试验旨在建立一种快速检测禽源沙门氏菌SYBR Green Ⅰ荧光定量PCR(qPCR)的方法,即根据沙门氏菌invA基因的保守序列设计引物,利用普通PCR方法扩增沙门氏菌invA基因保守基因片段,将其克隆到pMD18-T载体上,将获得的重组质粒pMD18-T-invA作为标准阳性模板.经qPCR条件优化后,进行特异性、灵敏性和重复性试验.结果显示,所建立的SYBR Green Ⅰ qPCR方法的Ct值与标准品在1.4～1.4 × 1010拷贝/µL范围内呈良好的线性关系,R2为0.9963,扩增效率为95％,检测下限为1.4拷贝/µL;与大肠埃希菌、金黄色葡萄球菌、链球菌、痢疾志贺菌、多杀性巴氏杆菌无交叉反应;该方法组内变异系数和组间变异系数均小于2.5％;对44份粪便样本和132份蛋液样本进行qPCR方法和常规PCR方法检测,结果显示该qPCR方法的阳性检出率分别为22.7％(10/44)、0.8％(1/132),常规PCR的阳性检出率分别为9.1％(4/44),0％(0/132).结果表明:试验成功建立禽源沙门氏菌qPCR检测方法,可为禽源沙门氏菌的快速检测提供技术支撑.
Construction and Application of Fluorescent Quantitative PCR Method for Avian Salmonella
To establish a rapid method for detecting Salmonella SYBR Green Ⅰ from poultry using fluorescence quantitative PCR(qPCR),primers were designed according to the conserved sequence of Salmonella invA gene.The conserved gene fragment of Salmonella invA gene was amplified by ordinary PCR and cloned into pMD18-T vector.The obtained recombinant plasmid pMD18-T-invA was used as a standard positive template.After optimization of qPCR conditions,specificity,sensitivity and repeatability tests were performed.The results showed that the Ct value of the established SYBR Green Ⅰ fluo-rescence quantitative PCR method had a good linear relationship with the standard in the range of 1.4 × 1010 copies/μ L,R2 was 0.9963,the amplification efficiency was 95％,and the detection limit was 1.4 copies/μ L.There was no cross reaction with Escherichia coli,Staphylococcus aureus,Streptococcus,Shigella dysenteriae and Pasteurella multocida.The intra-group coefficient of variation and inter-group coefficient of variation were less than 2.5％.44 fecal samples and 132 egg liquid samples were tested using qPCR and conventional PCR methods.The results showed that the positive detection rates of the qPCR method were 22.7％(10/44)and 0.8％(1/132),the positive detection rates of conventional PCR were 9.1％(4/44)and 0％(0/132),respec-tively.In summary,this experiment successfully established a qPCR detection method for avian Salmonella,which provided technical support for the rapid detection of avian Salmonella.
SalmonellaSYBR Green Ⅰ fluorescence quantitative PCRinvA genedetection
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