禽源大肠杆菌荧光定量PCR方法的构建及应用

Establishment and application of Real-Time Fluorescence Quantitative PCR Assay for Chicken Escherichia Coli

为建立一种灵敏、准确且快速检测禽源大肠杆菌的方法,根据禽源大肠杆菌uidA基因的一节特异性保守序列设计引物,建立用于禽源大肠杆菌的荧光定量PCR(qPCR)检验方法,并对其各方面进行评价。试验数据表明所建立的qPCR方法的Ct值与标准品在4.69×10^(2)~4.69×10^(9 )copies/μL范围内存在优良的线性关系,线性相关系数为R2=0.993;该方法标准曲线方程为y=-3.2786x+39.032,熔解曲线表现为单峰,不存在非特异性扩增。对重组质粒标准品的最低检测浓度为4.69×10^(2) copies/μL,是普通PCR方法的1000倍。该方法检测临床样本阳性率为68.3%(41/60),普通PCR阳性检出率为23.3%(14/60),阳性检出率高出45%。此次研究建立的检测禽源大肠杆菌的荧光定量PCR检测方法可用于禽源大肠杆菌的快速诊断,对于禽大肠杆菌病的监测具有重要意义。

Aimto establish a sensitive,precise and rapid Real-time fluorescence quantitative PCR means for the detection of chicken escherichia coli,we designed primers based on a specific conserved sequence of uidA gene,which is chicken escherichia coli’s specific gene.The uidA gene of Avian Pathogenic Escherichia Coli was constructed on 19T vector.We utilized the recombinant plasmid as a standard positive template to set up means for the detection of chicken escherichia coli and evaluated the specificity,Sensitiveness and repeatability of the method.The results showed that the Ct values of the established qPCR method showed a good linear relationship with those of the standard products in the range of 4.69×10^(2)~4.69×10^(9) copies/μL,the linear correlation coefficient was R^(2)=0.993 and the standard curve equation was y=-3.2786x+39.032.At the same time,the melting curve is unimodal distribution and non-specific amplification.The results of Sensitivity test showed that the method possessed high sensitivities and the minimum detection limit was 4.69×10^(2) copies/μL,which was 1000 times that of the conventional PCR method.We also found that the positive rate of this method was 45%higher than ordinary PCR through the analysis of 60 clinical samples.The SYBR Green I real-time quantitative PCR method for the detection of chicken Escherichia coli established in this study is able to be used for the rapid diagnosis of chicken Escherichia coli,which is of great significance for the detection of colibacillosis.