A probe-free four-tube real-time PCR assay for simultaneous detection of twelve enteric viruses and bacteria

• Twelve common diarrhea-associated pathogens can be simultaneously detected.
• Four-tube assay is sensitive and easy to operate without post-PCR gel electrophoresis step.
• The cost for the assay is approximately $5 per test for twelve pathogens.
• The whole process takes no more than 3 h and works with different real-time PCR machines.

ObjectivesWe aim to develop a multiplex real-time PCR assay to detect the most common pathogens causing community outbreaks of diarrhea.MethodsFour reaction systems of fluorescence dye-based real-time PCR assay were performed to amplify genes of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, Campylobacter jejuni, Yersinia enterocolitica, Vibrio parahaemolyticus, Salmonella spp., Escherichia coli, and Shigella spp. PCR products of each pathogen were identified by characteristic peaks in melting curves.ResultsThe assay was able to achieve detection limit of 50 copies/reaction for each individual virus target, and 140–500 CFU/mL for each individual bacterium target. A total of 122 clinical specimens from hospitalized children with acute diarrhea were used to evaluate the assay. The clinical sensitivity was very similar to that of reference methods. Norovirus genogroup II revealed the highest detectable rate (45/122, 36.9%). Coinfection was found in 28 out of 122 (23%) clinical specimens.ConclusionThis assay proved to be a cost-effective, sensitive and reliable method for simultaneous detection of enteric viruses and bacteria.