鸡毒支原体强弱毒株荧光定量PCR鉴别检测方法的建立

Development of a real-time PCR assay for differentiation of Mycoplasma galli septicumvirulent strains from attenuated strains

为建立一种能鉴别鸡毒支原体(MG)强、弱毒株的快速检测方法,本研究根据GenBank中MG强毒株和弱毒株的基因组序列,选取特异性保守区序列设计了2对引物和2条探针,分别用于强弱毒株和弱毒株的检测,优化反应条件,建立了能区分MG强、弱毒株的荧光定量PCR检测方法。该法特异性强,对鸡常见呼吸道病原体的反应均为阴性;灵敏度高,可检测到100拷贝/μL的模板;稳定性好,批内和批间试验Ct值的变异系数小。本研究建立的MG强、弱毒鉴别检测方法简便、快捷,为该病的防控与净化提供新方法、新思路。

To develop a rapid method for differentiation of virulent strains of Mycoplasma gallisep-ticum （MG）from the attenuated strains, two pairs of primers and two TaqMan probes were de- signed according to the specific coservative region of MG sequence in GenBank,and used to detect MG strains and attenuated strains,respectively. The reaction conditions were optimized and the re-al-time PCR was established. There was no cross-reactivity with the common respiratory patho-gens of chicken, the detection limit of the assay was 100 copies/μL of template, the coefficients of variation were both low for the intra-assay and inter-assay tests,indicating a good specificity, sensi-tivity and reliability. These results suggest that the differential assay is a simple and rapid method, and provide a new way for control and purification of MG.