锦鲤疱疹病毒TaqMan荧光定量PCR快速检测方法的建立及应用

Establishment and Application of the TaqMan Real-Time PCR Detection Assay for Koi Herpesvirus

为建立快速有效的锦鲤疱疹病毒病检疫方法,根据锦鲤疱疹病毒(KHV)聚合酶基因(Sph)的保守序列设计1对引物和相应的TaqMan探针,建立了一种快速检测锦鲤疱疹病毒病荧光PCR方法。用建立的检测方法对细胞培养物进行检测,并与常规PCR对比,结果荧光PCR灵敏度高于普通PCR,能检出的最低拷贝数为1.6×102/μL。应用该方法对样品进行检测,结果表明:所建立的荧光PCR检测方法4 h内可报告检测结果。该方法具有快速、灵敏、特异及重复性好等特点,适用于锦鲤疱疹病毒的快速检疫。

To establish a rapid and effective quarantine method of the Kio Hepesvims disease, a set of primers and TaqMan probe for Fluorescent Quantitation PCR were designed to detect the conservative se-quenee of Kio Hepesvims Sph genes. The eell eultures were deteeted by using the established quantitative PCR assay, and the results were eompared with those of the routine PCR. The quantitative PCR sensitivi-ty was higher than that of the routine PCR. The quantity of KHV DNA was 1.6 ×102 eopies/FL. A reli-able diagnostic result earl be obtained just within 4 h. The assay proved to be a rapid, sensitive, specific and repetitive method for rapid detection of Kio Hepesvims from fish in quarantine.