臭氧对赤点石斑鱼神经坏死病毒RNA破坏效果的评价方法的建立与应用

Development and Application of the LR RT-PCR Method for Evaluating Destructive Effects of Ozone on Red-spotted Grouper Nervous Necrosis Virus

本研究依据赤点石斑鱼神经坏死病毒(Red-spotted grouper nervous necrosis virus,RGNNV)RNA2的序列,在其3′端设计反转录引物,在5′端设计PCR扩增引物,构建了一种可以评价RNA链相对完整性的RT-PCR方法,命名为LR RT-PCR(Left reverse transcript Right amplify RT-PCR),并对该方法的引物浓度、Mg2+浓度、d NTPs浓度、退火温度4个重要参数进行了优化。结果显示,优化后的LR RT-PCR反应体系为:引物浓度0.2μmol/L、Mg2+浓度4 mmol/L、d NTPs浓度0.5 nmol、退火温度60℃。本研究建立的LR RT-PCR方法灵敏度高,可以检出1 pg的RGNNV RNA2标准品;该方法特异性强,与RSIV等鱼类常见病毒、鳗弧菌等常见水产病原菌以及石斑鱼RNA均不产生交叉反应。应用建立的LR RT-PCR方法对臭氧破坏RGNNV RNA2的效果进行评价,发现当臭氧浓度由0.3 mg/L依次增加为0.5、1.0、2.0 mg/L时,LR RT-PCR的扩增产物逐渐减少,直至消失。结果表明,建立的LR RT-PCR方法可以快速、准确地评价臭氧对RGNNV RNA2的破坏效果,适用于养殖场评估臭氧对RGNNV的消毒效果,并对RGNNV进行检测和监控。

Red-spotted grouper nervous necrosis virus （RGNNV） is one major pathogen of aquaculture. It mainly affects the larvae and juveniles of groupers. RGNNV is composed of two single stranded RNA （RNA1 and RNA2）. Ozone can inactivate RNA virus by degradation of viral genome. So treatment by ozone is a common measure to disinfect RGNNV in farms. But it has no efficient methods to evaluate the disinfection effect of ozone up to now. In this report, a special RT-PCR named as LR RT-PCR （Left reverse transcript Right amplify RT-PCR） was developed and optimized to evaluate degradation effects of RGNNV RNA2 by ozone. Based on the 3￠ end and 5￠ end of RGNNV RNA2, a special reverse transcription primer and a set of PCR primers were designed respectively. The concentration of primers, Mg2＋, dNTPs as well as annealing temperature of LR RT-PCR was optimized in this study. The sensitivity and specificity of LR RT-PCR also were confirmed. Finally the LR RT-PCR method was used to evaluate degradation effects of RGNNV RNA2 by ozone. With summarizing the results of the experiment, in 25 μl of reaction volume, the optimized parameters of LR RT-PCR were primers 0.2 μmol/L, Mg2＋ 4 mmol/L, dNTPs 0.5 nmol, Ex Taq 0.5 U and cDNA template 1 μl. The annealing temperature was 60℃. The sensitivity of LR RT-PCR was 1 pg to RGNNV RNA2 and there were no cross reactions with genomic RNA from healthy groupers, DNA from common bacteria and viruses of aquaculture. Comparative analysis of LR RT-PCR and routine RT-PCR was carried out to evaluate the destructive effects by ozone. As the concentration of ozone increased from 0.3 mg/L to 2 mg/L in solution of RGNNV RNA2, the amplified product of LR RT-PCR decreased and finally was undetected. Former reported routine RT-PCR method for RGNNV detection did not reflect above tendency. This report demonstrated that the LR RT-PCR can quickly and accurately evaluate disinfection effects of RGNNV by ozone and can be widely used in hatcheries of groupers.