进境动物源性饲料中沙门菌荧光定量PCR检测方法的建立及应用

Establishment and application of a real-time qPCR for the rapid detection of Salmonella spp. in imported animal-derived feedstuff

为建立快速鉴别诊断进境动物源性饲料中沙门菌的实时荧光定量PCR方法,以inv A基因为靶基因,通过构建p UCm-inv A重组质粒建立的标准曲线确定了检测体系的灵敏度,并利用特异性试验和稳定性试验评价体系的检测性能。结果显示,该检测体系对基因组DNA的检测灵敏度为17 copies/L;由标准曲线的相关系数可知,初始拷贝数的对数值与Ct值之间具有良好的重现性（R2=0.988）;进境动物源性饲料样品的检出率与国标法一致;30份模拟污染样品的检出率达100%。结果表明,建立的快速鉴别诊断方法具有检测限低、重复性好、定量准确、数小时内即可得出检测结果的优点,极大地提高了进境动物源性饲料中沙门菌检测的工作效率和口岸通关速度。

A real-time quantitative PCR method was established for rapid detection of Salmonella spp. from imported animal-derived feedstuff. The invAgene was used as the target gene. The standard curve was established by constructing pUCm-invA recombinant plasmid and the sensitivity of the detection method was determined. The detection performance of this method was evaluated by specificity and stability experiment. The results were as followed：l）the sensitivity of assay was 17 copies/μ;2） there existed a good reproducibility between the logarithmic value of the initial copy number and the Gvalue （Re=O. 988）;3）the detection results of clinical samples were consistent with the method of GB/T 13091-2002;4）the detection rate of 30 artificial contamination samples was lO0%.Therefore,this rapid diagnosis method have advantages of low detection limit and good repeatability,which can greatly impro- vethe detection efficiency of Salmonella spp. in daily imported animal-derived feedstuff quarantine work.