title: [Development of a rapid molecular typing method for Vibrio cholerae using melting curve-based multilocus melt typing].
abstract: OBJECTIVE
  To develop a high-throughput rapid method for Vibrio (V.) cholerae molecular typing based on Melting Curve-based Multilocus Melt Typing (McMLMT).
  
  
  METHODS
  Seven housekeeping genes of V.cholerae were screened out, and for each gene, the specific primers were designed for correspondent genes as well as 4 probes covering polymorphism loci of sequences. After optimizing all parameters, a method of melting-curve analysis following asymmetric PCR was established with dual-fluorescent-reporter in two reaction tubes for each gene. A set of 28 Tm-values was obtained for each strain and then translated into a set of code of allelic genes, standing for the strain's McMLMT type (MT). Meanwhile, sequences of the 7-locus polymorphism were typed according to the method of MLST. To evaluate the efficiency and reliability of McMLMT, the data were compared with that of sequence-typing and PFGE using BioNumerics software.
  
  
  RESULTS
  McMLMT method was established and refined for rapid typing of V. cholerae that a dozen of strains can be finished testing in a 3-hours PCR running using 96-well plates. 108 strains were analyzed and 28-Tm-values could be grouped and encoded according to 7 housekeeping gene to obtain the code set of allelic genes, and classified into 18 types (D = 0.723 3). Sequences of the 7 genes' polymorphism areas were directly clustered into the same 18 types with reference to MLST method. 46 of the strains, each represented a different PFGE type, could be classified into 13 types (D = 0.614 5) with McMLMT method and A- K groups at 85% similarity (D = 0.858 9) with PFGE method.
  
  
  CONCLUSIONS
  McMLMT method is a rapid high-throughput molecular typing method for batches of strains with a resolution equal to MLST method and comparable to PFGE group.
authors.affiliation: Department of Microbiology, Xiamen Centre for Disease Control and Prevention, Xiamen 361021, China.
authors.forename: Shijie
authors.initials: S
authors.lastname: Huang
authors.forename: Zanzan
authors.initials: Z
authors.lastname: Liu
authors.forename: Huixin
authors.initials: H
authors.lastname: Wen
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Qingge
authors.initials: Q
authors.lastname: Li
authors.affiliation: Email: fjxmhjw@163.com.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Huang
mesh_terms: D058885:Multilocus Sequence Typing; D016133:Polymerase Chain Reaction; D014734:Vibrio cholerae