Rapid and accurate diagnosis based on real-time PCR cycle threshold value for the identification of Campylobacter jejuni, astA gene-positive Escherichia coli, and eae gene-positive E. coli

Abstract

  We previously developed a multiplex real-time PCR assay (Rapid Foodborne Bacterial Screening 24 ver.5, RFBS24 ver.5) for simultaneous detection of 24 foodborne bacterial targets. Here, to overcome the discrepancy of the RFBS24 ver.5 and bacterial culture method (BC) results, we analyzed 246 human clinical samples from 49 gastroenteritis outbreaks by the RFBS24 ver.5 and evaluated the correlation between cycle threshold value (CT) of RFBS24 ver.5 and the BC results. The results showed that the RFBS24 ver.5 was more sensitive than BC for Campylobacter jejuni and Escherichia coli harboring astA or eae at 45.5–87.0% of positive predictive values (PPV) and 0.60–0.92 of kappa coefficient (KC), respectively. Their CTs were significantly different between BC-positive and -negative samples (p<0.01). All RFBS24 ver.5-positive samples were BC-positive under the lower limit of the 95% or 99% confidence interval (CI) for the CT of the BC-negative samples. We set the 95% or 99% CI lower limit to the determination CT (d-CT) for the discrimination of assured BC-positive results (d-CTs: 27.42–30.86), and the PPVs (94.7%–100.0%) and KCs (0.89–0.95) of these three targets were increased. Together, we concluded that the implication of d-CT-based approach would be a valuable tool for rapid and accurate diagnostic approach in the RFBS24 ver.5 system.