【摘要】 为了快速准确地诊断禽心包积液-肝炎综合征(hydropericardium-hepatitis syndrome, HHS),试验根据禽腺病毒血清4型(Fowl adenovirus serotype-4,FAdV-4)的六邻体(Hexon)基因保守序列设计特异性引物和探针，将重组酶介导等温核酸扩增技术(recombinase-aided amplification, RAA)与荧光探针结合，通过对反应条件(温度和时间)进化优化建立一种用于检测FAdV-4的实时荧光RAA方法，对该方法进行特异性、敏感性、重复性试验，并分别采用该方法、普通PCR方法和实时荧光定量PCR(real-time fluorescence quantitative polymerase chain reaction, RFQ-PCR)方法检测56份鸡肝脏样本，并计算该方法与其他两种方法的符合率。结果表明：建立的实时荧光RAA方法在40℃条件下反应15 min即可完成检测；仅可检测出FAdV-4,与禽流感病毒(Avian influenza virus, AIV)、新城疫病毒(Newcastle disease virus, NDV)、传染性支气管炎病毒(Infectious bronchitis virus, IBV)、传染性喉气管炎病毒(Infectious laryngotracheitis virus, ILTV)、鸡贫血病毒(Chicken anemia virus, CAV)和传染性法氏囊病病毒(Infectious bursal disease virus, IBDV)均无交叉反应，特异性较强；对FAdV-4的最低检测限为1×10~1拷贝/μL,是普通PCR方法的100倍，与RFQ-PCR方法相当，敏感性较高；重复性试验中的组内重复性试验和组间重复性试验的变异系数(coefficient of variation,CV)均小于10%,重复性良好；对56份临床样品进行检测，该方法与RFQ-PCR方法和普通PCR方法的符合率分别为100%、96.43%。说明成功建立了FAdV-4的实时荧光RAA检测方法，该方法操作简便、快速，特异性较强，敏感性较高，重复性良好，可用于HHS的早期诊断。更多还原

【Abstract】 In order to a rapid and accurate diagnosis of avian hydropericardium-hepatitis syndrome(HHS), in the test, specific primers and probes were designed according to the conserved sequence of the Hexon gene of Fowl adenovirus serotype 4(FAdV-4). By combining recombinase-aided amplification(RAA) with fluorescent probes, a real-time fluorescent RAA method for the detection of FAdV-4 was established by optimizing the evolution of reaction conditions(temperature and time). Specificity, sensitivity and reproducibility tests were carried out on the method. Fifty-six chicken liver samples were detected by this method; ordinary PCR and real-time fluorescence quantitative polymerase chain reaction(RFQ-PCR), and the coincidence rate between the method and the other two methods was calculated. The results showed that the established RF-RAA method could be detected after 15 min of reaction at 40 ℃. Only FAdV-4 could be detected; there was no cross-reactivity with Avian influenza virus(AIV), Newcastle disease virus(NDV), Infectious bronchitis virus(IBV), Infectious laryngotracheitis virus(ILTV), Chicken anemia virus(CAV) and Infectious bursal disease virus(IBDV), with strong specificity. The minimum detection limit for FAdV-4 was 1×10~1 copies/μL, which was 100 times higher than that of ordinary PCR methods; comparable to RFQ-PCR methods, it had high sensitivity. The coefficient of variation(CV) within groups and between groups in the repeatability test was less than 10%, and the repeatability was good. Fifty-six clinical samples were tested, and the coincidence rates of the method with the RFQ-PCR method and the ordinary PCR method were 100% and 96.43%, respectively. The results indicated that the RF-RAA method for the detection of FAdV-4 was successfully established, which was simple and rapid with strong specificity, high sensitivity and good repeatability, and could be used for the early diagnosis of HHS.更多还原