An in-house multiplex PCR for detection of S. typhi and S. paratyphi A: A comparison between detection method by agarose gel electrophoresis and lateral flow assay

Abstract

Background: Typhoid and paratyphoid still remain as public health problem in developing and underdeveloped countries caused by Salmonella Typhi and Salmonella Paratyphi. Culture method is the gold standard to diagnose typhoid and paratyphoid fever as well as carriers. However, the method is less sensitive and produces results within 2-7 days. Thus, the study aims to develop the multiplex PCR for simultaneous detection of S.Typhi, S.Paratyphi A and Salmonella genus in presence of internal amplification control (IAC) with detection method via agarose gel electrophoresis and compare with lateral flow assay. Methods: PCR was optimized with annealing temperature ranging between 55-75̊C. Other parameters such as concentration of labeled primers, MgCl2, dNTPs and Taq polymerase were also optimized and the detection was performed via 2% agarose gel electrophoresis. For lateral flow assay, the nitrocellulose membrane was dotted with biotin, anti-FITC, anti-Cy5, anti-Dig and anti-DNP and assembled with conjugate pad. The analytical sensitivity was carried out by agarose gel electrophoresis and lateral flow assay. Each amplicon was applied onto the dotted dipstick and the intensity of the dots was observed in 15 min. The test was validated with stool samples spiked with 25S. Typhi, 25 S. Paratyphi A and 25 otherSalmonella serovars and other bacteria. The test was also validated with nine stool samples of food handlers. Results: The multiplex PCR successfully developed to detect S. Typhi, S. Paratyphi A, Salmonella genus and IAC without any cross-reaction. The detection limit for S. Typhi and S. Paratyphi A were 0.16 ng/ml and 0.08 ng/ml respectively using lateral flow assay compared 0.63 nl/ul for both S. Typhi and S. Paratyphi A using agarose gel electrophoresis. The test was successfully validated with spiked stool samples of 75 bacterial isolates and 9 stool samples of food handlers during typhoid outbreak with both sensitivity and specificity of 100%. Conclusion: The findings suggest that the multiplex PCR-DNA dipstick is a potential assay as molecular diagnostics for detection of S. Typhi and S. Paratyphi A.