猪丹毒丝菌SYBR GreenⅠ荧光定量PCR检测方法的建立

Development of a SYBR GreenⅠ real-time PCR method for detection of Erysipelothrix rhusiopathiae

为建立一种快速、 敏感的猪丹毒丝菌检测方法, 本试验将猪丹毒丝菌的 grol 基因与载体TA/ Blunt- Zero 相结合构建重组质粒,以该重组质粒 DNA 为模板,筛选最佳反应条件,建 立 SYBR Green Ⅰ荧光定量 PCR 检测方法,并对该方法进行灵敏度、特异性和重复性测定。 结果表明,本研究建立的检测方法对在 2×10^(2)~2×10^(9)copies/ μL 浓度范围内的质粒标准品有良好的线性关系, 线性相关系数 R^(2)=0.999 5,标准曲线为 y=-3.231x+41.834,未见非特异性扩增。 该方法对质粒标准品最低检测浓度为 20 copies/ μL,敏感度比普通 PCR 提高了 1 000 倍。 用该方法检测猪支气管败血波氏杆菌(Bb)、猪链球菌 3 型(Ss3)、猪链球菌 2型(Ss2)、大肠杆菌(E.coli)、沙门菌(Salmonella)和多杀性巴氏杆菌(Pm)时,检测结果均为阴性,表明该方法具有良好的特异性。重复试验结果显示,组内和组间的变异系数均小于 2%,表明该方法具有良好的重复性。运用本试验建立的方法对 20 株疑似猪丹毒丝菌临床样品进行检测,阳性检出率为 60%,高于普通 PCR 检出率(50%)。 综上,本试验建立的检测方法灵敏度高、特异性强、重复性好,适合临床上快速诊断丹毒丝菌。

In order to establish a rapid and sensitive detection method for E.rhusiopathiae,grol gene was connected with TA/Blunt Zero vector to construct a recombinant plasmid.Using the recombinant plasmid DNA as the template,the optimal reaction conditions were screened,and a SYBR GreenⅠ fluorescence quantitative PCR detection method was established.The sensitivity,specificity and repeatability of the method were performed.The results showed that this method has a good linear relationship with plasmid standards within the concentration range of 2×10^(2)~2×10^(9)copies/μL,with a linear correlation coefficient of R^(2)=0.999 5.The standard curve is y=-3.231x+41.834,no nonspecific amplification was observed.The minimum detection limit for plasmid standards is 20 copies/μL,and the sensitivity is 1×103times higher than that of common PCR methods.The test results of Bordetella bronchiseptica,Streptococcus suis 3(Ss3),Streptococcus suis 2(Ss2),Escherichia coli(E.coli),Salmonella and Pasteurella multocida(Pm),indicating that the method has good specificity.The coefficient of variation within and between groups in the repeated experiment were less than 2%,indicating that the method had good repeatability.The method established in this experiment was used to detect 20 suspected E.rhusiopathiae clinical samples,and the positive detection rate was 60%,higher than the ordinary PCR detection method(50%).The method established in this experiment has high sensitivity,high specificity,and good reproducibility,which is suitable for rapid clinical diagnosis of E.rhusiopathiae infection.