Development Of Dna-Based Diagnostic Tests For The Detection Of Shigella Dysenteriae, Shigella Flexneri And Shigella Sonnei

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dc.contributor.authorMohamed Shakrin, Nik Noorul Shakira
dc.date.accessioned2018-06-12T02:40:16Z
dc.date.available2018-06-12T02:40:16Z
dc.date.issued2010-03
dc.description.abstractShigellosis or bacillary dysentery caused by Shigella spp. remains as significant health problem worldwide. Current detection and identification methods of the pathogens from stool specimens by culture method is relatively insensitive, generally time consuming (~ 2-5 days) and laborious. In this study, thermostabilised Polymerase Chain Reaction (PCR) assays were developed for the detection of ompA genes in Shigella dysenteriae, Shigella flexneri and Shigella sonnei. The tests contained specific primers for the detection of S. dysenteriae, S. flexneri and S. sonnei respectively, freezed-dried or thermostabilised PCR reagents with Internal Control (IC) and gel loading dye. Optimization of PCR parameters including annealing temperature (Ta °C) and MgCl2 concentrations was performed. In assessing specificity and sensitivity, the primers were challenged with known S. dysenteriae, S. flexneri, S. sonnei and other enteric pathogens. None of the DNA template from non-targeted organisms was amplified. Five pg/μl of IC was incorporated into the assays after optimization of IC DNA template concentration was done. Minimum concentration of genomic DNA that could be detected by the assays was 0.39 ng/μl for S. sonnei and and S. flexneri .As for the detection of S. dysenteriae was 0.78 ng/μl. At bacterial level, the sensitivity was found to be 4.0 x103 cfu/PCR reaction for the test developed for the detection of S. dysenteriae and 4.0 x104 cfu/PCR reaction for S. flexneri and S. sonnei. Thermostabilised PCR mix periodic stability assessment for each test was done at ambient temperature (25-26°C) and 4°C. Results suggested that the reagent is stable until 6 months of storage at both temperatures. Optimization of stool culture in GNB (Gram Negative broth) was done in order to measure optimum incubation time prior to PCR assay. The optimum time was found to be 8 to 12 hours for all tests. Preliminary evaluation of the developed tests was done using children seeded stool culture in GNB with S. dysenteriae, S. flexneri, S. sonnei and other enteric bacteria (n=50). The PCR results gave 100% for sensitivity and specificity, with 100% Positive Predictive Value (PPV) and Negative Predictive Value (NPV) for the test developed for the detection of S. dysenteriae and S. sonnei. As for the detection of S. flexneri, the sensitivity was found to be 100% and for specificity, 92.8%. Therefore, the PPV and NPV was 92.8% and 100% respectively. This study has developed simple, rapid, sensitive, specific and cost-effective tests with built-in control for the detection of S. dysenteriae, S. flexneri, and S. sonnei. However, further studies with a larger set of sample probably using adult stool need to be done to evaluate the true performance of the developed tests.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/5735
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectDevelopment of DNA-based diagnostic tests foren_US
dc.subjectthe detection of shigella dysenteriae, shigella flexneri and shigella sonneien_US
dc.titleDevelopment Of Dna-Based Diagnostic Tests For The Detection Of Shigella Dysenteriae, Shigella Flexneri And Shigella Sonneien_US
dc.typeThesisen_US
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