鸭甲肝病毒基因A型和C型双重RT-PCR检测方法的建立

Establishment of a duplex RT-PCR assay for detection of duck hepatitis A virus genotype A and genotype C

为建立同时检测基因A型和C型鸭甲肝病毒(DHAV-A,DHAV-C)的双重RT-PCR方法,本实验根据GenBank中DHAV-A和DHAV-C全基因组序列,针对DHAV-A的3C、3D和DHAV-C的2C区域分别设计了2对引物,通过对反应体系和反应条件的优化及特异性、灵敏度评价,建立了检测DHAV-A和DHAV-C的双重RT-PCR方法。该方法能够分别从DHAV-A、DHAV-C阳性样本中扩增出DHAV-A(259 bp)和DHAV-C(194 bp)的特异片段,而不与其它被检的无关病原发生非特异反应;对DHAV-A和DHAV-C的最低检出限分别为4.98×104拷贝/μL、1.68×104拷贝/μL;对2009年8月至2011年7月送检病料检出DHAV-A和DHAV-C的阳性率分别为20%(14/70)、70%(49/70);随机抽取的DHAV-C PCR阳性样本的病毒分离结果与双重RT-PCR检测结果的符合率为100%(16/16)。本研究结果显示,建立的双重RT-PCR方法具有良好的特异性和敏感性,可用于临床DHAV的快速鉴别诊断。

To develop a duplex RT-PCR assay for detecting both duck hepatitis A virus genotype A （DttAV-A） and genotypc C （DHAV-C）. Two pairs of primers were designed according to the genomic sequences of 3C and 3D gene region of DttAV-A and 2C gene of DHAV-C available in GenBank. The duplex RT-PCR assay was developed through optimization of reaction conditions and validation of specificity, sensitivity and repetitiveness of the method. The fragments of DttAV-A （259 bp） andDHAV-C （194 bp） were specific amplified from corresponding positive sample by the duplex RT-PCR, respectively, and no amplification for other virus. The sensitivity of the assay was 4.98 × 10^4 copies/μL for DHAV-A and 1.68 × 10^4 copies/μL lbr DHAV-C DNA. Seventy clinical samples collected from August 2009 to July 2011 were detected by the duplex RT-PCR and thepositive rates of DHAV-A and DHAV-C were 20% （14/70）, 70% （49/70）, respectively. Comparing with virus isolation of 16 DHAV-C positive samples detected by the duplex PCR assay, the duplex RT-PCR assay developed in this study is a sensitive and specific method for clinical differential diagnosis of the infection of duck hepatitis A virus genotype A or genotype C.