中国媒介生物学及控制杂志 ›› 2014, Vol. 25 ›› Issue (6): 489-491.DOI: 10.11853/j.issn.1003.4692.2014.06.001

• 论著 •    下一篇

一种区分田鼠型与非田鼠型鼠疫耶尔森菌的PCR-RFLP方法

郑霄1, 夏连续1, 海荣1, 张志凯1, 蔡虹1, 尤鑫2, 平静2, 李伟1   

  1. 1 中国疾病预防控制中心传染病预防控制所, 传染病预防控制国家重点实验室, 北京 102206;
    2 北京市疾病预防控制中心
  • 收稿日期:2014-07-25 出版日期:2014-12-20 发布日期:2014-12-20
  • 通讯作者: 李伟,Email: liwei@icdc.cn
  • 作者简介:郑霄,男,医学博士,主要从事传染病防控与研究工作,Email: zhengxiao@icdc.cn
  • 基金资助:

    卫生行业科研专项(201202021)

Development of PCR-RFLP for rapid differentiation of Yersinia pestis strains between Microtus and non-Microtus biotypes in China

ZHENG Xiao1, XIA Lian-xu1, HAI Rong1, ZHANG Zhi-kai1, CAI Hong1, YOU Xin2, PING Jing2, LI Wei1   

  1. 1 State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China;
    2 Beijing Center for Disease Control and Prevention
  • Received:2014-07-25 Online:2014-12-20 Published:2014-12-20
  • Supported by:

    Supported by the Special Fund for Health Sector, People's Republic of China (No. 201202021)

摘要:

目的 建立一种快速区分鉴别田鼠型与非田鼠型鼠疫耶尔森菌(鼠疫菌)的分子分型方法。方法 设计特异引物对93株不同来源、不同生物型鼠疫菌的天冬氨酸酶基因(aspA)进行PCR扩增,用限制性内切酶Hpy CH4Ⅳ对扩增产物进行限制性片段长度多态性(RFLP)分析;直接测序法检测aspA基因多态性。结果 aspA基因扩增产物经Hpy CH4Ⅳ酶切后呈现2种基因型:田鼠型鼠疫菌(38株)均为101、126 bp 2个条带;非田鼠型鼠疫菌(55株)均为227 bp单一条带。测序结果证实酶切识别位点发生突变导致带型差异。结论 初步建立了鼠疫菌PCR-RFLP(聚合酶链反应-限制性片段长度多态性)法aspA基因分型方案,通过Hpy CH4Ⅳ酶切带型差异区分高毒力的非田鼠型菌株与低毒力的田鼠型菌株,适用于疫源地流行病学调查。

关键词: 鼠疫耶尔森菌, 聚合酶链反应-限制性片段长度多态性, 分子分型

Abstract:

Objective To develop a simple genotyping method for rapid differentiation of Yersinia pestis strains between Microtus and non-Microtus biotypes. Methods Specific primers were designed and applied to amplify the aspartase gene (aspA) through polymerase chain reaction (PCR) among 93 Y. pestis strains of diverse origins and biotypes. The PCR products were genotyped by restriction fragment length polymorphism (RFLP) analysis using the restriction enzyme Hpy CH4Ⅳ. Genetic polymorphisms of aspA were determined by direct DNA sequencing. Results Among the 93 test strains, the amplified products of aspA digested with Hpy CH4Ⅳ showed two genotypes: Microtus biotype strains (38) all had two RFLP bands in sizes of 101 and 126 bp, respectively; and non-Microtus biotype strains (58) consistently had one single RFLP band in size of 227 bp. Direct sequencing data confirmed that mutation at the recognition site for restriction enzyme led to differences in the RFLP banding patterns. Conclusion A new genotyping method (aspA-PCR-RFLP) was established for rapid differentiation of Y. pestis strains between Microtus biotype with low virulence and non-Microtus biotype with high virulence according to different RFLP banding patterns. The proposed method can be used for epidemiologic surveillance in plague foci.

Key words: Yersinia pestis, Polymerase chain reaction-restriction fragment length polymorphism, Molecular typing

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