Abstract: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 1 μL. 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.