贵州省鼠伤寒沙门菌单相变异株的CRISPR基因型和遗传多样性研究北大核心CSCD

目的了解贵州省鼠伤寒沙门菌单相变异株规律的成簇间隔短回文重复序列(CRISPR)的基因型及遗传多样性,为贵州省沙门菌病的预防控制提供依据。方法对分离自2013—2018年贵州省7个市(州)的71株疑似鼠伤寒沙门菌单相变异株进行系统生化鉴定及血清分型,结合双重PCR法确定疑似菌株为鼠伤寒沙门菌单相变异株。提取鼠伤寒沙门菌单相变异株的DNA作为模板,进行CRISPR1和CRISPR2的PCR扩增,将阳性扩增产物进一步测序,获得菌株的CRISPR1和CRISPR2间隔序列,根据间隔序列的组成,确定CRISPR基因型,并使用Bionumerics软件对菌株进行遗传多样性分析。结果71株疑似鼠伤寒沙门菌单相变异菌株经系统生化鉴定、血清分型及双重PCR法鉴定为鼠伤寒沙门菌单相变异株,经CRISPR1和CRISPR2两个位点的PCR扩增和产物测序,71株菌共检测到163个间隔序列,其中CRISPR1有102个,CRISPR2有61个。联合CRISPR1和CRISPR2两个位点的间隔序列进行分析,发现71株菌被分为33个CRISPR基因型(TSTs),其中TST11和TST1为贵州省的优势基因型,分别占总菌株数的25.35%和12.68%。71株菌经Bionumerics软件聚类分析后被分为A、B、C、D 4个簇,A簇包含的菌株最多,B簇包含的基因型最多,不同来源的3株参考株独立成簇,D簇包含7个TSTs基因型。结论贵州省鼠伤寒沙门菌单相变异株具有优势的CRISPR基因型和遗传多样性的特点,可能有其他的感染来源。

CRISPR genotypes and genetic diversity of Salmonella typhimurium monophasic variant strains in Guizhou Province,China

This study was aimed at studying the genotypes and genetic diversity of the clustered regularly interspaced short palindromic repeats (CRISPR) of monophasic variant strains of Salmonella typhimurium, to support prevention and control of salmonellosis in Guizhou Province, China. A total of 71 suspicious monophasic S. typhimurium strains isolated from seven cities (prefectures) in Guizhou Province between 2013 and 2018 were identified through biochemical identification, serotyping and duplex PCR. The DNA of monophasic S. typhimurium strains was extracted by boiling, and was used as a template for PCR amplification of the CRISPR1 and CRISPR2 loci. The positive amplification products were further sequenced to obtain the CRISPR1 and CRISPR2 spacer sequences of each strain. The CRISPR genotypes were determined according to the composition of CRISPR1 and CRISPR2 spacer sequences. Genetic diversity analysis of strains was performed in Bionumerics software. Seventy-one suspicious strains were identified as monophasic S. typhimurium by biochemical identification, serotyping and duplex PCR. A total of 163 spacer sequences were detected in the 71 strains after PCR amplification and product sequencing of CRISPR1 and CRISPR2 loci. Among them, 102 spacer sequences at CRISPR1 loci and 61 spacer sequences at CRISPR2 loci were identified. Analysis of spacer sequences combined with CRISPR1 and CRISPR2 indicated that 71 strains were divided into 33 CRISPR genotypes, among which TST11 and TST1 were the dominant genotypes in Guizhou Province, accounting for 25.35% and 12.68% of the total strains, respectively. The 71 strains were divided into four clusters by Bionumerics software: clusters A and B contained the most strains and genotypes, respectively; cluster C was independently composed of the reference strains from different sources; and cluster D contained seven TSTs genotypes. The CRISPR genotypes of monophasic S. typhimurium strains isolated from Guizhou Province showed characteristics of dominant CRISPR genotypes and genetic diversity, thus potentially suggesting other sources of infection.