| Summary: | A multiplex rapid detection system, based on a PCR-lateral flow biosensor (mPCR-LFB) was developed to identify <i>Salmonella</i> Typhi and <i>Salmonella</i> Paratyphi A from suspected carriers. The lower detection limit for <i>S</i>. Typhi and <i>S</i>. Paratyphi A was 0.16 and 0.08 ng DNA equivalent to 10 and 10<sup>2</sup> CFU/mL, respectively. Lateral flow biosensor was used for visual detection of mPCR amplicons (stgA, SPAint, ompC, internal amplification control) by labeling forward primers with fluorescein-isothiocyanate (FITC), Texas Red, dinitrophenol (DNP) and digoxigenin (DIG) and reverse primers with biotin. Binding of streptavidin-colloidal gold conjugate with the amplicons resulted in formation of a red color dots on the strip after 15–20 min of sample exposure. The nucleic acid lateral flow analysis of the mPCR-LFB was better in sensitivity and more rapid than the conventional agarose gel electrophoresis. Moreover, the mPCR-LFB showed 100% sensitivity and specificity when evaluated with stools spiked with 100 isolates of <i>Salmonella</i> genus and other bacteria. A prospective cohort study on stool samples of 1176 food handlers in outbreak areas (suspected carriers) resulted in 23 (2%) positive for <i>S</i>. Typhi. The developed assay has potential to be used for rapid detection of typhoid carriers in surveillance program.
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