Rapid and Simple Universal Genotyping Method Based on Multiple-Locus Variable-Number Tandem-Repeat Analysis Using Single-Tube Multiplex PCR and Standard Gel Electrophoresis.

We developed a multiplex PCR method based on multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing of and isolates. The method amplifies seven VNTRs and does not require a sequencing capillary or fluorescent dyes. The amplification products are simply loaded on a standard agarose gel for electrophoresis, and the banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: the reference (ECOR) collection ( = 72), O1:K1 isolates causing neonatal meningitis ( = 38), extended-spectrum beta-lactamase-producing fecal isolates belonging to the worldwide sequence type 131 (ST131) clone ( = 38), Shiga toxin-producing (STEC) isolates of serogroups O157:H7 ( = 21) and O26 ( = 16, 8 of which belonged to an outbreak), 27 isolates (22 isolates, including 5 epidemic strains), and 8 reference strains. The performances were compared to those of multilocus sequence typing (MLST), the DiversiLab automated repetitive element palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). We found 66 different profiles among the isolates in the ECOR collection. Among the clonal group O1:K1 isolates, 14 different profiles were identified. For the 37 STEC isolates, we found 23 profiles, with 1 corresponding to the 8 epidemic strains. We found 19 profiles among the 27 isolates, with 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone and to distinguish the O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows the simple, fast, and inexpensive typing of / isolates that can be carried out in any laboratory equipped for molecular biology and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE. Fast typing methods that can easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted with outbreaks or performing epidemiological studies. Highly discriminatory universal methods, like PFGE, optical mapping, or WGS, are expensive and/or time-consuming. MLST is useful for phylogeny but is less discriminatory and requires sequencing facilities. PCR methods, which are fast and easy to perform, also have drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack the discriminatory power to differentiate isolates during outbreaks. MLVA combines the advantages of PCR methods with a high discriminatory power but in its standard form requires sequencing capillary electrophoresis. The method that we have developed combines the advantages of standard PCR (simple, fast, and inexpensive) with the high discriminatory power of MLVA and permits the typing of all isolates (either intestinal or extraintestinal pathogenic isolates as well as commensal isolates).