventional PCR and 76.31% for real-time PCR with substantial correlation (0.718). The ELISA assay detected porcine RVA in 10,5% (8/78) of fecal samples, whereas the detection rates of both SYBR Green real-time PCR and conventional PCR assays were 50% (29 of 58) and 30.1% (24 of 63), respectively. A moderate correlation (0.546) was found between conventional PCR and real-time PCR; low (0.056) between the conventional PCR and ELISA; absent (0.0) between the real-time PCR and ELISA. Our findings suggest that detection of Group A porcine rotavirus in fecal samples by use of the real-time PCR assay may be fast and efficient increasing the repertoire of tests established to improve sensitivity for epidemiology and clinical diagnosis.

Strongyloides stercoralis (S. stercoralis) is an intestinal nematode mainly present in tropical areas of the world. Most infections with this parasite are asymptomatic, but in immunosuppressed patients S. stercoralis may disseminate throughout the body and cause gastrointestinal symptoms as well as shock, neurological malfunctions and septicemia. When patients are treated with immunosuppressive therapy, chronic infections may be reactivated. In some instances infection with S. stercoralis may be fatal. Entamoeba histolytica (E. histolytica) is a pathogenic amoeba that can induce both intestinal and extraintestinal infections. Infections with E. histolytica most commonly occur in developing countries. Differentiation between the pathogenic E. histolytica and the related apathogenic amoeba Entamoeba dispar (E.dispar) is not possible by microscopic examination. This master thesis, performed at the Swedish Institute for Communicable Disease Control (SMI), aimed at establishing a qualitative method for the detection of S. stercoralis with real-time PCR as it is a fast, accurate and highly sensitive technique for qualitative or quantitative genomic analysis. This would provide an improved preparedness for the diagnostic unit when physicians suspect infection with S. stercoralis. Another aim of the thesis was to establish a qualitative real-time PCR method for differentiation between E. dispar and E. histolytica. The current method for differentiation between these two species at SMI is traditional gel-based PCR. A transfer from PCR to real-time PCR would contribute to a less time consuming and more efficient diagnostic procedure. A TaqMan probe and primers specific for the gene encoding 18S ribosomal RNA (18S rRNA) for S. stercoralis were acquired and tested for use with the same PCR program and reagents as other real-time PCR methods performed at SMI. Primer and probe concentrations were optimized. The test of analytical specificity indicated that the primer/probe system was specific for S. stercoralis. The amplification efficiency and analytical sensitivity was also determined. Two systems specific for E. histolytica and E. dispar respectively targeting the gene encoding 18S rRNA were acquired and tested in the same manner. All optimized systems of the real-time PCR methods proved to be compatible with the desired instrument, PCR program and reagents. Weak signals were obtained with the E. dispar specific system when template of E. histolytica was added. With the knowledge of how to correctly interpret the result data, accurate diagnosis could still be obtained despite this issue. If the result from an investigation of accuracy is satisfactory, validation and subsequent implementation of the method for differentiation between E. histolytica and E. dispar is recommended. The optimization of the S. stercoralis specific method indicates that validation can be initiated.

Thesis (MScMedSc)--Stellenbosch University, 2011.
ENGLISH ABSTRACT: Background: The conventional sequence analysis is the most common method used for the detection of drug-resistant mutants. Due to its sensitivity limitations, it is unable to detect these mutants when comprising less than 20% (minor populations) of the total virus population in a sample. However, real-time PCR-based assays offer a rapid, sensitive, specific and easy detection and quantification of such mutants. The HIV-1 variants harbouring the K103N mutation are associated with resistance to nevirapine (NVP) and efavirenz (EFV). The persisting drug-resistant mutants decay slowly to low levels, and therefore they are called minor drug-resistant mutants. Consequently, they affect subsequent treatment with the drugs of the relevant class. Objectives: The objective of this study was to design two TaqMan real-time PCR-based assays called selective-polymerase chain reaction (SPCR), namely the total viral copy SPCR assay and the K103N-SPCR assay. The former detects HIV-1 of subtype C reverse transcriptase sequences, whereas the latter detects K103N drug-resistant variants in these sequences. Design and Methods: In developing the SPCR assays, sets of appropriate primers and probes for the HIV-1 subtype C reverse transcriptase (RT) were developed to use in the K103N-specific reaction and the total copy reaction. Twelve DNA plasmid standards with sequence diversity were constructed for the assay from two HIV-1subtype C samples known to harbour the K103N mutation (AAC or AAT) in our Department‟s Resistance Databank. Their RT regions were amplified, cloned and verified with sequencing. Site-directed mutagenesis was used to induce mutations at 103 amino acid position in some of these clones to generate more standards with either one of the three codons (AAA, AAC and AAT). The two assays were optimized and validated, and a standard curve was generated for each assay using 10-fold serial dilution (5x107-5x100 DNA copy/μL) of a K103N-mutant plasmid standard. The optimized and validated SPCR assays were used to screen 40 nested PCR products of previously genotyped patient samples for minor K103N variants. Results: Two sensitive and reproducible selective real-time PCR (SPCR) assays, with cut-offs of 8.23 and 10.33 and a detection limit of 0.01% for the K103N resistance variants, were successfully developed. The assays detected a prevalence of 25.64-46.15% for the K103N resistance mutation in 39 patient samples. The genotyping (population sequencing) missed 40-53.85% of these variants. Conclusion: In conclusion, sensitive and reliable selective real-time PCR assays to detect and quantify minor K103N variants of HIV-1 in nested PCR products were successfully developed. The assay had a lower detection limit of 0.01%.
