title: Identification of Campylobacter jejuni and determination of point mutations associated with macrolide resistance using a multiplex TaqMan MGB real-time PCR.
abstract: OBJECTIVE
  The aim of the study was to develop a multiplex real-time PCR method to identify Campylobacter jejuni containing mutations commonly associated with macrolide resistance.
  
  
  RESULTS
  A multiplex fluorescence real-time PCR assay was developed based on TaqMan minor groove binder (MGB) probes. The VS1-MGB probe was designed based on the VS1 gene and was used to identify Camp. jejuni. The 23S rDNA-MGB probe was designed to distinguish macrolide resistance mutations in 23S rDNA, while 57D-MGB and 74D-MGB were designed to detect resistance mutations in ribosomal protein L4. The specificity and accuracy of our method were identical to the conventional biochemical tests, mapA PCR, minimum inhibitory concentration (MIC) determination and DNA sequencing. The linear detection limit of the method was 0·03 ng genomic DNA and three colony formation unit (CFU) per reaction. In 6 of 18 cases, the nature of Erythromycin resistance could be correctly determined from natural isolates; absence of the tested mutations was demonstrated in the remaining four resistant isolates.
  
  
  CONCLUSIONS
  A multiplex TaqMan MGB real-time PCR assay with high specificity and accuracy was developed to simultaneously identify Camp. jejuni and detect the gene mutations associated with macrolide resistance.
  
  
  CONCLUSIONS
  This multiplex method can potentially simplify the identification of Camp. jejuni and determine macrolide resistance due to mutations in 23S rDNA or ribosomal protein L4. This method has a potential for application in different research areas and molecular surveillance.
authors.affiliation: National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei, China.|MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: H
authors.initials: H
authors.lastname: Hao
authors.affiliation: National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.affiliation: National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: X
authors.initials: X
authors.lastname: Kuang
authors.affiliation: National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: M
authors.initials: M
authors.lastname: Dai
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: G
authors.initials: G
authors.lastname: Cheng
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: D
authors.initials: D
authors.lastname: Peng
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: L
authors.initials: L
authors.lastname: Huang
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: I
authors.initials: I
authors.lastname: Ahmad
authors.affiliation: National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: N
authors.initials: N
authors.lastname: Ren
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Liu
authors.affiliation: MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.affiliation: National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei, China.|MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Yuan
mesh_terms: D016123:Campylobacter jejuni / Q000187:drug effects* / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D004917:Erythromycin / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D017354:Point Mutation; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D018942:Macrolides; D012338:RNA, Ribosomal, 23S; D004917:Erythromycin
keywords: C. jejuni; MGB; macrolide resistance; point mutation; real-time PCR