Fimbriae-C Salmonella typhi Primers as Clinical Detection of Typhoid Disease by PCR Methods

Abstract

Salmonella typhi is bacteria causes typhoid disease in human. The mortality rate of typhoid disease in Indonesia is increasing. The most common detection method currently used is serological test. However, this method is often resulted in less accurate, less sensitive and specific detection. The previous research has successfully discovered fim-C-S. typhi gene which is able to code protein to contribute in adherent or colonization in human epithelial cell. The information of fim-C-S. typhi gene is used to develop more specific and more accurate detection method for S. typhi bacteria by amplification, characterization, specificity and sensitivity assay, and continue with clinical assay. The PCR result was the amplified 0.2 kilo base (kb) DNA fragment using the fimbriae-C-S. typhi primer pairs. The specificity assay was conducted by comparing the amplicon in size of 0.2 kb S. typhi with Gram negative bacteria genome with similar characteristics. The results indicated the fimbriae-C-S. typhi primers can differentiate amplicon in size of 0.2 kb S. typhi against Enterobacter aerogenes, Klebsiella pneumonia, Vibrio cholera, Escherichia coli, and Pseudomonas aeruginosa. However, they cannot differentiate S. typhimurium which have higher homology in their genome. Sensitivity assay was conducted by determining the detection level of fim-C-S. typhi primers to S. typhi genome. The sensitivity assay showed the detected fimbriae-C primers S. typhi with 1.295 x 10-39 μg/mL DNA chromosome. The clinical assay was evaluated by comparing the PCR product of suspected patient blood, the isolated bacteria S. typhi genome served as positive control, and uninfected human blood as negative control. The PCR product from 10 suspected patient bloods showed the band size 0.2 kb equals to the size of band produced by the PCR product from isolated S. typhi bacteria genome. Meanwhile, the band was not detected in the uninfected blood. Band size 0.2 kb from the suspected patient blood sample indicated positive infection of S. typhi bacteria. The above results demonstrated the developed clinical detection for typhoid disease using fimbriae-C-S. typhi primer pairs.