Development of DNA-based diagnostic tests for the detection of Shigella dysenteriae, Shigella flexneri, and Shigella sonnei
Loading...
Date
2010-03
Authors
Mohamed Shakrin, Nik Noorul Shakira
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Shigellosis 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 (Ie) and gel loading dye.
Optimization ofPCR parameters including annealing temperature (Ta 0c) and MgCb
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/J!I 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/J!l for S. sonnei and and S. flexneri .As
for the detection of S. dysenteriae was 0.78 ng/f..tl. At bacterial level, the sensitivity
was found to be 4.0 x I 03 cfulPCR reaction for the test developed for the detection of
S. dysenteriae and 4.0 xlO4 cfulPCR reaction for S. flexneri and S. sonnei.
Thennostabilised peR 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 peR 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 peR 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 have 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
pertonnance of the developed tests.
Description
Keywords
Shigellosis or bacillary dysentery caused by Shigella spp. , remains as significant health problem worldwide