鹦鹉热衣原体real-time quantitative PCR检测方法的研究

目的]为鹦鹉热衣原体的快速、准确检测奠定基础。方法]根据鹦鹉热衣原体主要外膜蛋白(MOMP)基因序列设计一对特异性引物,以SYBR GreenⅠ为荧光染料,建立了鹦鹉热衣原体Real-time quantitative PCR检测方法。根据检测结果,计算样品的批内和批间变异系数(CV)。结果]以提取的鹦鹉热衣原体DNA为模板,用引物Cps-1和Cps-2进行PCR扩增,得到长100 bp的片段。扩增产物回收纯化后,连接到pGEM-T Easy载体上并转化到大肠杆菌DH5α,菌落PCR检测筛选阳性克隆,培养后提取质粒DNA。当模板浓度范围为1.78×102~1.78×108拷贝/μl时,标准曲线相关系数达0.998;批内和批间变异系数(CV%)分别为1.30%~4.59%和5.72%~9.87%。结论]为鹦鹉热衣原体的感染、流行调查等提供了重要的技术参考。

Study on the Detection Method of Chlamydophila Psittaci by Using Real-time Quantitative PCR

Objective] The aim was to provide a basis for the fast,accurate detection of Chlamydophila psittaci Method] A pair of specific primers was designed on the basis of the gene sequence of major outer membrane protein(MOMP) of C.psittaci,and with SYBR GreenⅠas fluorescent dye,the real-time quantitative PCR detection method of C.psittaci was established.According to detection result,the intra-and inter-assay variation coefficient(CV) were calculated.Result] With the extraction DNA of C.psittaci as template and PCR amplification was made with Cps-1 and Cps-2 primer and a fragment with 100 bp length was gotten.The amplified products,after being recovered and purified,were connected to pGEMT Easy vector and transferred into coli DH5α.The colony was detected by PCR to screen out the positive clones.After cultivation,their plamid DNA was extracted.When the template concn.ranged from 1.78×102 to 1.78×108 copies /μl,the correlation coefficient of standard curve was 0.998,and the intra-and interassay variation coefficients were 1.30%-4.59% and 5.72%-9.87% resp.Conclusion] The important reference for the infection and prevalence investigation on C.psittaci was provided.