禽腺病毒血清4型实时荧光RAA检测方法的建立

Establishment of real-time fluorescence RAA detection method for Fowl adenovirus serotype 4

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。

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 C.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 1x10'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 RFQPCR 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.