Evaluation of a real-time multiplex PCR for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./EIEC, and Yersinia enterocolitica in fecal samples.

Abstract

Conventional diagnosis of infectious diarrhea caused by bacteria is time-consuming, labor-intensive, and has a suboptimal sensitivity. We have therefore developed a multiplex real-time polymerase chain reaction (PCR) for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./enteroinvasive Escherichia coli (EIEC), and Yersinia enterocolitica in fecal samples. No cross reactivity between the different pathogens was observed, and the multiplex setup of the assay did not have an impact on the sensitivity of the PCR. The analytical sensitivity was 87CFU/mL for C. jejuni, 61CFU/mL for Shigella spp./EIEC, 5,528CFU/mL for Salmonella spp., and 1,306CFU/mL for Y. enterocolitica. An extensive validation of the assay was performed by testing 1,687 patient samples by both PCR and with conventional techniques. The use of PCR increased the overall clinical sensitivity from 78 to 100% (p < 0.0001), the specificity was 99.4% for the PCR, compared with 99.9% for conventional culture. The novel PCR assay allows for rapid, sensitive, inexpensive, and high-throughput testing of the most common bacterial causes of gastroenteritis.