AFRIKAANSE OPSOMMING: Agtergrond: Konvensionele volgorde bepaling analise is die mees algemeenste metode wat gebruik word vir die opsporing van middel-weerstandige mutasies, maar weens beperkte sensitiwiteit is dit nie moontlik om hierdie mutante op te spoor wanneer dit minder as 20% (minderheids populasie) van die totale viruspopulasie in `n monster uitmaak nie. Nietemin, kwalitatiewe PKR-gebaseerd toetse bied vinnige, sensitiewe, spesifieke en makliker opsporings en kwantifisering van sulke mutante aan. MIV-1 variante wat die K103N mutasie bevat word geassosieer met weerstand teen nevirapine (NVP) and efavirenz (EFV). Volhoudende middel-weerstandige mutasies vergaan stadig na laer vlakke en word daarom na minderheids middel weerstandige mutasies verwys. Gevolglik affekteer dit opvolgende behandeling met die middel van die relevante klas. Doelwitte: Die doel van die studie was om twee TaqMan kwantifiserende PKR gebaseerde selektiewe polymerase ketting reaksies (SPKR), naamlik totale virale kopie SPKR en K103N-SPKR te ontwikkel. Die voormalige toets het die MIV-1 subtipe C omgekeerde transkriptase volgorde bepaal, waar K103N die middel-weerstand variante in hierdie volgorde opspoor. Ontwerp en Metodes: `n Geskikte stel inleiers en peiler was ontwikkel vir die MIV-1 subtipe C omgekeerde transkriptase (OT) vir gebruik in die K103N-spesifieke en die totaal kopie reaksie. Twaalf DNS plasmied standaarde met volgorde diversiteit was saamgestel vir die toets vanaf twee MIV-1 subtipe C monsters wat volgens ons Departement se weerstand databasis geklassifeer is vir die besit van die K103N mutasie (AAC of AAT). Die OT streke was geamplifiseer, gekloneer en geverifieer deur volgorde bepaling. Punt-gerigte mutagenese is gebruik om `n mutasie by die amino suur posisie 103 van sekere klone te induseer om meer standaarde te genereer wat een van die drie kodons (AAA, AAC en AAT) bevat. Die twee toetse is geoptimiseer en gevalideer en `n standard kurwe is genereer vir elk van die toetse deur die gebruik van tienvoud serie verdunnings (107-1 DNS kopie/μL) van `n algemene K103N-mutante plasmied standard. Die geoptimiseerde en gevalideerde SPKR toets was gebruik om vir die minderheids K103N variante in 40 “nested” PKR produkte van voorheen gegenotipeerde pasiënt te soek. Resultate: Twee sensitiewe en herproduseerbare selektiewe kwantitiewe PKR toetse met `n ΔCt afsnypunt van 8.23 en `n deteksie limiet van 0.006% was ontwikkel vir die K103N weerstand variant. Die toets het `n voorkomsyfer van 25.6 % vir die K103N weerstand mutasie in 40 pasiënt monsters bepaal, waar genotipering (populasie volgorde ) 40% van hierdie variante nie opgespoor het nie. Gevolgtrekking: `n Sensitiewe en betroubare selektiewe kwantitatiewe PKR toets vir die opspoor en kwantifisering van die minderheids K103N variante van MIV-1 in PKR produkte was ontwikkel. Hierdie toets het `n laer opsporings limiet van 0.01%.
Poliomyelitis Research Foundation (PRF)
National Research Fund (NRF)
National Health Laboratory Service Research Trust (NHLS RT)

Brazil is the largest citrus producer in the world and has a large global citrus market share. However, several diseases affect the crop, being postbloom fruit drop (PFD) one of them. PFD has gained importance in São Paulo State for the displacement of citrus areas to regions with weather conditions more favorable for this disease. The accurate identification of the causal agent of the PFD has been performed and it was renamed as Colletotrichum abscissum. The origin of the initial inoculum is still an enigma for PFD epidemics and the hypotheses that the initial inoculum could be present in propagation material have been discussed but it has never been demonstrated. The objective of this work was to detect and quantify Colletotrichum abscissum from citrus leaves of budwood increase block and citrus nursery plants by qPCR. Four commercial citrus farms from São Paulo State, Brazil with budwood increase block and citrus nursery plants of Pera and Valencia sweet orange varieties were used for this work. C. abscissum was detected in budwood increase block and in nursery plant in both varieties (Valencia and Pera) at the four farms sampled. Out of 122 budwood increase block samples, 89 (73%) were positive for C. absicissum. From nursery plants, out of 175 samples, 129 (73%) were detected with the pathogen. The majority of the positive samples of budwood increase blocks and nursery plants contained 10 to 200 and 10 to 400 conidia of C. absicissum, respectively. With the methods used was not possible to isolate the fungus from vegetative material. This finding suggests a new long distances dispersion type of C. abscissum in the cycle of postbloom fruit drop by propagation material. Confirmation of C. abscissum in budwood increase block and nursery plants would lead to update regulations for the production of certified citrus nursery trees and searching for new control strategies of the pathogen.
O Brasil é o maior produtor de citros do mundo e possui uma grande participação no mercado global de citros. No entanto, várias doenças afetam a cultura, sendo uma delas a podridão floral dos citros (PFC). PFC ganhou importância no Estado de São Paulo pelo deslocamento de áreas de citros para regiões com condições climáticas mais favoráveis para a doença. A identificação precisa do agente causal do PFC foi realizada, tendo sido renomeado como Colletotrichum abscissum. A origem do inóculo inicial ainda é um enigma para as epidemias de PFC e as hipóteses do que o inóculo inicial poderia estar presente no material de propagação já foram discutidas, mas nunca foram demonstradas. O objetivo deste trabalho foi detectar e quantificar Colletotrichum abscissum em folhas de borbulheiras e mudas de citros por meio de qPCR. Neste trabalho, foram utilizadas quatro fazendas comerciais de citros do Estado de São Paulo, Brasil, com borbulheiras e viveiros de mudas de citros das variedades laranja Pera e Valência. C. abscissum foi detectado em borbulheiras e em mudas em ambas as variedades (Valência e Pêra) nas quatro fazendas amostradas. Das 122 amostras de folhas de borbulheiras, 89 (73%) foram positivas para C. absicissum. Das 175 amostras de folhas de mudas de citros, 129 (73%) foram detectadas com o patógeno. A maioria das amostras positivas de borbulheiras e mudas de citros continham 10 a 200 e 10 a 400 conídios de C. absicissum, respectivamente. Com os métodos utilizados, não foi possível isolar o fungo do material vegetativo. Esta descoberta sugere um novo tipo de dispersão a longas distâncias de C. abscissum no ciclo de podridão floral dos citros por meio do material de propagação. A confirmação de C. abscissum nas borbulheiras e mudas de citros levaria à atualização da regulamentação para a produção de mudas de citros certificadas e à busca de novas estratégias de controle do patógeno.

