qPCR method for virus titer testing of infectious spleen and kidney necrosis virus

Infectious spleen and kidney necrosis virus(ISKNV)is the causative agent causing high mortality and significant economic losses to Chinese perch and it is listed by OIE.Now an effective virus inactivated vaccine has been developed and it is necessary to determine virus titer in vaccine development process.Because the traditional cytopathic effect(CPE)method is time-consuming and laborious,it is essential to establish a rapid,accurate method for testing ISKNV titer.The development and validation of a TaqMan qPCR assay for determination of ISKNV titers were reported in the present study.This method used specic primers and probe designed to amplify a small genomic fragment of ISKNV 007 gene.The method was specific and reproducible as well as sensitive with the minimum detectable limit of 10 viral copies.At the same time cytopathic effect(CPE)method was compared and analyzed to qPCR.The results showed that the titers obtained by qPCR correlated well with TCID₅₀ as indicated by linear regression and linear equation was y=1.076x+0.545(R²=0.998 6).Y stands for Log gene copies concentration and X stands for Log TCID₅₀ of virus.The results showed that the real-time PCR method can replace the CPE method in evaluating the titer of ISKNV vaccine,which will shorten vaccine producing cycle and provide convenience for vaccine production.