锦鲤疱疹病毒潜伏感染实时荧光定量PCR检测方法的建立

为建立一种特异和敏感的检测锦鲤疱疹病毒(koi herpesvirus,KHV)潜伏感染的方法,以KHV Sph基因为靶序列设计特异性引物,通过条件优化建立Real–Time PCR检测方法,并且对该方法进行特异性和敏感性评估及临床样品应用研究。结果显示:建立的方法与鲤痘疱疹病毒、金鱼造血器官坏死病病毒和鳗鲡疱疹病毒均无交叉反应,特异性强;最低检出率为42拷贝/μL,灵敏度高。用本方法对63份临床样品进行检测,有锦鲤疱疹病毒病(KHVD)病史的18份样品的检测结果全为阳性,阳性率为100%;从有临床症状的鱼中得到的15份样品的检测结果全为阳性,阳性率为100%;30份无任何临床症状鱼中的6份被检测为阳性,阳性率为20%。本试验中建立的水生动物疱疹病毒潜伏感染快速检测方法,可为潜伏感染KHV的临床检测提供一种快速、敏感和特异的检测手段,可为KHVD的分子流行病学调查和预防提供参考。

Method of Real-Time PCR for the detecting latent infection of koi herpesvirus

To establish a rapid, sensitive and specific approach for detecting the latent infection of koi Herpesvirus (KHV), a Real-Time PCR test was designed through specific primers designed to target KHV Sph gene. Under the optimized reaction conditions, the results showed that this method was specific for latent detection of KHV without cross reaction to other fish pathogenic viruses, including carp pox herpes virus (CyHV-1), goldfish herpes virus (CyHV-2) and eel herpes virus (AngHV-1). The lowest limitation of detection concentration by Real-Time PCR was 42 copies per 1μL. A total of 63 clinical samples were detected by Real-Time PCR. The result showed that 18 samples out of which had koi herpesvirus disease (KHVD) history, all samples were positive; 15 samples out of which had in clinical signs and the positive rate was 100%; 30 samples out of which had no any clinical symptoms, and 6 out of the 30 samples were positive, the positive rate was 20%. In conclusion, the approach provides a rapid, sensitive and reliable molecular tool for detecting latent infection of koi Herpesvirus.