This study was carried out to analyze tomato samples and tomato seeds, purchased from different food markets of Turkey randomly, for the presence of genetic modification by using PCR method as it allows more specific detection. The DNAs of collected samples were isolated according to CTAB DNA extraction protocol and also with extraction kits. Screening tests of tomatoes were done by targeting 35S promoter, NOS terminator and NptII kanamycin resistance gene with eight different primer sets. Real time PCR is used to confirm 35S and NOS positives results obtained from conventional PCR. In this study, it was observed that 14 out of 35 seed samples, and 14 out of 40 fresh tomato samples which were screened had at least one transgenic element of 35S promoter, NOS terminator and NPTII kanamycin resistance gene indicating the possible presence of genetic modifications. After screening, gene specific studies were carried out for PG, sam-k indicating F type ripening delayed tomato and the 35 1 N lines respectively and cry1Ac genes inserted in 5345-1 insect resistant tomato line. PG and sam-k specific primers were not amplified in any of the samples investigated whereas 18 out of 75 samples were cry1Ac positive and 1 out of 75 samples was sam-k positive. Positives were confirmed by sequence analysis. Additionally, construct specific primers specific to 5345-1 and 35 1 N lines were designed. PCR amplicons indicate the existence of the construct sequence. In order to verify the results, PCR products were sent to sequence analysis

Amblyomma americanum, the lone star tick, is an indigenous tick species in southern Indiana that harbors a diverse group of pathogenic and nonpathogenic microorganisms, including Borrelia lonestari, the putative agent for the southern tick associated rash illness (START) and a spotted fever group rickettsial endosymbiont. The purpose of this study was to implement the real-time polymerase chain reaction (real-time PCR) as a molecular technique to examine the microbial diversity in A. americanum ticks by estimating abundances of different microorgansisms. A SYBR Green real-time PCR assay was designed to detect and quantify B. lonestari in A. americanum ticks, and a previously published TaqMan real-time PCR assay, designed to detect (not quantify) Rickettsia species in ticks, was validated for the detection and quantification of the spotted fever group rickettsial endosymbiont in A. americanum ticks. Many pitfalls associated with real-time PCR were experienced in this study, such as difficulties in assay design and problems with contamination, and appropriate modifications are recommended to laboratories routinely performing real-time PCR.
Department of Biology

The Simbu serogroup within the genus Orthobunyavirus belongs to the family Peribunyaviridae and comprises 32 recognised three-segmented negative-sense single-stranded RNA viruses, divided into two phylogenetic clades. Some members of this group of arthropod-borne viruses, cosmopolitan distributed, cause neurologic disease in humans as well as reproductive and neurologic disease in domestic animals, however, definitive diagnosis always requires laboratorial confirmation. Few real-time RT-PCR assays have been developed for the molecular diagnosis of Simbu serogroup orthobunyaviruses. There are two published methods with broad detection capacity, utilising either a SYBR Green based chemistry able to recognise viruses from both clades, which is not absolutely specific, or a TaqMan based chemistry that recognises only clade B viruses. A novel group-specific TaqMan-based real-time RT-PCR assay was developed, optimised and laboratory validated for the broad detection of the Simbu serogroup orthobunyaviruses. The published genomic data of the Simbu serogroup members were evaluated, and a conserved region, situated in the segment encoding the nucleocapsid protein, was selected to design a universal primer set and a pair of differently labelled hydrolysis probes, which allowed for the distinction between the two phylogenetic clades of the Simbu serogroup. Seven prototype Simbu serogroup isolates were used for the development of the assay, namely Akabane orthobunyavirus, Simbu orthobunyavirus, Shuni orthobunyavirus, Sathuperi orthobunyavirus, Shamonda orthobunyavirus, Ingwavuma virus and Sabo virus. The primer and probe concentrations in the reaction were optimised. Amplification efficiency was determined for each one of the viruses: AKAV (99%), SIMV (96%), SHUV (96%), SATV (97%), SHAV (84%), INGV (93%) and SABOV (110%). A panel constituted of genetically related, causative agents of abortion in ruminants and arthropod-borne viruses was selected for in vitro specificity analysis, and in silico analysis was also performed. The assay was shown to be specific, as no cross-reactions were observed either in vitro or in silico, and sensitive, with a 95% limit of detection ranging from 10-0,39 to 10-3,61 TCID50/reaction, for the detection of Simbu serogroup viruses. The repeatability of the assay was evaluated for both probes detection, using the intra- and inter-run standard deviations and coefficient of variation. This work resulted in a manuscript in submission process to a peer-reviewed journal. In addition, a comprehensive review of the viruses of the Simbu serogroup was carried out, including sites of viral isolation and seroconversion, and a map was generated using a geographic information system tool.
