Development and validation of a bacterial gastrointestinal multiplex RT-PCR assay for use on a fully automated...

PCR-based enteric multiplex panels represent a rapid and reliable alternative to conventional "classical" phenotypic stool diagnostics. The aim of this study was to establish a laboratory-developed non-commercial multiplex Real-Time-PCR panel for the detection of the most important bacterial stool pathogens, Salmonella spp., Shigella spp., Yersinia enterocolitica/ pseudotuberculosis and Campylobacter jejuni/coli. on the "open" cobas omni Utility Channel (UC) of the cobas 6800 system (Roche). The aim was to replace the laborious phenotypical stool diagnostics with a high throughput Real-Time PCR method. The respective primers and probes were designed to cover conserved genomic regions of the pathogens and validated using Ultramer oligonucleotides, positive stool material and reference strains. To further validate the multiplex PCR-assay, the following parameters were evaluated: analytical-sensitivity and -specificity, cross-reactivity, linearity and inter- and intra-assay variance as well as limit of detection (LOD). In addition a retrospective analysis of culture positive and negative samples from daily routine was performed using 745 native stool samples. The Gastro assay was linear over a 5-log-unit and within the expected dynamic range with amplification efficiencies ranging from 94.6% to 120%. In addition, all targets showed excellent coefficients of repeatability (≤ 1.11%), intermediate precision (≤ 1.02%) and total variance (≤ 1.39%). In terms of analytical sensitivity the assay demonstrated detection limits ranging from 7.83 copies per reaction to 14.4 copies per reaction. The assay showed excellent agreement with culture methods (>95%) and a 100% sensitivity and specificity after resolution of discrepant results. The multiplex-PCR assay provides a comprehensive, rapid and sensitive alternative to conventional methods for the detection of the major bacterial stool pathogens in diagnostic laboratories.