鸡毒支原体与鸡滑液囊支原体双重PCR检测方法的建立

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。

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

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.