Academic literature on the topic 'Diagnostic Assay Development'

Detta projekt var ett samarbete mellan Aplex Bio AB och Scilifelab. Projektets mål var att utveckla en molekylär diagnostisk panel med förmågan att detektera och diskriminera mellan 14 olika typer av patogener. Projektet innehåller 4 kapitel med fokus på olika mål. I första kapitlet utvecklades en metod för att karakterisera emissioner av fluorescerande nanopartikel kluster. Den första utvärderade metoden utnyttjade klick-kemi för att binda nanopartiklarna till makrostrukturer uppbyggda av amplifierat DNA. Den andra utvärderade metoden skapade aggregerade komplex av nanopartiklar med amplifierat DNA för att utvärdera partiklarnas emissioner. I kapitel 2 av projektet användes azid-funktionaliserade nanopartiklar levererade av Aplex Bio AB för att tvärbinda DBCO modifierade oligonukleotider. Sedan utvecklades en hybridiserings baserad metod för att kvantifiera relativa mängden oligonukleotider på partiklarna. Denna metod användes för att reproducerbart funktionalisera partiklar och utveckla nanopartikel-sonder som kan binda till DNA genom hybridisering. I kapitel 2 utvärderades även hur effektivt och specifikt de utvecklade nanopartikel-sonderna hybridiserar till DNA. I kapitel 3 utvärderades amplifiering av syntetiska ssDNA sekvenser valda från genetiska markörer av 14 patogener, DNA amplifierades med metoden RCA. Målet var att utvärdera specificiteten av amplifieringen. Specifik amplifiering av varje DNA sekvens i panelen var en förutsättning för att möjliggöra detektion och diskriminering av alla patogener i panelen. I kapitel 4 var målet att utveckla en kostnadseffektiv metod för att funktionalisera nanopartiklar med oligonukleotid sekvenser. För att göra detta användes DBCO-NHS-ester reagens och amin-modifierade oligonukleotider. Förverkligande av detta projekt skulle skapa en diagnostisk panel med potentialen att påverka det diagnostiska fältet på en global skala. När detta projekt är fullt utvecklat kan panelen modifieras för detektion av önskade DNA/RNA sekvenser vilket möjliggör ett mångfalt av applikationer, detta skulle göra panelen konkurrerande med dagens diagnostiska metoder då den kan användas i existerande mikroskopiuppsättningar.<br>This work was a collaboration between Aplex Bio AB and Scilifelab with the aim of developing a molecular assay capable of detecting and discriminating between 14 different pathogenic targets. There are 4 chapters with focus on different goals. In chapter one a method of evaluating emissions of fluorescent nanoparticle clusters was developed. The first approach of evaluating nanoparticle emissions was to utilize click chemistry to bind nanoparticles to macroscale structures of amplified DNA targets. The second evaluated approach was the formation of aggregated complexes of nanoparticles and amplified DNA targets. The second chapter of the thesis used azide functionalized nanoparticles supplied by Aplex Bio AB to utilize azide groups as crosslinkers and use them to functionalize the nanoparticles with DBCO oligos. A hybridization-based method was then developed to quantify relative oligo densities on the nanoparticles, enabling reproducible oligo functionalization of nanoparticles, producing nanoparticle probes that can bind to DNA. The final task of chapter 2 was evaluating the binding efficiency and specificity of the developed nanoparticle probes. The third chapter of the thesis evaluated amplification of synthetic ssDNA sequences corresponding to genetic markers of 14 pathogenic targets using RCA. The goal was to confirm specificity of chosen padlock probes and corresponding synthetic targets for each pathogen. Specific amplification of each target was a prerequisite to enable detecting and discriminating between the 14 pathogenic targets. In chapter 4 the goal was to develop a cost-effective method of oligo functionalization for nanoparticles. This chapter evaluated two main approaches of using DBCO-NHS-ester reagents to perform DBCO modification of amine-oligos. The realization of this work would develop an assay that has the potential to impact the field of diagnostics on a global scale. When fully developed, the molecular assay can be modified to detect any RNA/DNA targets which enables numerous applications, making the assay a competitive diagnostic tool which can be implemented in existing microscopy systems.

