犬圆环病毒荧光RAA检测方法的建立及初步应用

Establishment and preliminary application of a real-time recombinase-aid amplification assay for detection of canine circovirus

为建立一种高效、简便的犬圆环病毒(CCV)的检测方法,本研究针对CCV Rep基因设计了特异性引物和探针,同时构建了基于该病毒Rep基因的重组质粒标准品pCCV-Rep,经过优化后确定了CCV重组酶介导核酸等温扩增(RAA)检测方法引物、探针的最适浓度均为10μmol/L,在39℃恒温条件下20 min即可完成检测。利用该方法检测犬圆环病毒、狂犬病病毒、犬细小病毒、犬瘟热病毒、犬副流感病毒、犬腺病毒等其他常见的犬源病毒,结果显示除CCV外,该方法对其他病毒均无交叉反应,特异性较强;将p CCV-Rep分别稀释为10~6拷贝/μL~10~0拷贝/μL后作为模板,进行敏感性试验,结果显示该方法最低检出限可达10~2拷贝/μL,敏感性较高;批内批间重复性试验结果均表明该方法具有良好的重复性。采用该方法与常规PCR方法同时检测82份犬粪便拭子,结果显示,该RAA方法检出阳性样品5份,检测结果与常规PCR一致。本实验首次建立的CCV荧光RAA检测方法简便、高效、特异、灵敏,为CCV的临床筛查与流行病学研究提供了可行技术。

In order to establish an assay to diagnose canine circovirus efficiently and easily, specific primers, probes and recombinant standard plasmids named p CCV-Rep containing the Rep gene of canine circovirus were designed. By screening the primers and optimizing the reaction conditions, a real-time recombinase-aid amplification(RAA) assay was established. The detection can be completed within 20 minutes at 39℃ and no cross-reactivity with rabies virus, canine parvovirus, canine distempervirus, canine parainfluenza virus and canine adenovirus were observed. Sensitivity test with serial dilutions of pCCV-Rep ranging from 106 copies/μL-100copies/μL showed the minimum detection of pCCV-Rep was 102 copies/μL. Repeatability tests in intra-and inter-assay indicated the assay had good repeatability. 82 fecal swabs were collected to evaluate the clinical performance and 5 samples were detected to be positive, which was consistent with the results from PCR detection. Overall, the CCV real-time RAA assay established was specific, sensitive, and efficient. It could be used for clinical screening and epidemiological surveillance of CCV.