RESUMO - O serogrupo Simbu pertence ao género Orthobunyavirus, família Peribunyaviridae, e é constituído por 32 vírus de RNA tri-segmentado de cadeia simples e polaridade negativa, divididos em duas clades filogenéticas. Alguns membros deste grupo de vírus transmitidos por artrópodes, com distribuição cosmopolita, causam doença neurológica em humanos bem como doença reprodutiva e neurológica em animais domésticos, no entanto, o diagnóstico definitivo requer sempre confirmação laboratorial. Poucos ensaios de RT-PCR em tempo real têm sido desenvolvidos para o diagnóstico molecular destes vírus, ainda assim, existem dois que se destacam pela capacidade de detecção em largo espectro. Um deles, com sistema de detecção de fluorescência baseado na utilização de SYBR Green, é capaz de detectar vírus das duas clades, mas não é absolutamente específico, e o outro, baseando-se na utilização de sondas TaqMan, só detecta vírus de uma das clades. Um ensaio de RT-PCR em tempo real grupo-específico, inédito, com sondas TaqMan, foi desenvolvido, optimizado e caracterizado em termos laboratoriais, para a detecção em largo espectro dos orthobunyavirus do serogrupo Simbu. Os dados publicados referentes ao genoma dos membros do serogrupo foram analisados, e uma região conservada, situada no segmento codificante da proteína da nucleocápdise, foi eleita para o desenho de um par de primers específicos universal, bem como de duas sondas de hidrólise específicas, distintamente marcadas, e que, portanto, permitem a diferenciação entre as clades filogenéticas. Sete isolados de referência foram utilizados no desenvolvimento do ensaio, nomeadamente Akabane orthobunyavirus, Simbu orthobunyavirus, Shuni orthobunyavirus, Sathuperi orthobunyavirus, Shamonda orthobunyavirus, Ingwavuma virus e Sabo virus e, consequentemente, as concentrações dos primers e sondas na reação foram optimizadas. A eficiência de amplificação foi determinada para cada um dos vírus: AKAV (99%), SIMV (96%), SHUV (96%), SATV (97%), SHAV (84%), INGV (93%) e SABOV (110%). Um painel constituído por vírus geneticamente relacionados, agentes causais de aborto em ruminantes e transmitidos por artrópodes foi seleccionado no sentido de avaliar a especificidade do ensaio in vitro, tendo sido também efectuada uma análise in silico. O ensaio é específico, visto que não foram observadas reacções cruzadas quer in vitro quer in silico, e sensível, com um limite de detecção de 95% entre 10-0,39 a 10-3,61 TCID50/reacção, para a detecção de vírus do serogrupo Simbu. A repetibilidade foi avaliada para a detecção com ambas as sondas, pelo cálculo do desvio padrão intra- e inter-corridas bem como do coeficiente de variação. Este trabalho originou um manuscrito em processo de submissão para uma revista cientifíca. Além disso, foi levada a cabo uma revisão bibliográfica do serogrupo Simbu, incluindo locais de isolamento viral e seroconversão, e um mapa inédito desta distribuição foi gerado.
N/A

Verticillium wilt, caused by Verticillium dahliae Kleb, is one of the most economically important diseases of woody hosts such as ash (Fraxinus spp.), sugar maple (Acer saccharum), and redbud (Cercis canadensis). The causal agent has a broad host range, including not only woody hosts but also important vegetable and field crops, and it is distributed worldwide. Diagnosis of V. dahliae in infected woody hosts is often based on the occurrence of vascular discoloration and time-consuming isolation. However, not all woody hosts exhibit vascular discoloration symptoms, and not all vascular discoloration symptoms are due to infection by V. dahliae. In this study, real-time PCR-based assays were evaluated and employed for rapid and accurate detection of V. dahliae in different woody hosts. DNA was extracted in large quantities from presumptively infected woody hosts by collecting drill-press shavings from sample tissue, bead-beating, and extracting using a CTAB method. Six published primer sets were evaluated against genomic DNA of V. dahliae as well as selected negative controls, and two sets (VertBt-F/VertBt-R and VDS1/VDS2) showed promise for further evaluation using DNA extracts from field samples. The VertBt primers amplified a species-specific 115-bp fragment of the expected size, while the VDS primers amplified the expected specific 540-bp fragment. However, the VertBt primer set exhibited higher sensitivity in detection of V. dahliae even in asymptomatic trees. The PCR-based methods developed here could be used as rapid tools for pathogen detecting and monitoring, thus informing plant pathogen management decisions.

Association of Mycobacterium avium subspecies paratuberculosis (MAP) with Crohn's disease (CD) and not with ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), has been vigorously debated in recent years. This theory has been strengthened by recent culture of MAP from breast milk, intestinal tissue and Blood from patients with active Crohn's disease. Culture of MAP from clinical samples remained challenging due to the fastidious nature of MAP including its lack of cell wall in infected patients. The advent of real time PCR has proven to be significant in infectious disease diagnostics. In this study, real time reverse transcriptase PCR (RT-PCR) assay based on targeting mRNA of the IS900 gene unique to MAP has been developed. All variables included in RNA isolation, cDNA synthesis and real time PCR amplification have been optimized. Oligonucleotide primers were designed to amplify 165 bp specific to MAP and the assay demonstrated sensitivity of 4 genomes per sample. In hope this real time RT-PCR may aid in the detection of viable MAP cells in Crohn's disease patients, a total of 45 clinical samples were analyzed. Portion of each sample was also subjected to 12 weeks culture followed by standard nested PCR analysis. The samples consisted of 17 cultures (originated from 13 CD, 1 UC and 3 NIBD subjects), 24 buffy coat blood (originated from 7 CD, 2 UC, 11 NIBD and 4 healthy subjects) and 4 intestinal biopsies from 2 CD patients. Real time RT-PCR detected viable MAP in 11/17 (65%) of iii suspected cultures compared to 12/17 (70%) by nested PCR including 77% and 84% from CD samples by both methods, respectively. Real time RT-PCR detected MAP RNA directly from 3/7 (42%) CD, 2/2 (100%) UC and 0/4 healthy controls similar to results following long term culture incubation and nested PCR analysis. Interestingly, real time RT-PCR detected viable MAP in 2/11 (13%) compared to 4/11 (26%) by culture and nested PCR in NIBD patients. For tissue samples, real time RT-PCR detected viable MAP in one CD patient with the culture outcome remains pending. This study clearly indicates that a 12-hr real time RT-PCR assay provided data that are similar to those from 12 weeks culture and nested PCR analysis. Consequently, use of real time In our laboratory, we previously demonstrated a possible downregulation in the Interferon-gamma receptor gene (IFNGR1) in patients with active Crohn's disease using microarray chip analysis. In this study, measurement of RNA by real time qRT-PCR indicated a possible downregulation in 5/6 CD patients compared to 0/12 controls. The preliminary data suggest that downregulation in INFGR1 gene, and the detection of viable MAP in CD patients provides yet the strongest evidence toward the linkage between MAP and CD etiology.
