化脓隐秘杆菌TaqMan实时荧光定量PCR检测方法的建立

本试验旨在建立检测化脓隐秘杆菌(Arcanobacterium pyogenes,A.pyogenes)特异、灵敏的TaqMan实时荧光定量PCR检测方法。根据GenBank公布的化脓隐秘杆菌溶血素(pyolysin,PLO)基因高保守序列,设计特异性引物和探针建立检测体系,用于化脓隐秘杆菌的快速检测,并对该方法的特异性和灵敏度进行检测。结果显示,本试验建立的TaqMan实时荧光定量PCR方法仅对化脓隐秘杆菌的检测结果为阳性;该方法最低检测DNA浓度为77.6 fg,最低检测细菌浓度为63 CFU/mL。采用本研究建立的方法检测23份林麝临床病例样品,共鉴定出16株化脓隐秘杆菌,与API Coryne生化鉴定方法的结果相同。本研究为化脓隐秘杆菌的检测提供了一种灵敏、特异、快速的检测方法,其可用于化脓隐秘杆菌的诊断和流行病学调查。

Establishment of TaqMan Real-time PCR Assay for Detection of Arcanobacterium pyogenes

The study was aimed to establish a specific and sensitive TaqMan Real-time PCR assay for detection of Arcanobacterium pyogenes (A.pyogenes).Based on the conservative sequence of pyolysin (PLO) gene of A.pyogenes published in GenBank, specific primers and TaqMan probes were designed. The TaqMan Real-time PCR assay was established, and the specificity and sensitivity were tested.The specificity test results showed that only A.pyogenes exhibited typical curves.The detection sensitivity of this assay was 77.6 fg genomic DNA per 20 μL reaction, and 63 CFU/mL for pure cultures.16 out of 23 clinical samples were positive detected by the TaqMan Real-time PCR assay, which were consistent with API Coryne identification.The TaqMan Real-time PCR assay developed in this study was specific and sensitive for detection of A.pyogenes, and it could be used for identification and epidemiological investigation of A.pyogenes.