Identification of Campylobacter jejuni and determination of point mutations associated with macrolide resistance using a multiplex TaqMan MGB real-time PCR

Abstract

Campylobacter jejuni, one of the most important food-borne pathogens, develops resistance to macrolides by acquiring point mutations in the 23S rDNA gene (e.g. A2075G or A2074C) and in ribosomal protein L4 (e.g. G74D or G57D). In this study, a multiplex fluorescence real-time PCR assay, based on TaqMan minor groove binder (MGB) probes, was developed to identify C. jejuni containing mutations associated with macrolide resistance. The VS1-MGB probe was designed based on the VS1 gene and was used to identify C. jejuni. The 23S rDNA-MGB probe was designed to distinguish macrolide resistance mutations in 23S rDNA, whilst 57D-MGB and 74D-MGB were designed to detect resistance mutations in ribosomal protein L4. The specificity and accuracy of the established method were 100% identical to the classic biochemical method, mapA PCR, minimum inhibitory concentration (MIC) determination and DNA sequencing. The linear detection limitation of the method was 0.03ng/reaction genomic DNA and 3CFU/reaction bacterial colonies. In conclusion, this study provides a novel method that can specifically identify C. jejuni and accurately detect the gene mutations associated with macrolide resistance.