|990-1||Genotyping of Yersinia pseudotuberculosis based on high resolution melting analysis (HRMA) for multiple locus VNTR analysis (MLVA)|
|Autores:||Roberto Antonio de Souza (FCFRP-USP - Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP) ; Juliana Pfrimer Falcão (FCFRP-USP - Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP) |
Introduction: Yersinia pseudotuberculosis is a widespread enteric pathogen that can infect a wide range of animals, including humans. The aim of this work was to genotype Y. pseudotuberculosis strains using a method based on high-resolution melting analysis (HRMA) for multiple locus VNTR analysis (MLVA).
Materials and methods: Forty-one Y. pseudotuberculosis strains isolated from bovine and bubaline in the southern region of Brazil was studied. A total of 13 VNTR loci were analyzed by HRMA. Real-time PCR cycling was performed on a 7500 Fast Real-Time PCR System apparatus. The reaction was performed in 20 μL reaction mixtures that contained 10 μL of MeltDoctor™ HRM Master Mix, 1.2 μL of each primer at 5 μM, 4 μL of genomic DNA at 5 ng/μL and 3.6 μL of DNase- and RNase-free distilled water. The PCR conditions were as follows: 1 cycle of 95ºC for 10 min and 40 cycles that consisted of 95ºC for 15 s and 55ºC for 1 min. The HRMA step was performed immediately after PCR cycling. The amplicons were heated to 95ºC for 10 s and were then cooled to 60ºC for 1 min. The melting curves were generated by increasing the temperature from 60 to 95ºC in 1.6ºC/s increments and fluorescence was detected at each 0.1 s. The normalized melting curves generated were analyzed with the 7500 software program v2.0. For each VNTR locus, different melting profiles were assigned as distinct alleles and the combination of the 13 alleles provided the MLVA type for each strain. The population structure was assessed by eBURST v3.0 program.
Results: A total of 19 different MLVA types were identified by HRMA, 16 of which composed by the Y. pseudotuberculosis of the bioserogroup 2/O:3 and three composed by the Y. pseudotuberculosis of the bioserogroup 1/O:1a. All the Y. pseudotuberculosis 2/O:3 were clustered in a clonal complex, as well as, all the Y. pseudotuberculosis 1/O:1a were clustered in another one.
Conclusion: The Y. pseudotuberculosis strains of the same bioserogroup showed to be genetically very similar which is indicative of a common evolutionary origin. This fact suggests that two different clonal populations have been circulating among the livestock in the southern region of Brazil.
Palavras-chave: genotyping, high-resolution melting analysis (HRMA), multiple locus VNTR analysis (MLVA), Yersinia pseudotuberculosis