荧光定量聚合酶链式反应检测乳及乳制品中结核分枝杆菌方法的建立与评价

建立一种快速检测乳及乳制品中结核分枝杆菌的荧光定量聚合酶链式反应法.根据结核分枝杆菌保守序列设计特异引物,建立结核分枝杆菌的实时荧光定量聚合酶链式反应技术(polymerase chain reaction,PCR)检测法.对该方法的灵敏度、特异度和重复性进行考察.结果表明:建立的实时荧光定量PCR方法标准曲线线性关系良好,得到标准曲线方程为y=-3.29x+38.60 (R2=0.998 0);敏感性高,可达1×102 copies/μL;抗干扰能力强,仅结核分枝杆菌出现特异性扩增曲线;重复性良好,分别用不同质量浓度的标准质粒进行组内和组间平行实验,批内和批间的变异系数均小于1％.因此,实时荧光定量PCR法能准确快速检测乳及乳制品中结核分枝杆菌.

Establishment and Evaluation of Real-Time Fluorescence Quantitative PCR Method for Mycobacterium tuberculosis in Milk and Dairy Products

A real-time fluorescence quantitative polymerase chain reaction (PCR) method was established for the rapid detection of Mycobacterium tuberculosis in milk and dairy products. According to the conserved sequence of Mycobacterium tuberculosis, specific primers were designed for establishing the PCR assay. The methodology was validated with respect to sensitivity, specificity and repeatability. The standard curve of the real-time PCR method had a good linear relationship with a correlation coefficient (R2) of 0.998 0, and its equation was fitted as y = ? 3.29x + 38.6. This method had a high sensitivity of 1 × 102 copies/μL and high anti-interference capacity, gave specific amplification curves for Mycobacterium tuberculosis and exhibited good repeatability. The intra- and inter-assay coefficients of variation (CV) obtained with varied concentrations of the standard plasmid were both smaller than 1%. To conclude, the real-time PCR method allows fast and accurate detection of Mycobacterium tuberculosis in milk and dairy products.