林麝源化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌三重实时荧光定量PCR检测方法的建立

Development of Triplex TaqMan Real-time PCR for Detection of Arcanobacterium pyogenes,Mannheimia haemolytica and Streptococcus pneumoniae from Musk Deer

为快速准确检测化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌,分别根据化脓隐秘杆菌plo基因、溶血性曼氏杆菌ICEMh1基因和肺炎链球菌pspC基因的特异性序列设计引物和探针,以重组质粒a.p-m.h1-s.p为标准品,通过优化反应条件,建立一种检测化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌三重实时荧光定量PCR方法,对其特异性、灵敏度和可重复性进行测试。结果表明,建立的方法对标准品阳性质粒的最低检测量为20拷贝,标准曲线在1.25×10^6 copies/μL^1.0×101 copies/μL范围内具有良好的线性关系。对2016年-2018年在陕西省收集的病料中化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌检出率分别为66.67%、5.5%和44.44%。建立的三重实时荧光定量PCR方法能快速、准确和稳定地检测化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌。

Triplex TaqMan real-time PCR was developed to simultaneously detect Arcanobacterium pyogenes,Mannheimia haemolytica and Streptococcus pneumoniae in musk deers.The specific primers and probes were designed in the specific regions based on the plo gene of Arcanobacterium pyogenes,ICEMh1 gene of Mannheimia haemolytica and pspC gene of Streptococcus pneumoniae.The sensitivity,specificity and reproducibility of the assay were evaluated.The results showed that the assay has high specificity and good reproducibility for the detection of Arcanobacterium pyogenes,Mannheimia haemolytica and Streptococcus pneumoniae.The standard curve established using the recombinant plasmid a.p-m.h1-s.p as a standard has a good linear relationship in the range of 1.25×10^6 to 1.0×101 copies/μL.The limit of quantitation was 20 copies of positive plasmid standard.18 clinical samples were examined by this real-time PCR.The results showed that the detection rates of plo,ICEMh1 and pspC genes were 66.67%(13/18),5.5%(1/18)and 44.44%(8/18)respectively.This study provided a method for rapid,accurate and stable detection of Arcanobacterium pyogenes,Mannheimia haemolytica and Streptococcus pneumoniae.