基于荧光定量PCR的鳜传染性脾肾坏死病毒滴度检测方法

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

针对传染性脾肾坏死病毒(ISKNV)的ORF007基因,设计特异性引物及TaqMan探针,建立了ISKNV的实时荧光定量PCR方法。采用CPE法对连续10倍稀释的ISKNV培养液的滴度进行了测定,同时采用荧光定量PCR法测定病毒拷贝数。结果显示,荧光定量PCR测定的病毒拷贝数与CPE法测定的病毒滴度具有良好的线性关系,其线性方程为y=1.076x+0.545(R2=0.998 6),其中y为基因组拷贝数浓度的对数,x为病毒滴度TCID50的对数。研究表明,荧光定量PCR法可替代CPE法应用于ISKNV疫苗抗原的定量,大大缩短了疫苗制备的时间,为疫苗生产提供了方便。

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. 076 x ＋ 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.