Thesis (MSc)--Stellenbosch University, 2015.<br>ENGLISH ABSTRACT: The nepoviruses are a group of nematode-transmitted plant viruses that are distributed worldwide and infect a wide range of plant species, including grapevine. Most of the nepoviruses are foreign to South Africa and to date, only Grapevine fanleaf virus (GFLV) is present. The Department of Agriculture, Forestry and Fisheries (DAFF), as the official National Plant Protection Organisation (NPPO) of South Africa, is committed to prevent the importation and spread of plant pathogens by administering the Agricultural Pests Act, 1983 (Act No. 36 of 1983). Effective measures are implemented by which the introduction of agricultural pests may be prohibited to safeguard the agricultural environment. One of the core functions of DAFF is to render a routine plant health diagnostic service for imported plants and plant products to prevent exotic pathogens from entering the country. The objective of this study was to develop a diagnostic assay for the detection of nepoviruses in grapevine. The project aimed to produce antibodies by recombinant DNA technology against bacterially expressed viral coat protein of a specific nepovirus [Tomato ringspot virus (ToRSV)] and subsequently develop a DAS-ELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) assay for the detection of the virus. The coat protein (CP) was successfully isolated from imported ToRSV-infected grapevine material. Two expression systems were utilised for expression of the ToRSV-CP, the GST gene fusion system and an Agrobacterium-mediated expression system. The GST gene fusion system was unsuccessful as insufficient soluble protein expression prevented the production of antibodies and thus the development of the DAS-ELISA assay. Tissue print immunoassay (TPIA) initially showed positive results for transient expression of the fusion protein in tobacco plants, but further confirmation proved to be inconclusive. The project also aimed to develop a real-time PCR assay for the specific detection and relative quantification of GFLV, based on a conserved region of the RNA-2 genome. A partial GFLV-RNA-2 from a South African isolate of grapevine was sequenced and used for the design of specific primers. The quantitative real-time PCR assay based on SYBR green technology proved to be sensitive in detecting levels as low as 0.11ng/reaction in infected plants, making it a highly effective diagnostic tool for the detection of GFLV.<br>AFRIKAANSE OPSOMMING: Die nepovirusse is 'n groep van nematode-oordraagbare plant virusse wat wêreldwyd versprei word en 'n wye verskeidenheid van plantspesies infekteer, insluitend wingerd. Die meeste van die nepovirusse is uitheems aan Suid-Afrika en tot op datum is net Wingerd netelblaar virus (GFLV) teenwoordig. Die Departement van Landbou, Bosbou en Visserye (DAFF), as die amptelike Nasionale Plant Beskermings Organisasie (NPBO) van Suid-Afrika, is daartoe verbind om die invoer en verspreiding van plantpatogene te voorkom deur administrasie van die Wet op Landbouplae, 1983 (Wet No. 36 van 1983). Doeltreffende maatreëls word geïmplementeer waardeur die invoer van landbouplae verbied word om sodoende die landbou-omgewing te beskerm. Een van die kernfunksies van DAFF is om 'n roetine plant gesondheid diagnostiese diens vir ingevoerde plante en plantprodukte te lewer om te verhoed dat eksotiese patogene die land binnedring. Die doel van hierdie studie was om 'n diagnostiese toets vir die opsporing van nepovirusse in wingerd te ontwikkel. Die projek was daarop gemik om antiliggame te vervaardig deur rekombinante DNA-tegnologie teen bakterieël-uitgedrukte virale mantelproteïen van 'n spesifieke nepovirus [Tomato ringspot virus (ToRSV)] en vervolgens ‘n DASELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) toets vir die opsporing van die virus te ontwikkel. Die mantelproteïen (CP) is met sukses geïsoleer vanaf ingevoerde ToRSV-besmette wingerdmateriaal. Twee uitdrukking stelsels is gebruik vir uitdrukking van die ToRSV-CP, die “GST gene fusion” stelsel en 'n Agrobacterium-bemiddelde uitdrukking stelsel. Die “GST gene fusion” stelsel was egter onsuksesvol aangesien onvoldoende oplosbare proteïen uitdrukking die produksie van antiliggame en dus die ontwikkeling van die DAS-ELISA toets verhoed het. “Tissue print immunoassay” (TPIA) het aanvanklik positiewe resultate getoon vir tydelike uitdrukking van die fusie proteïen in tabakplante, maar verdere bevestiging was onoortuigend. Die projek was ook daarop gemik om ‘n in-tyd polimerase ketting reaksie (PKR) toets vir die spesifieke opsporing en relatiewe kwantifisering van GFLV, gebaseer op 'n gekonserveerde volgorde van die RNA-2 genoom, te ontwikkel. 'n Gedeeltelike GFLV-RNA-2 nukleïensuurvolgorde van 'n Suid-Afrikaanse wingerd isolaat is bepaal en gebruik vir die ontwerp van spesifieke inleiers. Die kwantitatiewe in-tyd PKR toets gebaseer op SYBR groen tegnologie was sensitief genoeg om vlakke van so laag as 0.11ng/reaksie in geïnfekteerde plante op te spoor, wat dit 'n hoogs effektiewe diagnostiese hulpmiddel vir die opsporing van GFLV maak.

