title: [Development of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae non-O1/O139 serogroups].
abstract: OBJECTIVE
  To develop methodology of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae (V. cholerae) serogroups non-O1 and non-O139.
  
  
  METHODS
  The outer membrane protein gene (ompW) specific for V. cholerae, as well as O antigen rfb genes specific for both O1 and O139, were used for the design of the PCR primers. Both multiple PCR and real-time SYBR green PCR systems were used to detect both O1 and O139. Specific rfb genes and ompW were developed to evaluate their specificity, limit of detection, reproducibility and consistency.
  
  
  RESULTS
  We established multiple PCR and real-time SYBR green PCR methods. According to the specific electrophoretic bands (multiple PCR) and the specific melt curve temperature (real-time SYBR green PCR), both methods could specifically detect the non-O1, non-O139 V. cholerae, and to differentiate them from O1,O139 V. cholerae, other five Vibrios and 3 intestinal bacteria. The detection limits were 7 × 10(4) cfu/ml (multiple PCR) and 7 × 10² cfu/ml(real-time SYBR green PCR), with statistically significant difference seen (P < 0.05). For the reproducibility of real-time SYBR green PCR, the external coefficient variation ranging from 0.22% to 0.92% while the internal coefficient variation ranging from 0.27% to 1.41%. 370 strains of non-O1, non-O139 V. cholerae, were detected, with both consistency rates as 100% .
  
  
  CONCLUSIONS
  Both multiple PCR and real-time SYBR green PCR could detect non-O1, non-O139 V. cholerae, rapidly, specifically, and reproducibly, that could all be used for the detection and identification of non-O1, non-O139 under different conditions.
authors.affiliation: State Key Laboratory for Infectious Diseases Prevention and Control, National Institute of Communicable Disease Control and Prevention, Chinese Center of Disease Control and Prevention, Beijing 102206, China.
authors.forename: Fengjuan
authors.initials: F
authors.lastname: Li
authors.affiliation: State Key Laboratory for Infectious Diseases Prevention and Control, National Institute of Communicable Disease Control and Prevention, Chinese Center of Disease Control and Prevention, Beijing 102206, China.
authors.forename: Biao
authors.initials: B
authors.lastname: Kan
authors.affiliation: State Key Laboratory for Infectious Diseases Prevention and Control, National Institute of Communicable Disease Control and Prevention, Chinese Center of Disease Control and Prevention, Beijing 102206, China. Email: wangduochun@icdc.cn.
authors.forename: Duochun
authors.initials: D
authors.lastname: Wang
mesh_terms: D005798:Genes, Bacterial; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D041242:Vibrio cholerae O139 / Q000235:genetics / Q000302:isolation & purification*; D044163:Vibrio cholerae non-O1 / Q000235:genetics / Q000302:isolation & purification*