鸭甲肝病毒2型TaqMan实时荧光定量PCR方法的建立

Establishment TaqMan Based Real-time Fluorescent Quantitative PCR of Duck Hepatitis A Virus Type 2

研究利用全基因合成技术合成DHAV-2全基因组序列,并以此为标准品建立检测DHAV-2的TaqMan实时荧光定量PCR方法。结果表明:建立的方法敏感性高,最低检测限仅为33.7拷贝/μL;特异性强,对鸭甲肝病毒1型(DHAV-1)、鸭甲肝病毒3型(DHAV-3)、禽流感病毒(Avian influenza virus,AIV)、番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)、新型鸭呼肠孤病毒(Novel duck reovirus,N-DRV)、鸭源禽Ⅰ型副黏病毒(Duck-origin avian paramyxovirus type 1,APMV-1)和禽坦布苏病毒(Avian Tembusu virus,ATmV)均无交叉扩增;重复性佳,组内和组间变异系数最高分别为1.68%和2.19%。对2018年福建地区临床收集的128份鸭源病料进行检测,结果未检测DHAV-2感染阳性。研究结果为DHAV-2的快速检测试剂盒研究及储备DHAV-2检测技术奠定了基础。

In this study,the duck hepatitis A virus type 2(DHAV-2)genome plasmids was synthesized and then used as positive plasmids,to establish a TaqMan based real-time fluorescent quantitative PCR platform for diagnose of DHAV-2 infection.After conditional optimization,the established real-time fluorescence quantitative PCR method for detection of DHAV-2 had a high sensitivity and the minimal limit detection was 33.7 copies/μL.There was strong specificity and no cross-reaction with the pathogens of common infectious diseases in ducks,such as DHAV-1,DHAV-3,AIV,MDRV,N-DRV,APMV-1 and ATmV.The repeatability was excellent,and the coefficient of variation for intra-and inter-group repeat tests was less than 1.68%and 2.19%,respectively.One hundred and twenty-eight clinical samples collected at 2018 in Fujian province were detected by the TaqMan based real-time fluorescent quantitative PCR technology,no positive sample was found.In conclusion,this study lay foundation for rapid detection kit and reserve detection technology of DHAV-2 infection.