The aim of this project was to develop a serological assay based on recombinant antigens derived from the OvHV-2 genome and validation of the ELISA test using clinical serum samples. Eight OvHV-2 genes were selected for expression in three bacterial systems. Four genes could in at least one system. Three genes were expressed in an <i>in vitro</i> transcription-translation system (Ov8, OV8.5 and orf65). Protein purification, using epitope-tag affinity purification and conventional chromatography, was unsuccessful. Due to the lack of recombinant antigen expression, crude antigen preparations from cultured AIHV-1 and OvHV-2 infected cells were analysed. Antigens suitable for the detection of MCF virus specific serum antibodies could not be extracted from an OvHV-2 infected cell line but AIHV-1 WC11 antigen could be used to differentiate MCF-positive and –negative sera. An ELISA based on this crude antigen was established, designated WC11-ELISA. Initial validation revealed over 93% concordance between WC11-ELISA and commercial ELISA, while a comparison with serological assays, a PCR test and final veterinary evaluations, showed good agreement between tests. The WC11-ELISA showed 57% agreement to PCR while commercial CI-ELISA showed 66% agreement for the same samples. This indicates the value of the ELISA as an economical, rapid and specific serological assay for the detection of antibodies against MCFV. Potentially antigenic components of the ELISA lysates were analysed by western blotting in comparison with other bovine herpesviruses, and fractionation by gel filtration showed clear antigenic bands in the eluent. Proteomic analysis of antigenic bands identified proteins encoding viral thymidine kinase (orf21), a virus capsid protein (orf25) and a viral antigen (orf54) which encodes a dUTPase.

Toxoplasma gondii (T. gondii) is a ubiquitous parasite that infects warm-blooded animals and humans. In humans, T. gondii causes encephalitis in AIDS patients, and there is no drug that can eliminate T. gondii infection. T. gondii specifically manipulates the intermediate host’s behaviour favouring its transmission to the definitive feline host. Human T. gondii seropositivity has also been associated with mental disorders. T. gondii has two aromatic amino acid hydroxylases, TgAaaH (1 and 2), that convert phenylalanine to tyrosine, and tyrosine to L- DOPA, the latter being the rate-limiting step of dopamine biosynthesis. Based on this and elevated dopamine levels in brain tissue cysts and infected dopaminergic cells, it has been hypothesised that TgAaaH has a role in altering brain neuromodulation and potentially subsequently in the behavioural changes observed. As TgAaaH genes encode a signal peptide, the location of the enzyme was examined. TgAaaH was localised to outside the parasite both membrane- bound to parasites within the parasitophorous vacuole based on immunofluorescence, fractionation, and trypsin susceptibility of released parasites. Another possible role of TgAaaH in cyst wall generation was examined by testing for dopa-oxidase activity to convert L-DOPA to dopa- quinone. Dopa-oxidase activity was not detectable in infected fibroblasts, yet it remains possible that parasite produced L-DOPA is metabolised to dopaquinone by host cell enzymes such as within feline gut endothelial cells where oocysts are formed. This, and our finding that host cell dopa-decarboxylase is required for dopamine biosynthesis, suggest that the product of parasite-produced L-DOPA may be dependent upon the type of cell infected (ie. dopamine in catecholaminergic cells and dopaquinone in endothelial cells). The limitation of studying the bradyzoite stages of infection due to proliferation of tachyzoite stages was resolved by development of a new culture system with depleted tryptophan. This method was then applied to develop a novel throughput assay to identify bradyzoite inhibitors. The validity of this assay was evaluated using tachyzoite and bradyzoite specific inhibitors. This assay will help in finding an anti-toxoplasma drug for curing of infection.

