动物医学进展2025，Vol.46Issue(8)：12-18,7.
金黄色葡萄球菌血浆凝固酶基因实时荧光定量PCR检测方法的建立及应用
Establishment and Application of Real-time Fluorescence Quantitative PCR Assays for Detection of S.aureus coa Gene
摘要
Abstract
In order to establish an efficient and sensitive real-time fluorescence quantitative PCR method for the detection of coa gene of Staphylococcus aureus,and to provide technical support for disease diagnosis of S.aureus infection and food contamination detection.The specific primers were designed according to the sequences of coa gene,the target fragment was amplified by PCR and ligated into pMD-18T vector,and then transferred into Escherichia coli DH5α competent cells to construct positive recombinant plasmid.The real-time fluorescence quantitative PCR method for rapid detection of S.aureus coa gene was established by optimizing the primer concentration and annealing temperature.And the sensitivity,specificity and repeat-ability of the real-time fluorescence quantitative PCR method were verified.The results of this study showed that the standard curve y=-3.478x+44.8 was established using the recombinant plasmid as the standard substance,and the linear correlation coefficient was R2=0.999,showing a good linear relationship.The optimal primer concentration was 0.15 μmol/L and the optimal annealing temperature was 54.3℃.The minimum detectable limit was 5.76 × 103 copies/μL.The sensitivity was 100-fold higher than that of con-ventional PCR.The method showed a good specificity and no amplification signal for Staphylococcus epi-dermidis,Escherichia coli,Bacillus subtilis,Streptococcus porcinus and Pseudomonas aeruginosa.The co-efficients of variation of inter-group and intra-group repeatability test results were less than 2％,which showed good repeatability.The real-time fluorescence quantitative PCR method proved to possess high sen-sitivity,specificity and good repeatability based on the coa gene of S.aureus,the 56 clinical isolates of S.aureus were detected,38 isolates were plasma coagulase positive and the positive rate was 67.86％(38/56),which can be used for the detection and epidemiological investigation of coagulase-positive S.aureus.关键词
金黄色葡萄球菌/血浆凝固酶基因/荧光定量PCRKey words
S.aureus/coa gene/Real-time fluorescence quantitative PCR分类
农业科技引用本文复制引用
李雪婷,付晓佳,王越,韩淋,徐莹,王羽,郭海勇..金黄色葡萄球菌血浆凝固酶基因实时荧光定量PCR检测方法的建立及应用[J].动物医学进展,2025,46(8):12-18,7.基金项目
吉林省科技发展计划项目(20170101027JC) （20170101027JC）
吉林省教育厅科学技术研究规划项目(JJKH20230519KJ) （JJKH20230519KJ）
