鸭甲肝病毒1型和3型一步法双重RT-PCR鉴别检测方法的建立及其初步应用

Establishment and preliminary application of a one-step dual RT-PCR method for the differentiation of duck hepatitis A virus types 1 and 3

为建立一种快速鉴别不同基因型鸭甲肝病毒(DHAV)的检测方法,根据DHAV-1和DHAV-3特异性序列,分别设计合成鉴别检测引物,经一步法双重RT-PCR反应条件的优化,建立了鉴别DHAV-1和DHAV-3的一步法双重RT-PCR方法,并应用该方法对2016—2021年山东地区采集的58份临床病料样品进行病原学检测分析。结果显示:该方法能够分别扩增出DHAV-1的230 bp和DHAV-3的502 bp的特异性条带,而DTMUV、NDV、AIV-H9、FADV、DEV、GPV均无任何扩增条带。该方法最低可检测到0.62 ng DHAV-1 RNA和0.59 ng DHAV-3 RNA。该方法与病毒分离方法的符合率达100%。该方法检测58份临床病料样品,共检测出阳性样品27份,阳性率达46.55%,其中2016—2021年DHAV-1阳性率分别为33.33%,25.00%,22.22%,12.50%,11.11%,0.00%;DHAV-3阳性率分别为8.33%,16.67%,22.22%,37.50%,33.33%,37.50%。表明建立的DHAV-1和DHAV-3一步法双重RT-PCR鉴别检测方法具有良好的特异性、敏感性、准确性和临床适用性;同时发现山东地区DAHV的优势流行毒株已由DHAV-1转变为DHAV-3。

This study aims to establish a rapid detection method for duck hepatitis A virus(DHAV)compatible with the epidemic law of DHAV in Shandong.In this study,the identification and detection primers were designed and synthesized according to the specific sequences of DHAV-1 and DHAV-3.After the optimization of one-step dual RT-PCR reaction conditions,a one-step dual RT-PCR method capable of differentiating DHAV-1 from DHAV-3 was established and used to detect the 58 clinical specimen collected in Shandong from 2016 to 2021.The results showed that the one-step dual RT-PCR method could amplify 230 bp and 502 bp specific bands for DHAV-1 and DHAV-3,respectively with no amplification bands for DTMUV,NDV,AIV-H9,FADV,DEV and GPV.The detection sensitivity of DHAV-1 and DHAV-3 was 0.62 ng and 0.59 ng respectively.The coincidence rate with the virus isolation method was 100%.Out of 58 clinical samples,27 samples were detected as positive with the positive rate of 46.55%.The positive rates of DHAV-1 from 2016 to 2021 were 33.33%,25.0%,22.22%,12.50%,11.11%and 0.00%,respectively.The positive rates of DHAV-3 were 8.33%,16.67%,22.22%,37.50%,33.33%and 37.50%,respectively.The results showed that the established one-step dual RT-PCR for differentiation of DHAV-1 from DHAV-3 has good specificity,sensitivity,accuracy and clinical applicability.The dominant epidemic strain of DHAV in Shandong appears to have changed from DHAV-1 to DHAV-3.