M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology

Dissertação para obtenção do Grau de Mestre em Biotecnologia
Brettanomyces bruxellensis is a major threat to wine industry due to its spoilage ability characterized by high production of volatile phenols, mainly 4-ethylphenol. The horse sweat odor, characteristic of this phenol, causes large economic losses to wineries. A better understanding of the behavior of this yeast and better detection methods may lead to a decrease in 4-ethylphenol incidence in red wines worldwide. In the present work, we studied: (i) the ability of B. bruxellensis to enter the viable but nonculturable state by using both vital staining and plate counts to distinguish between viable and culturable cells; (ii) the production of 4-ethylphenol at different growth phases; (iii) the improvement of selective culture media; (iv) the application of a Real-Time PCR protocol for the rapid detection of B. bruxellensis. The existence of a viable but nonculturable state was evidenced during growth in synthetic medium ranging from 2% in strain ISA 2211 to 71% in strain ISA 1791 of the viable cells. The production rate of 4-ethylphenol was maximum when the precursor p-coumaric acid was added during exponential growth and decreased in stationary phase with incubation time. The developed selective medium presented recovery rates higher than the general purpose medium GYP and selectivity similar to DBDM. Response time lasted from 3 to 5 days while DBDM colonies appeared only after 12 days or more of incubation. Real-Time PCR showed to be an easy and faster method for a highly selective detection, taking 3 hours to obtain a positive response. The detection threshold was 700 cells/mL which may be decreased using sample concentration by centrifugation. However, results were 3.7 times higher than the viable counts, probably due to the DNA of dead or lysed cells. Collectively, this work represented a step forward in understanding the spoiling behavior of this yeast species and enabled the development of better detection methods for B. bruxellensis.

The research during this doctorate focused on the development of commercial genetic tests in the context of personalized medicine. In the first part of this project two assays were developed that allow allelic discrimination of two informative single nucleotide polymorphisms (SNPs) near the IL28B locus, which are useful for the management of patients with chronic hepatitis C infection. Hepatitis C is a significant health problem worldwide, with approximately 170 million infected people. For many years the standard of care (SOC) for HCV therapy has consisted in the administration of pegylated interferon α and ribavirin (PEG-IFN/RBV). With this therapy, however, around 80% of patients infected by genotype 2 or 3 of HCV and only 50% of those infected by genotype 1 or 4 reached a sustained virological response (SVR), which indicates complete eradication of the virus. In the last years, several studies have demonstrated that, among others, host genetic factors, such as different genotypes of rs12979860 and rs8099917 SNPs located upstream of the IL28B gene, determine the probability of attaining SVR. Patients homozygous for the favorable allele in both SNPs (CC for rs12979860 and TT for rs8099917) had a two- to three-fold higher likelihood to achieve spontaneous viral clearance in acute infections or SVR after treatment compared to patients heterozygous or homozygous for the unfavorable alleles (CT or TT for rs12979860 and TG or GG for rs8099917). Recently developed direct-acting antiviral agents (DAAs) inhibiting proteases involved in viral replication have been shown to significantly increase SVR rates in patients independently of the viral genotype, when combined with standard therapy. Two of those DAAs, boceprevir and telaprevir, were approved by the FDA in 2011 and many others are currently under investigation in order to develop an IFN-free SOC. Although the IL28B SNP genotype may have less effect on viral kinetics in the new combination therapies, there is evidence that it will continue to be relevant also in the context of IFN-free regimens. Therefore, genotyping of IL28B SNPs prior to therapy will remain fundamental to avoid ineffective treatments that are not only long and costly but, most importantly, are associated with significant side effects (e.g., flu-like syndrome, hematologic abnormalities and adverse neuropsychiatric events). The objective of this study was the development of two real-time PCR based tests for rs12979860 and rs8099917 genotyping using TaqMan® probes. Primers and probes that specifically bind the target region were designed. Probes were labeled with different fluorophores for each allele. Primer specificity to the target genomic sequence was determined using Blast and the probability of unwanted folding and secondary structures was checked with mfold. Starting from samples with a known genotype, positive controls for both assays were prepared by cloning the targeted gene region into a plasmid vector. Correct insertion of the target sequence into the plasmid was checked with Sanger sequencing. The reaction protocol was optimized by testing and comparing several amplification conditions with different master mixes and primer/probe concentrations. The reaction protocols were optimized to run on the following real-time PCR systems: Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR System, Applied Biosystems 7500 Fast and 7500 Fast Dx Real-Time PCR Systems, Applied Biosystems 7300 Real-Time PCR System, Bio-Rad Dx Real-Time System and Bio Rad CFX96™ Real-Time PCR Detection System. Both IL28B diagnostic devices were demonstrated to have high diagnostic specificity and sensitivity, 99.50% and 99.48% for rs12979860 and rs8099917, respectively, by analyzing around 200 samples that had been