A novel high-resolution melting analysis-based method for Yersinia pseudotuberculosis genotyping

Yersinia pseudotuberculosis is an enteric pathogen that is environmentally widespread and is known to cause human and animal infections. The development of a fast and inexpensive typing system is necessary to facilitate epidemiological studies of Y. pseudotuberculosis infections. In this study, we aimed to develop a method of Y. pseudotuberculosis genotyping based on determining differences in single-nucleotide polymorphisms (SNPs) using a high-resolution melting analysis (HRMA). Using a set of nine primer pairs, ten SNPs were screened from sequences in the 16S rRNA, glnA, gyrB and recA sequences of 12 Y. pseudotuberculosis strains that were deposited in the GenBank database. The genetic diversity of a collection of 40 clinical Y. pseudotuberculosis strains was determined using the HRMA method and the multilocus sequence typing (MLST) technique was used for comparison. Different melting profiles were found in five out of a total of nine analyzed fragments. A phylogenetic tree was constructed from the nucleotides that were identified in the nine analyzed fragments, and the tree demonstrated that Y. pseudotuberculosis strains were separated into two groups. The first cluster was composed of strains from the 1/O:1a serogroup and the second of strains from the 2/O:3 serogroup. The separation into two clusters based on distinct bio-serogroups of Y. pseudotuberculosis was consistent with the results in the MLST database. The simple and highly reproducible HRMA assay developed by us may be used as a rapid and cost-effective method to genotype Y. pseudotuberculosis strains of O:1 and O:3 serogroups and it can complement sequence-based methods facilitating epidemiological studies of this Yersinia species.