3型鸭甲型肝炎病毒逆转录环介导等温扩增技术快速检测方法的建立

Development of a reverse transcription loop-mediated isothermal amplification for duck hepatitis A virus type 3 detection

为建立一种3型鸭甲型肝炎病毒(DHAV-3)的快速检测方法,本研究针对DHAV-3 VP1基因序列中的8个不同区段设计了3对特异性引物,并对反应体系进行优化,通过加入羟基萘酚蓝(HNB)实现对反应结果的可视化观察,建立了检测DHAV-3的逆转录环介导等温核酸扩增(RT-LAMP)方法,采用已建立的方法对人工感染雏鸭的血清样品和病死鸭肝组织样品提取的核酸进行了检测。结果显示,该方法仅对DHAV-3的RNA进行扩增,而对1型鸭甲型肝炎病毒(DHAV-1)以及鸭常见的病原RNA均无扩增反应;可以检测出10拷贝/μL的DHAV-3 RNA,其敏感性为RT-PCR的1 000倍;最低能够检测到相当于1.1个DELD50的DHAV-3;能够检测到人工感染雏鸭血清和病死鸭肝组织中的DHAV-3;上述结果表明该方法能够特异、灵敏、快速地检测DHAV-3,而且使用方便。

In order to establish a rapid detection assay of duck hepatitis A virus type 3 （DHAV-3）, the reverse transcription loop-mediated isothermal amplification （RT-LAMP） was developed with three pair primers targeting VP1 gene sequence of DHAV-3 for the detection of DHAV-3, and the amplification result was visualized by adding the dye of HNB. DHAV-3 in the serum and liver tissue samples fi：om the artificial infection ducklings was detected by using the established assay. The results showed that the assay only reacted with DHAV-3 and had no cross-reaction with DHAV-I and other related duck pathogens. The detection limit of RT-LAMP assay was 10 copies/ixL of the virus, which was 103 fold higher than that of conventional RT-PCR. The assay had the ability to detect 1.1 DELDs0 of DHAV-3 and the virus in the serum and liver tissue samples from the artificial infection ducklings; The method is rapid, specific and sensitive for detecting of DHAV-3.