previously genotyped with a reference IVD. Using different concentrations of clinical samples, the assays were shown to work with DNA concentrations of 2 to 250 ng/µL. A reagent shelf-life of 12 months was determined following a stability study that assessed assay performances at different time points. At the end of this study, the final assays REALQUALITY RS-IL28B rs12979860 and REALQUALITY RQ-IL28B rs8099917 were notified with the Ministry of Health and placed on the market as CE-IVD commercial kits. By allowing simultaneous genotyping of both IL28B SNPs, these devices represent one of the most complete systems available for HCV patients management. Moreover, both kits have been shown to be highly sensitive and robust even under suboptimal conditions (after several freeze-thaw cycles of the amplification mix and with degraded DNA samples). The second part of this project focused on the development of two assays for the detection of the complete panel of mutations found in exon 9 of the CALR gene and for the detection and semi-quantification of the two most frequent mutations (W515L and W515K) in the MPL gene. Recently discovered somatic mutations in the gene encoding for calreticulin (CALR) have been demonstrated – together with JAK2 and MPL mutations – to be the driver mutations responsible for a subclass of myeloproliferative neoplasms (BCR-ABL1-negative MPN). To this group of MPNs belong polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF). Although mutations in these three genes are not disease-specific, they have been shown to be mutually exclusive and present in the vast majority of patients with these types of neoplasms. According to relative mutation frequencies the V617F mutation in exon 14 of JAK2 is at the first place, being present in almost all PV cases (the remaining cases carry mutations in exon 12 of this gene) and in most cases of ET and PMF. CALR mutations are the second most common genetic alterations in ET and PMF, whereas the mutations in codon 515 of exon 10 of the MPL gene are the third most frequent mutation encountered in ET and PMF. Mutations in JAK2, CALR and MPL are considered to have high diagnostic and prognostic value, and are used to monitor disease progression towards myelofibrosis and leukemia. In 2014, a new revision of the World Health Organization (WHO) criteria for diagnosis of BCR-ABL1-negative MPNs has been proposed including CALR mutations as major criterion for ET and PMF together with the above-mentioned JAK2 (V617F) and MPL mutations. Compared to patients with JAK2 V617F and MPL mutations, carriers of CALR mutations have lower hemoglobin levels, lower leukocyte counts, higher platelet counts and improved thrombosis-free survival. Since tests for the identification, semi-quantification and absolute quantification of the JAK2 V617F allele are already available at AB ANALITICA, this study focused on the development of tests for detection of mutations in the CALR and MPL genes. Different molecular biology technologies were used for these two assays. The test for CALR mutations detection was based on end-point PCR and agarose gel electrophoresis, whereas the assay for analysis of the two most common MPL mutations (W515L and W515K) used real-time PCR. Primers for both assays and TaqMan® MGB probes of the MPL W515L/K assay were designed and checked as described above. Plasmid positive controls were prepared using commercial cloning kits. The protocol of the CALR MUTATION assay was optimized by testing several amplification settings and conditions, different master mix, primer concentration and number of amplification cycles, as well as by refining the visualization step (variation of agarose type, agarose density, DNA-intercalating agent, loading volumes). The MPL W515L/K assay was tested on the Applied Biosystems StepOnePlus™ Real-Time PCR System with different master mixes and concentrations of primers and probes. The diagnostic specificity and sensitivity of CALR MUTATION assay were determined on a total of 65 clinical samples: 36 wild-type samples and 29 mutated samples containing the two most common CALR mutations (Type-1 and Type-2) as well as the so-called rare CALR mutations with a sequence length that differs by at least 4 base pairs from the wild-type sequence. The assay was able to correctly discriminate all samples tested, hence the test was assigned a diagnostic specificity and sensitivity of 100%. In addition, the assay correctly typed samples in a DNA concentration range of 25 to 100 ng/reaction and the measured detection limit was 10% of mutated allele on wild-type background. Although the stability study is still on-going, a shelf-life of 6 months was assigned to the device according to the European Standard EN 13640:20021. The primers for optional Sanger sequencing included with the device were shown to be highly specific for the target region. During the development of the assay for detection of W515L/K mutations in MPL, a cut-off of 1.5% of mutated allele was established. This cut-off allowed the correct identification of all tested wild-type samples (11 in total). The assay was able to detect and correctly semi-quantify 1/1 sample with the W515K mutation and 4/4 samples with the W515L mutation. When using samples with other mutations in or close to codon 515, no amplification signal of the mutated sequence was generated, due to the high specificity of the probes for the W515L and W515K mutations. As a conclusion of this study, the completed device GENEQUALITY CALR MUTATION was notified with the Ministry of Health and placed on the market as the first commercial CE-marked IVD device for detection and identification of mutations in exon 9 of the CALR gene. Concerning the MPL W515L/K assay, the design of new probes for the detection of all mutations in codon 515 of MPL was decided and is currently on going.