Improved diagnosis, prognosis and disease follow-up is a fundamental procedure and a constant challenge in medicine.  Among the different molecular biomarkers, proteins are the essential regulatory component in blood; hence, by developing enhanced specific and sensitive molecular tools will gives great insight into the different processes in disease treatment.  In this thesis, we build on the proximity ligation assay to develop and apply new adaptable methods to facilitate protein detection. In paper I, I present a variant of the proximity ligation assay (we call PLARCA) using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer. PLARCA detected femtomolar levels of these proteins in patient samples, which was considerably below the detection threshold for ELISA. In paper II, we developed and adapted a new method into the in situ PLA methods for detection and identification of extracellular vesicles (EVs) using flow cytometry as readout (a method we call ExoPLA).  We identified five target proteins on the surface of the Evs and using three colors, we identified the EV using flow cytometer. In paper III, we aim to improve the efficiency of in situ PLA by creating and developing new designs and versions of the assay we called Unfold probes Through comparison of detection of protein using in situ PLA versus Unfold probes, we observed considerable decrease in non-specific signals, and also a lower detection threshold. In paper IV, we describe the development of a solid phase proximity extension (sp-PEA) assay for protein detection and quantification. We compared detection of IL-8, TNF-alpha, IL-10 and IL-6 using spPEA and PEA; spPEA demonstrations over 2 orders of magnitudes in the lower detection concentrations by decreased in background noise.

Background: Respiratory diseases are common worldwide, which are caused by various respiratory viruses. As symptoms caused by these viruses are similar, laboratory diagnosis is essential to distinguish the virus. Conventionally, respiratory viruses are isolated by cell culture with a panel of cell lines. However, handling of several cell lines is labour intensive, and the turnaround time of conventional culture is long. In previous study, the use of human colon adeno-carcinoma (Caco-2) in conventional culture was investigated. The study has proven that Caco-2 is generally susceptible to the eight common respiratory viruses, i.e. Adenovirus, Influenza A and B, Respiratory Syncytial virus, Parainfluenza virus 1, 2,3 and 4. As turnaround time of conventional culture is long; therefore, in this study, rapid shell vial culture using Caco-2 cells were evaluated. Moreover, the application of Caco-2 shell vial culture on recovering human metapneumovirus (hMPV) was also investigated. Materials and methods: This study consisted of four stages. First, recovery of viruses by conventional culture and shell vial culture of Caco-2 were compared. Specimens were added to conventional culture and shell vial simultaneously. For conventional culture, formation of CPE was examined daily and IF staining was performed when CPE was indicated; meanwhile, shell vial culture were incubated for seven days and stained with IF to detect infected cells. In stage two, the effect of incubating shell vial culture in rolling drum was investigated. Shell vials inoculated with the same specimen in duplicate were incubated in rolling drum and without rolling drum simultaneously. IF staining was performed in day 2, and results were obtained. For those which are IF negative in day 2, second shell vial was further incubated to seven days before harvest. In the next stage, a large batch of samples was used to evaluate on the use of Caco-2 shell vial culture in day 2 and day 7. Lastly, Caco-2 shell vial and conventional culture and LLC-MK2 conventional culture were tested for isolation of hMPV. Results: Compared to Caco-2 conventional culture, recovery rate of shell vial culture was elevated slightly. When experimenting on the effect of incubation in rolling drum, results showed that recovery rate was raised in shell vial with rolling drum in day 2, moreover, the percentage of positive cells were increased significantly (p value < 0.05). Furthermore, in the evaluation of Caco-2 shell vial in day 2 and day 7, 75% of samples were isolated in day 2 while 85% were recovered in day 7. Lastly, in the investigation on recovery of hMPV, 53%, 42% and 17% hMPV positive cases were isolated by Caco-2 shell vial, Caco-2 conventional culture and LLC-MK2 conventional culture respectively. Conclusion: First, although recovery rate by shell vial and conventional culture were similar, turnaround time was reduced from a week to a few days by shell vial culture. Therefore, Caco-2 shell vial culture is a more efficient than Caco-2 conventional culture in isolating respiratory viruses. The study also showed that incubation of shell vial in rolling drum able to increase the number of positive cells. Furthermore, in this study, Caco-2 cells were also shown to be more efficient in isolating hMPV when compare to LLC-MK2 cells.<br>published_or_final_version<br>Microbiology<br>Master<br>Master of Medical Sciences