A rapid and specific PCR- ELISA for detecting Salmonella typhi

Salmonella continues to be a major food borne pathogen for animals and humans. A sensitive and specific PCR–ELISA technique was developed to detect Salmonella typhi. The assay was based on the incorporation of digoxigenin-labeled dUTP and a biotin-labeled primers specific for rfbE gene during PCR amplification. The labeled PCR products were bound to streptoavidin-coated wells of a microtiter plate and detected by ELISA. The specificity of the PCR was determined using 4 strains of Entrobacteriaceae family including Escherichia coli, Klebsiella, Proteus, and Enterobacter and 2 strains of salmonella genus including paratyphi A and entritidis. Among all the strains, only Salmonella typhi was positive. The PCR-ELISA detecting system was able to increase the sensitivity of assay up to 100 fold, compared with a conventional PCR. The detection limit in PCR-ELISA was 2.5ρg in genomic DNA and 20 cells in direct manner per reaction. The entire procedure took about 100 minutes. For further confirmation of the test, internal biotin labeled probe was designed for rfbE gene and detected with streptavidin. We have developed a rapid and simple PCR-ELISA protocol suitable for routine analysis of viable Salmonella typhi.