血清3型鸭甲型肝炎病毒实时荧光定量RT-PCR检测方法的建立与初步应用

Establishment and application of real-timeRT-PCR to detect Duck hepatitis A virus serotype 3

关键词:  血清3型鸭甲型肝炎病毒  实时荧光定量RT-PCR

Key words:  Duck hepatitis A virus serotype 3  real-time RT-PCR

摘要:

通过对Genbank公布的鸭甲型肝炎全序列比对，在3型鸭甲肝炎病毒（DHVA-3）5，非编码区的保守区设计一对检测引物和一条特异性TaqMan探针，经过反应条件和反应体系的优化，建立了一种能够特异性检测DHAV–3的实时荧光定量RT-PCR方法。试验结果显示，该方法能够特异性地检测出DHAV-3，而与血清1型鸭肝炎病毒（DHAV-1）、鸭瘟病毒、新城疫病毒、禽流感病毒(H9)、呼肠孤病毒、传染性支气管炎病毒无交叉反应。以目标序列质粒经体外转录合成的RNA作为阳性标准品，在1.8?108～1.8?103copies/μL的浓度范围内标准曲线线性关系较好，相关系数为0.992。敏感性试验表明，最低检测限为36copies。用该方法检测尿囊液中病毒拷贝数与ELD50滴度测定进行比较，表明二种方法检测结果呈正相关。本研究所建立的检测方法特异性好、灵敏度高且方便操作，为血清3型鸭甲型肝炎病毒的快速诊断和流行病学调查提供了技术手段，也会疫苗生产中毒价测定提供了一种参考方法。

Abstract:

By comparing genome sequences of Duck hepatitis A virus delivered by GenBank, primers and probe were designed based on conserved region of the 5' untranslated region (UTR) of DHAV-3. The reaction conditions were optimized for sensitivity and specificity. Results show that, the detection assay has good specificity for DHAV-3, and there is no cross reaction with Duck hepatitis A virus serotype 1, Duck plague virus, Newcastle disease virus, Avian Influenza Virus(H9), Duck reovirus, Avian Infectious Bronchitis Virus Standard positive template was prepared by the transcription of recombinant plasmid in vitro. The correlation coefficient is 0.992, when the template was diluted to 1.8?108～1.8?103 copies/μL, and the detection limit of the assay was 36copies. Compared the real-time RT-PCR with ELD50 in the detection of DHAV-3, the virus copies was positively correlated with virus titer in duck allantoic fluid, The developed method could be used for rapid diagnosis and epidemiological survey of DHAV-3, and also for the detection of virus titer in vaccine production as a reference method.