ABSTRACT
<p><b>OBJECTIVE</b>To develop a high-throughput rapid method for Vibrio (V.) cholerae molecular typing based on Melting Curve-based Multilocus Melt Typing (McMLMT).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Subject(s)
Multilocus Sequence Typing , Polymerase Chain Reaction , Vibrio choleraeABSTRACT
Objective To investigate the genotype and epidemiology of plasmid‐mediated AmpC β‐lactamases produced by the clinical strains of Escherichia coli and Klebsiella pneumoniae .Methods A total of 176 clinical nonrepetitive cefoxitin non‐sensitivity isolates of Escherichia coli and Klebsiella pneumoniae was collected from July 2011 to August 2012 .Polymerase chain reaction (PCR) for AmpC enzyme gene amplification and DNA sequencing were carried out for genotype of AmpC beta‐lactamases .Results The results of PCR showed that the positive rate of ampC of the 176 strains of Escherichia coli and Klebsiella pneumoniae AmpC was 18 .2% ,mainly DHA type ,counting for 59 .4% ,CIT counting for 37 .5% ,EBC counting for 3 .1% .The positive rate of ampC of Escherichia coli was 11 .4% ,mainly CIT type ,counting for 77 .8% ,the positive rates of DHA type and EBC type both were 11 .1% .The positive rate of ampC of Klebsiella pneumoniae were 23 .7% ,mainly DHA type ,counting for 78 .3% ,CIT type count‐ing for 21 .7% .The results of DNA sequencing showed that there were 18 strains DHA‐1 type and 1 strain ampC gene type of Morganella morganii in DHA type strains ,the concordance rate was 97 .0% ,10 CIT type strains was CMY‐2 type ,1 strain was CMY‐42 ,one strain was CMY‐4 type ,EBC type was ampC gene type of Enterobacter cloacae ,the concordance rate was 99 .0% .A total of 32 strains of gene sequencing were registered as KJ127248 - KJ127279 in GenBank .Conclusion The main genotypes of plasmid‐mediated ampC enzyme produced by Escherichia coli and Klebsiella pneumoniae were CMY‐2 and DHA‐1 respectively .