中国媒介生物学及控制杂志 ›› 2012, Vol. 23 ›› Issue (2): 108-110.

• 论著 • 上一篇    下一篇

TaqMan-MGB探针实时荧光定量PCR检测恙虫病东方体方法的建立

付秀萍, 贺金荣, 张景山, 王景泉   

  1. 中国疾病预防控制中心传染病预防控制所人兽共患病室, 北京102206
  • 收稿日期:2011-10-08 出版日期:2012-04-20 发布日期:2012-04-20
  • 作者简介:付秀萍(1977-),女,硕士,助理研究员,从事立克次体疾病预防与控制研究。Email: fuxiuping313@sina.com
  • 基金资助:

    国家重点基础研究发展计划(973计划)资助(2010CB530200)

Establishment of Real-time PCR assays for detection of Orientia tsutsugamushi 56 kD gene with a TaqMan-MGB probe

FU Xiu-ping, HE Jin-rong, ZHANG Jing-shan, WANG Jing-quan   

  1. National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
  • Received:2011-10-08 Online:2012-04-20 Published:2012-04-20
  • Supported by:

    Supported by the National Basic Research Program of China(973 Program)(No. 2010CB530200)

摘要:

目的 建立敏感、特异、定量Real-time PCR方法,用于恙虫病东方体的检测。方法 根据恙虫病东方体56 kD外膜蛋白基因序列设计引物和探针,建立实时荧光定量PCR检测方法。结果 建立的TaqMan-MGB探针具有良好的特异性,建立的荧光定量PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.99),灵敏性评估发现每个20 μlPCR反应管中只要有26个拷贝的目的基因即可被检测到,即最低检出浓度为2拷贝/μl,并且具有较好的重复性。结论 建立的荧光定量PCR方法具有很高的特异性和敏感性,可用于恙虫病东方体感染的快速检测。

关键词: 恙虫病东方体, 实时荧光定量PCR, TaqMan-MGB探针

Abstract:

Objective To develop a highly specific and sensitive Real-time PCR assay to detect Orientia tsutsugamushi. Methods A pair of primers and a TaqMan-MGB probe were designed according to the 56 kD outer membrane gene sequence. Results A linear relationship between the threshold cycle (Ct) of the quantitative Real-time PCR and the DNA copy number was demonstrated (r=0.99). The standard curve showed that 26 copies target genes per reaction could be detected by this method. The lowest detection limit of this method was 2 copies per μl. The method showed high species specificity and good reproducibility. Conclusion These results suggested that the Real-time PCR with TaqMan-MGB assay is highly specific and sensitive for the detection of O. tsutsugamushi, which might be applied for the diagnosis of this infection.

Key words: Orientia tsutsugamushi, Real-time PCR, TaqMan-MGB probe

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