Multiplex real-time PCR for detection of Campylobacter, Salmonella, and Shigella

Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacter spp., Salmonella spp., and Shigella spp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96±0.05°C for invA, 85.56±0. 28°C for ipaH, and 89.21± 0.24°C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 10⁴ CFU g^-1; however, the limit of detection was 10³ CFU g ^-1. This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens.