Development of a Novel Real-Time Polymerase Chain Reaction Assay to DetectEscherichia albertiiin Chicken Meat

Abstract

Escherichia albertii is an emerging enteropathogen. Several foodborne outbreaks of E. albertii have been reported in Japan; however, foods associated with most outbreaks remain unidentified. Therefore, polymerase chain reaction (PCR) assays detecting E. albertii specifically and sensitively are required. Primers and probe for real-time PCR assays targeting E. albertii-specific gene (EA-rtPCR) was designed. With 74 strains, including 43 E. albertii strains and several of its close relatives, EA-rtPCR specifically amplified E. albertii; therefore, the sensitivity of EA-rtPCR was then evaluated. The detection limits were 2.8 and 2.0–3.2 log colony-forming unit (CFU)/mL for E. albertii culture and enriched chicken culture inoculated with the pathogen, respectively. In addition, E. albertii was detected from 25 g of chicken meat inoculated with 0.1 log CFU of the pathogen by EA-rtPCR. The detection of E. albertii from chicken meat by EA-rtPCR was also evaluated by comparing with the nested-PCR assay, and 28 retail chicken meat and 193 dissected body parts from 21 chicken carcass were tested. One and three chicken meat were positive in the nested-PCR assay and EA-rtPCR, respectively. Fourteen carcasses had at least one body part that was positive for EA-rtPCR, and 36 and 48 samples were positive for the nested-PCR assay and EA-rtPCR, respectively. A total of 37 strains of E. albertii were isolated from seven PCR-positive samples obtained from six chicken carcass. All E. albertii isolates harbored eae gene, and were classified as E. albertii O-genotype (EAOg)3 or EAOg4 by EAO-genotyping. The EA-rtPCR developed in this study has potential to improve E. albertii detection in food and advance research on E. albertii infection.