A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China

Abstract

Objective: Antimicrobial resistance (AMR) has become a global concern and is especially severe in China. To effectively and reliably provide AMR data, we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology, and screened multiple AMR genes in broiler fecal samples.

Methods: A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection. A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.

Results: The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction. The sensitivity rate, specificity rate, positive predictive value, negative predictive value and correct indices were 99.30%, 98.08%, 95.31%, 99.79%, and 0.9755, respectively. Utilizing this assay, we demonstrate that AMR genes are widely spread, with positive detection rates ranging from 0 to 97.07% in 273 broiler fecal samples. blaCTX-M, blaTEM, mcr-1, fexA, cfr, optrA, and intI1 showed over 80% prevalence. The dissemination of AMR genes was distinct between the two farms.

Conclusion: We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples. The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.

Keywords: Antimicrobial resistance; Antimicrobial resistance gene; Broiler fecal sample; High-throughput real-time PCR array.