DEVELOPMENT OF THERMOSTABILIZED MULTIPLEX PCR FOR RAPID DETECTION AND SPECIATION OF SHIGELLA SPECIES
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Date
2012-10
Authors
SUVASH CHANDRA, OJHA
Journal Title
Journal ISSN
Volume Title
Publisher
Pusat Pengajian Sains Perubatan Universiti Sains Malaysia
Abstract
Shigella, the causative agent of shigellosis or “bacillary dysentery” has emerged as a significant public health problem worldwide. In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Routine microbiological identification of Shigella from stool samples are relatively inefficient, time consuming, labour intensive and the diagnosis often remain obscure due to the presence of low numbers of causative organisms, competition from other commensal organisms and
inappropriate sample collection. To date, no rapid diagnostic methods are available commercially. Hence, the present study was focused on developing a multiplex PCR assay for the diagnosis of Shigella species which is rapid, simple and sensitive diagnostic test. Primers were newly designed for the multiplex PCR development because it was necessary to ensure they melted and annealed with equal efficiency and also the amplicons generated were close enough in size that amplification efficiency would not be hindered but that they differed enough in size and could be differentiated on an agarose gel. This assay simultaneously detects four genes namely invC of Shigella genus, rfc of S. flexneri, wbgZ of S. sonnei and rfpB of S. dysenteriae and one internal control gene (ompA). The specificity of the primers were confirmed by BLAST analysis and the amplified PCR products were confirmed by DNA sequencing. This combination of gene was chosen to address several issues. This combination would allow both Shigella genus and species differentiation and
invC gene is common within the Shigella species. The incorporated internal control successfully validated the reliability of the PCR assay to exclude false negative result. The multiplex PCR results showed 100% concordance to the conventionally confirmed strains by culture methods. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% accuracy and specificity. The sensitivity of the assay at genomic level was 100 pg and 1 ng for multiplex PCR and thermostabilized multiplex PCR respectively. However, the
sensitivity of the assay was 5.4 x 104 CFU/ml at bacterial level, with approximately similar detection levels observed for spiked fecal samples. The thermostabilized multiplex PCR could detect <104 CFU following preincubation for 4 h in Gram negative broth. A thermostabilzed multiplex PCR assay was developed and an accelerated stabilty test was evaluated at 37°C ± 2°C, room temperature and 4°C. The thermostabilized multiplex PCR mixture stored at 4°C was stable up to 2 years by the accelerated stability test. The diagnostic
evaluation was determined using a total of 200 clinical stool specimens. The developed thermostabilized multiplex PCR assay is robust and can give information about four genes that are essential for the identification of the Shigella species. This information would be useful in rapid diagnosis of shigellosis to control outbreaks of this contagious disease and to prevent further complication. The diagnosis would be helpful in reducing the morbidity and mortality caused by shigellosis.
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Keywords
Medical Microbiology