畜牧与饲料科学 ›› 2019, Vol. 40 ›› Issue (2): 12-15.doi: 10.12160/j.issn.1672-5190.2019.02.004

• 著者文摘 • 上一篇    下一篇

鸡毒支原体与鸡滑液囊支原体双重PCR检测方法的建立

杨斌[1];朝洛门[2];陈志鹏[2];苏日娜[2];张月梅[1];宋越[1];萨娜[2];萨日盖[2];萨茹丽[2];赵世华[1]   

  1. [1]内蒙古自治区农牧业科学院兽医研究所,内蒙古呼和浩特010031;[2]内蒙古农业大学动物科学学院,内蒙古呼和浩特010018
  • 收稿日期:2019-01-11 出版日期:2019-02-28 发布日期:2019-08-19
  • 通讯作者: 杨斌-内蒙古自治区农牧业科学院兽医研究所,内蒙古呼和浩特010031
  • 作者简介:杨斌(1984—),男,助理研究员,博士,主要研究方向为动物群体普通病防控及疫病诊断技术;通讯作者:赵世华(1962-),男,研究员,主要研究方向为兽医传染病防控。
  • 基金资助:
    国家重点研发计划项目(2016YFD0500800);内蒙古自治区自然科学基金项目[2016BS(LH)0301]。

Development of a Duplex PCR Assay for Simultaneous Detection of Mycoplasma gallisepticum and Mycoplasma synoviam

YANG Bin[1];Chaoluomen[2];CHEN Zhi-peng[2];Surina[2];ZHANG Yue-mei[1];SONG Yue[1];Sana[2];Sarigai[2];Saruli[2];ZHAO Shi-hua[1]   

  1. [1]Insititute of Veterinary Medicine,Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot 010031,China;[2]College of Animal Science,Inner Mongolia Agricultural University,Hohhot 010081,China
  • Received:2019-01-11 Online:2019-02-28 Published:2019-08-19

摘要: 为建立能同时检测鸡毒支原体(Mycoplasma gallisepticum, MG)和鸡滑液囊支原体(Mycoplasma synoviae,MS)的双重PCR诊断方法,该研究根据GenBank 中登录的MG gapA基因序列和MS heat shock ATP-dependent protease基因序列,设计2对特异性引物,通过对PCR扩增条件的优化,建立了能够同时检测MG和MS的双重PCR诊断方法。特异性检测结果显示,该方法能够扩增出729 bp的MG和309 bp的MS特异性片段,对禽巴氏杆菌、大肠杆菌、鸡白痢沙门菌、副鸡禽杆菌核酸扩增均为阴性;敏感性检测结果显示,对MG和MS DNA的最低检出量均为5×10^-2 ng/μL;临床样品的检测结果显示,所建立的双重PCR方法可同时有效地检测出MG、MS混合感染和单独感染。该研究建立的鸡毒支原体与鸡滑液囊支原体双重PCR方法具有良好的特异性、敏感性、重复性,为快速、高效检测MG和MS提供了技术支持。

关键词: 鸡毒支原体;鸡滑液囊支原体;双重PCR

Abstract: To develop a rapid PCR assay for simultaneous detection of Mycoplasma gallisepticum (MG) and Mycoplasma synovial (MS), two pairs of primers were designed and synthesized according to the published sequences of the gapA gene of MG and the heat shock ATP-dependent protease gene of MS in GenBank, and a duplex PCR protocol for detection of MG and MS was developed by optimizing conditions of PCR reaction. The specific fragments of 729 bp for MG and 309 bp for MS were simultaneously amplified in the duplex PCR, but no PCR products were amplified for Pasteurella multocida,Escherichia coli,Samonella pullorum and Avibacterium paragallinarum. The detection limit was 5×10^-2 ng/μL for both of MG and MS. Detection results from clinical samples demonstrated that the co-infection of MG and MS as well as single infection of MG or MS could be found by using the established duplex PCR assay. In conclusion, the duplex PCR assay has good specificity, sensitivity and repeatability, which provides effective technical support for clinical diagnosis of MG and MS co-infection in veterinary clinic.

中图分类号: