CRISPR genotyping and regional distribution characteristics ofYersinia pestis in natural plague focus of Tibet Autonomous Region
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目的 探究西藏自治区鼠疫自然疫源地鼠疫耶尔森菌（以下简称鼠疫菌）规律成簇间隔短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）基因型及其地区分布特征。 方法 选取西藏自治区鼠疫自然疫源地内不同时间、地区、宿主、媒介体内分离的125株代表性鼠疫菌株作为实验菌株，采用苯酚-氯仿混合抽提法提取鼠疫菌DNA。应用3对CRISPR引物（针对YPa、YPb、YPc 3个位点）分别对实验菌株DNA进行PCR扩增，经测序确定鼠疫菌CRISPR基因型。 结果 125株鼠疫菌均具有YPa、YPb、YPc 3个CRISPR位点，共发现18种间区序列（spacer），其中YPa位点8种、YPb位点6种、YPc位点4种；新发现spacer 2种，分别为b52、c14。经CRISPR基因分型，共有G1、G7、G7-b4′′′、G7-b52、G7-c2-、G8、G22、G22-a4-、G22-b4′′′、G22-c14 10种基因型，其中6种为新发现基因型。125株实验菌株中，以G7型为主要基因型，占65.6%（82/ 125），在西藏自治区下辖的6个地级市和1个地区均有分布；其次为G22、G7-c2-型，分别占14.4%（18/ 125）、11.2%（14/125），其中G22型分布于那曲市、昌都市、拉萨市及阿里地区，G7-c2-型分布于日喀则市和山南市。 结论 西藏自治区鼠疫自然疫源地鼠疫菌CRISPR位点遗传多态性较高，不同基因型的鼠疫菌株具有明显的地区分布特征。 Objective To study the clustered regularly interspaced short palindromic repeats (CRISPR) genotype of Yersinia pestis and its regional distribution characteristics in natural plague focus of Tibet Autonomous Region. Methods A total of 125 representative Yersinia pestis strains isolated from natural plague focus in Tibet Autonomous Region at different times, regions, hosts and vectors were selected as experimental strains, and the phenol chloroform mixed extraction method was used to extract Yersinia pestis DNA. Three pairs of CRISPR primers (for YPa, YPb, YPc locus) were used to amplify the DNA of the experimental strains, and the CRISPR genotype of Yersinia pestis was determined by sequencing. Results All 125 strains of Yersinia pestis had three CRISPR locus: YPa, YPb, and YPc. A total of 18 spacer were found, including 8 in YPa loci, 6 in YPb loci, and 4 in YPc loci. Two new types of spacers had been discovered, namely b52 and c14. CRISPR typing revealed 10 genotypes, including G1, G7, G7-b4''', G7-b52, G7-c2-, G8, G22, G22-a4-, G22-b4''', and G22-c14, of which 6 were newly discovered genotypes. Among the 125 experimental strains, G7 was the main genotype, accounting for 65.6% (82/125), which was distributed in 6 prefecture level citys and 1 region of Tibet Autonomous Region. Next were G22 and G7-c2- genetypes, accounting for 14.4% (18/125) and 11.2% (14/125), respectively. G22 gene type was distributed in Nagqu, Changdu, Lhasa citys, and Ngari Prefecture, while G7-c2- genetype was distributed in Shigatse and Shannan cities. Conclusion The CRISPR locus of Yersinia pestis in natural plague focus of Tibet Autonomous Region is highly polymorphic, and the Yersinia pestis strains with different genotypes have obvious regional distribution characteristics.
Objective To study the clustered regularly interspaced short palindromic repeats (CRISPR) genotype of Yersinia pestis and its regional distribution characteristics in natural plague focus of Tibet Autonomous Region. Methods A total of 125 representative Yersinia pestis strains isolated from natural plague focus in Tibet Autonomous Region at different times, regions, hosts and vectors were selected as experimental strains, and the phenol chloroform mixed extraction method was used to extract Yersinia pestis DNA. Three pairs of CRISPR primers (for YPa, YPb, YPc locus) were used to amplify the DNA of the experimental strains, and the CRISPR genotype of Yersinia pestis was determined by sequencing. Results All 125 strains of Yersinia pestis had three CRISPR locus: YPa, YPb, and YPc. A total of 18 spacer were found, including 8 in YPa loci, 6 in YPb loci, and 4 in YPc loci. Two new types of spacers had been discovered, namely b52 and c14. CRISPR typing revealed 10 genotypes, including G1, G7, G7-b4''', G7-b52, G7-c2-, G8, G22, G22-a4-, G22-b4''', and G22-c14, of which 6 were newly discovered genotypes. Among the 125 experimental strains, G7 was the main genotype, accounting for 65.6% (82/125), which was distributed in 6 prefecture level citys and 1 region of Tibet Autonomous Region. Next were G22 and G7-c2- genetypes, accounting for 14.4% (18/125) and 11.2% (14/125), respectively. G22 gene type was distributed in Nagqu, Changdu, Lhasa citys, and Ngari Prefecture, while G7-c2- genetype was distributed in Shigatse and Shannan cities. Conclusion The CRISPR locus of Yersinia pestis in natural plague focus of Tibet Autonomous Region is highly polymorphic, and the Yersinia pestis strains with different genotypes have obvious regional distribution characteristics.
Yersinia pestisClustered regularly interspaced short palindromic repeatsGenotyping
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