L’attività di ricerca di questo Dottorato ha avuto come obiettivo quello di sviluppare test genetici di interesse commerciale nell’ambito della medicina personalizzata. La prima parte di questo progetto ha riguardato lo sviluppo di due saggi per la discriminazione allelica di due polimorfismi a singolo nucleotide (SNP) vicini al locus di IL28B che svolgono un ruolo fondamentale nella gestione dei pazienti con epatite C cronica. L’epatite C è uno dei maggiori problemi di salute pubblica a livello globale; si stima infatti che vi siano circa 170 milioni di individui affetti da epatite C al mondo. Per molti anni la terapia definita come “standard of care” (SOC) per la cura dell’epatite C si è basata sulla somministrazione combinata di interferone alfa peghilato e ribavirina (PEG-IFN/RBV). Con questa terapia, però, circa l’80% dei pazienti infettati dai genotipi 2 e 3 di HCV e solo il 50% di quelli infettati dai genotipi 1 e 4 erano in grado di raggiungere una risposta virologica sostenuta (SVR) con l’eradicazione completa del virus. Negli ultimi anni, diversi studi hanno dimostrato in modo indipendente che fattori genetici legati all’ospite, quali il genotipo dei polimorfismi rs12979860 e rs8099917 a monte del gene IL28B, sono correlati alla probabilità di raggiungere SVR. Si è infatti osservato che i pazienti omozigoti per l’allele favorevole in entrambi i polimorfismi (CC per rs12979860 e TT per rs8099917) raggiungevano una clearance virale spontanea nelle infezioni acute oppure una SVR dopo il trattamento in quelle croniche, con una probabilità 2-3 volte maggiore rispetto ai pazienti eterozigoti o omozigoti per l’allele sfavorevole (CT o TT per rs12979860 e TG o GG per rs8099917). Recentemente, l’uso di nuovi agenti antivirali ad azione diretta (DAA) che inibiscono le proteasi coinvolte nel ciclo replicativo di HCV, in combinazione con la terapia standard ha permesso di ottenere un aumento significativo dei tassi di SVR nei pazienti indipendentemente dal genotipo virale dell’infezione. Due di questi, boceprevir e telaprevir, sono stati approvati dall’FDA nel 2011 e molti altri sono attualmente in fase di sviluppo per arrivare a definire una nuova terapia SOC non più basata sull’utilizzo di interferone. Nonostante l’effetto dei genotipi dei polimorfismi di IL28B sulla cinetica virale sembri risultare indebolito dall’uso di questi potenti agenti antivirali nella terapia, dati sperimentali indicano che le varianti genetiche di questi SNP rimarranno fortemente informative anche nel contesto dei futuri regimi IFN-free. Pertanto, la genotipizzazione degli SNP di IL28B prima della terapia è fondamentale allo scopo di evitare un trattamento inefficace caratterizzato non solo da tempi lunghi e costi elevati ma soprattutto da pesanti effetti collaterali (come ad esempio sindrome di tipo influenzale, anormalità ematologiche ed eventi avversi neuropsichiatrici). Lo scopo di questo studio è stato quindi sviluppare due test per la genotipizzazione di rs12979860 e rs8099917 basandosi sul metodo della real-time PCR con sonde TaqMan®. Sono stati disegnati i primer e le sonde per l’ibridazione specifica con la sequenza target di DNA. Le sonde sono state marcate con fluorofori specifici per ciascun allele. La specificità dell’appaiamento dei primer alla sequenza genomica di interesse è stata verificata mediante il tool bioinformatico di allineamento Blast mentre l’assenza di strutture secondarie o folding indesiderati è stata confermata con il programma mfold. Utilizzando campioni clinici con genotipo noto degli SNP di IL28B, i controlli positivi di entrambi i saggi sono stati preparati mediante clonaggio della regione target all’interno di un vettore plasmidico. La corretta inserzione della sequenza target nel plasmide è stata accertata mediante sequenziamento di Sanger. Il protocollo di reazione è stato ottimizzato confrontando tra loro diverse condizioni sperimentali con cui sono state testate diverse master mix di amplificazione e concentrazioni di primer e sonde. I protocolli di reazione sono stati ottimizzati per funzionare con i seguenti strumenti: Applied Biosystems StepOne™ e StepOnePlus™ Real-Time PCR System, Applied Biosystems 7500 Fast e 7500 Fast Dx Real-Time PCR Systems, Applied Biosystems 7300 Real-Time PCR System, Bio-Rad Dx Real-Time System e Bio Rad CFX96™ Real-Time PCR Detection System. Sensibilità e specificità diagnostiche sono state calcolate analizzando per ciascun saggio circa 200 campioni precedentemente genotipizzati con un IVD di riferimento, e sono risultate rispettivamente pari al 99,50% per rs12979860 e al 99,48% per rs8099917. Il range di concentrazioni del DNA entro il quale le performance del test rimangono invariate è stato determinato usando campioni di DNA a diverse concentrazioni. Si è visto che entrambi i saggi assegnano correttamente il genotipo a campioni con concentrazioni di DNA comprese tra 2 e 250 ng/µL. In seguito ad uno studio di stabilità durante il quale sono state misurate le performance del test a vari intervalli di tempo, è stato possibile attribuire ai reagenti di entrambi i saggi una shelf-life di 12 mesi. I test messi a punto in questo studio, REALQUALITY RS-IL28B rs12979860 e REALQUALITY RQ-IL28B rs8099917, sono stati infine notificati al Ministero della Salute e immessi nel mercato come kit commerciali CE-IVD. Questi dispositivi, permettendo la genotipizzazione dei due SNP di IL28B nella stessa seduta, rappresentano uno dei sistemi commerciali più completi per la gestione dei pazienti con HCV. Inoltre, entrambi i kit hanno dimostrato di essere molto sensibili ed affidabili anche in condizioni non ottimali (ad esempio dopo ripetuti cicli di gelo-scongelo della mix di amplificazione oppure utilizzando campioni degradati). La seconda parte di questo progetto ha avuto come scopo lo sviluppo di due saggi, uno per la rilevazione delle mutazioni note nell’esone 9 di