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Dissertations / Theses on the topic "Detection PCR real-time"
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Malatji, Dikeledi Petunia. "Detection of Babesia rossi genotypes using real-time PCR." Diss., University of Pretoria, 2011. http://hdl.handle.net/2263/31138.
Full textAbstract:
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.<br>Dissertation (MSc)--University of Pretoria, 2011.<br>Veterinary Tropical Diseases<br>MSc<br>Unrestricted
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Landgraf, Maria. "Detection of food relevant filamentous fungi by real time PCR." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=98023946X.
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Afshari, Kashanian Elisa. "Detection of celery (Apium graveolens) in food with Real-Time PCR." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7130.
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<p>Directive EC 2003/89/EC of the European Parliament and of the Council states that certain</p><p>ingredients and products derived there of known to cause allergen reactions must always be</p><p>declared. Furthermore labelling is mandatory irrespective of the amount included. The National</p><p>Food Administration therefore needs methods for monitoring the presence of allergens in food.</p><p>Methods already exist for most of the allergens on the EU-list, but an operational method for</p><p>celery (Apium graveolens) is missing.</p><p>A specific DNA-method was developed, based on TaqMan Real-Time PCR with the celery</p><p>mannitol dehydrogenase gene as target sequence. The analysis was started with homogenisation</p><p>of the sample followed by extraction of DNA. The Real-Time PCR method was shown to be</p><p>specific for celery, producing a 113 bp fragment with two celery varieties and negative results</p><p>with other closely selected species commonly present together with celery in food products (12</p><p>samples). The detection limit was 2-20 pg DNA, which corresponds to 1-7 haploid genome</p><p>copies. When evaluated with model samples of celery in meat, a detection limit of less than</p><p>0,01 % was determined. When used to analyse food products from the market, six out of seven</p><p>products declared to contain celery were correctly identified as positive.</p>
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Muradrasoli, Shaman. "Detection and Quantification of Variable Viral RNA by Real-Time PCR Assays." Doctoral thesis, Uppsala universitet, Klinisk virologi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9193.
Full textAbstract:
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.
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Leopold, Luciana Eleanor Dittmer Dirk Peter. "Development of real-time PCR assays for the quantitative detection of herpesviruses." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,1487.
Full textAbstract:
Thesis (M.S.)--University of North Carolina at Chapel Hill, 2008.<br>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.
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Elfaitouri, Amal. "Development of Real-Time PCR Based Methods for Detection of Viruses and Virus Antibodies." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7320.
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Lee, Yu-yan, and 李羽殷. "Detection of influenza C virus in pediatric respiratory specimens by real-time PCR." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/193539.
Full textAbstract:
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.<br>published_or_final_version<br>Microbiology<br>Master<br>Master of Medical Sciences
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Chia, Helena Nien-Hwa 1982. "Development of tissue printed nitrocellulose cards/arrays for real time PCR amplification and detection." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/32828.
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Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2004.<br>Includes bibliographical references (leaf 21).<br>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.<br>by Helena Nien-Hwa Chia.<br>S.B.
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Malan, Stefanie. "Real time PCR as a versatile tool for virus detection and transgenic plant analysis." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/1921.
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Thesis (MSc (Genetics))--University of Stellenbosch, 2009.<br>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.<br>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.
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Amiri, Mehdi. "Real-time PCR detection and PFGE typing of Pseudomonas aeruginosa from cystic fibrosis patients." Doctoral thesis, Università Politecnica delle Marche, 2016. http://hdl.handle.net/11566/243098.
Full textAbstract:
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.