实验动物沙门氏菌TaqMan MGB探针荧光定量PCR检测方法的建立及初步应用研究OA
Establishment and preliminary application of a TaqMan MGB probe-based quantitative PCR assay for detecting Salmonella spp.in laboratory animals
目的 建立实验动物沙门氏菌的 TaqMan MGB 探针荧光定量 PCR 检测方法.方法 针对沙门氏菌 HisJ 基因设计特异性引物和探针,建立沙门氏菌 TaqMan MGB 探针荧光定量 PCR 检测方法,验证该方法的灵敏度、特异性和稳定性,并对采集的 90 例 SPF 级小鼠肠内容物样本进行检测.结果 沙门氏菌TaqMan MGB 探针荧光定量 PCR 检测方法具有高度特异性,对大肠埃希菌、鼠棒状杆菌、嗜肺巴斯德杆菌、铜绿假单胞菌、支气管鲍特杆菌、肺炎克雷伯杆菌均无交叉反应,检测灵敏度达到 10 copies/μL.标准曲线显示104～109 copies/μL 浓度范围内具有良好的线性关系,相关系数 R2=0.997 1,斜率为-3.236 5,PCR 扩增效率为103.69%.应用本方法检测90 例 SPF 级小鼠肠内容物样本,结果显示沙门氏菌阴性.结论 TaqMan MGB探针荧光定量 PCR 方法具有灵敏、特异、稳定的特点,适用于实验动物沙门氏菌的快速检测.
Objective To establish a TaqMan minor groove binder(MGB)probe-based quantitative PCR(qPCR)assay for the detection of Salmonella spp.in laboratory animals.Methods Specific primers and a TaqMan MGB probe were designed to target the HisJ gene of Salmonella spp.in the assay.The specificity,sensitivity,and stability of the assay were determined.Using this method,we tested 90 intestinal content samples collected from specific pathogen-free(SPF)mice.Results The TaqMan MGB probe-based qPCR assay demonstrated high specificity,showing no cross-reactivity with Escherichia coli,Corynebacterium murium,Pasteurella pneumotropica,Pseudomonas aeruginosa,Bordetella bronchiseptica,or Klebsiella pneumoniae.The limit of detection was 10 copies/μL.Within the concentration range of 104～109 copies/μL,the standard curve exhibited excellent linearity,with a correlation coefficient(R2)of 0.997 1,a slope of-3.236 5,and an amplification efficiency of 103.69%.Using this assay,all 90 intestinal content samples from SPF mice tested negative for Salmonella spp.Conclusions The TaqMan MGB probe-based qPCR assay is a sensitive,specific,and reproducible method suitable for the rapid detection of Salmonella spp.in laboratory animals.
陈亮;于萌;郑志红
中国医科大学实验动物部,辽宁省转基因动物研究重点实验室,沈阳 110122中国医科大学实验动物部,辽宁省转基因动物研究重点实验室,沈阳 110122中国医科大学实验动物部,辽宁省转基因动物研究重点实验室,沈阳 110122
农业科技
实验动物沙门氏菌TaqMan MGB探针荧光定量PCR检测
laboratory animalSalmonella spp.TaqMan MGB probequantitative PCRdetection
《中国比较医学杂志》 2026 (18)
67-74,8
国家重点研发计划(2024YFF0728802).
评论