CALR, l’altro per la rilevazione e la semi-quantificazione delle due mutazioni più frequenti di MPL (W515L e W515K). Le mutazioni somatiche nel gene codificante per la calreticulina (CALR) scoperte recentemente, assieme a quelle nei geni JAK2 e MPL, sono ritenute essere le mutazioni “driver” alla base della sottoclasse di neoplasie mieloproliferative (MPN) BCR-ABL1-negative. La policitemia vera (PV), la trombocitemia essenziale (ET) e la mielofibrosi primaria (PMF) fanno parte di questo gruppo di MPN. Le mutazioni in JAK2, CALR e MPL, pur non essendo specifiche per la malattia, sono presenti nella maggior parte dei pazienti con tali neoplasie in modo mutuamente esclusivo. La mutazione V617F nell’esone 14 di JAK2 è la più frequente in tutte le patologie considerate, essendo presente nella quasi totalità dei pazienti con PV (nei rimanenti casi si riscontrano mutazioni nell’esone 12 di JAK2) e nella maggior parte dei pazienti con ET o PMF. Le mutazioni di CALR sono presenti con una frequenza inferiore rispetto a JAK2 V617F nelle ET e PMF. Infine, le mutazioni nel codone 515 dell’esone 10 di MPL sono quelle presenti con la minor frequenza nelle ET e PMF. Le mutazioni in questi tre oncogeni, oltre che per la diagnosi e la prognosi, vengono anche utilizzate per monitorare la progressione della malattia verso forme più aggressive, quali leucemia e mielofibrosi. I criteri della WHO (World Health Organization) per la diagnosi delle MPN BCR-ABL1-negative, in particolare ET e PMF, sono attualmente in stato di revisione a seguito della proposta avanzata nel 2014 per l’inserimento delle mutazioni di CALR tra i criteri diagnostici maggiori accanto alle mutazioni di JAK2 (V617F) e MPL già presenti. È stato inoltre osservato che le mutazioni di CALR, rispetto a JAK2 V617F e alle mutazioni di MPL, sono associate ad un fenotipo caratterizzato da livelli inferiori di emoglobina, minor numero di leucociti, maggior numero di piastrine e maggiore sopravvivenza priva di trombosi. Siccome AB ANALITICA ha già sviluppato dei test per l’identificazione, la semi-quantificazione e la quantificazione assoluta dell’allele JAK2 V617F, questo studio aveva l’obiettivo di sviluppare due test per la rilevazione delle mutazioni in CALR ed MPL al fine di completare il pannello delle principali mutazioni associate alle MPN BCR-ABL1-negative. Dovendo rilevare mutazioni di tipo diverso (inserzioni e/o delezioni in CALR e due mutazioni puntiformi in MPL), si è scelto di usare per i due saggi due differenti tecnologie, vale a dire end-point PCR con elettroforesi in gel d’agarosio per le mutazioni di CALR e real-time PCR per le mutazioni W515L e W515K di MPL. Seguendo il modello descritto in precedenza, sono stati disegnati i primer di entrambi i test e le sonde TaqMan® MGB per il saggio di MPL W515L/K ed è stato verificato il loro appaiamento specifico alla regione target. I controlli positivi plasmidici di entrambi i sistemi sono stati preparati utilizzando kit commerciali per il clonaggio. Il protocollo del saggio per la rilevazione delle mutazioni di CALR è stato ottimizzato dopo aver testato diverse condizioni sperimentali sia per la reazione di amplificazione (variando master mix, concentrazione dei primer, numero di cicli di amplificazione) che per la visualizzazione (variando tipo di agarosio, densità del gel, agente intercalante del DNA, volumi da caricare nel gel). Una fase preliminare di messa a punto del saggio per la rilevazione delle mutazioni di MPL è stata effettuata sullo strumento StepOnePlus™ (Applied Biosystems) testando diverse master mix e concentrazioni di primer e sonde. Sensibilità e specificità diagnostiche del saggio delle mutazioni di CALR sono state calcolate utilizzando un totale di 65 campioni clinici di cui 36 wild-type e 29 mutati. Tutte le mutazioni analizzate in questo studio (Tipo-1, Tipo-2 e mutazioni rare) sono state precedentemente determinate mediante sequenziamento bidirezionale con cui si è visto che alterano la lunghezza della sequenza di almeno 4 paia di basi rispetto alla sequenza wild-type. Il saggio ha dimostrato di saper discriminare correttamente lo status di tutti i campioni testati (mutato vs wild-type), perciò gli è stata attribuita un’accuratezza (osservato/atteso) del 100%. Testando diluizioni seriali di allele mutato in un background di allele wild-type è stato identificato il limite di rilevabilità del saggio pari al 10% di allele mutato. Il test ha inoltre dimostrato di discriminare correttamente campioni con concentrazioni di DNA tra 25 e 100 ng/reazione. Nonostante lo studio di stabilità sia attualmente in corso, è stata attribuita ai reagenti una shelf-life di 6 mesi secondo la norma europea EN 13640:20021. I primer per il sequenziamento di Sanger (facoltativo) che consente l’identificazione della mutazione sono risultati altamente specifici per la regione target. Nelle prove preliminari del saggio per la rilevazione delle mutazioni W515L/K di MPL, è stato stabilito un cut-off dell’ 1,5% di allele mutato con cui sono stati correttamente discriminati 11/11 campioni wild-type. Il saggio è stato in grado di rilevare e semi-quantificare correttamente 1/1 campione con la mutazione W515K e 4/4 campioni con la mutazione W515L. Utilizzando però campioni con una mutazione diversa nel codone 515 o nelle sue vicinanze non è stato riscontrato alcun segnale di amplificazione relativo alla sequenza mutata, indice di una elevata specificità delle sonde per le due mutazioni W515L e W515K. In conclusione, questo studio ha portato allo sviluppo del dispositivo GENEQUALITY CALR MUTATION che è stato notificato al Ministero della Salute ed è il primo kit CE-IVD che permette di rilevare ed identificare le mutazioni nell’esone 9 del gene CALR. Per quanto riguarda il saggio di MPL W515L/K, è stato deciso ed è attualmente in corso il disegno di nuove sonde per la rilevazione di tutte le mutazioni nel codone 515 di MPL.