传染性脾肾坏死病毒、鳜鱼蛙病毒和鳜弹状病毒三重PCR检测方法的建立

Establishment of multiple PCR assay for detecting infectious spleen and kidney necrosis virus,Siniperca chuatsi ranairidovirus and Siniperca chuatsi rhabdovirus

【目的】建立可同时检测传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)、鳜鱼蛙病毒(Siniperca chuatsi ranairidovirus,SCRIV)和鳜弹状病毒(Siniperca chuatsi rhabdovirus,SCRV)的三重PCR检测方法,为鳜鱼等养殖品种流行病学调查提供方法支撑。【方法】根据ISKNV MCP基因、SCRIV MCP基因和SCRV N基因设计3对特异性引物,对PCR扩增反应中的退火温度和引物用量进行优化,建立可同时检测ISKNV、SCRIV和SCRV的三重PCR方法。为验证该方法的敏感性和特异性(备测病毒为IPNV、GCRV、KHV、SGIV、NNV、TiLV和SVCV),对22份疑似感染ISKNV、SCRIV和SCRV的样品分别进行单一和三重PCR检测。【结果】成功建立了三重PCR检测方法,利用该方法可同时检测ISKNV、SCRIV和SCRV,特异性较好,对IPNV、GCRV、KHV、SGIV、NNV、TiLV、SVCV等无扩增;该方法敏感性好,对3种病毒核酸的检测下限均为0.01 ng/μL;利用该方法和3种病毒单一PCR方法同时对22份临床样品进行检测,结果显示2种方法吻合率为100%,其中ISKNV阳性率为27%、SCRIV阳性率为41%、SCRV阳性率为9%、ISKNV和SCRIV混合感染阳性率均为9%,无ISKNV、SCRIV和SCRV混合感染。【结论】建立的三重PCR检测方法具有特异性强、灵敏度高等特点,可对这3种病毒进行快速鉴别诊断。

【Objective】This study established a method for simultaneous PCR detection of infectious spleen and kidney necrosis virus(ISKNV),Siniperca chuatsi ranairidoviru(SCRIV)and Siniperca chuatsi rhabdovirus(SCRV).【Method】Three pairs of specific primers were designed based on MCP gene in ISKNV,MCP gene in SCRIV and N gene in SCRV,and annealing temperature and primer dosage in PCR amplification reaction were optimized.A multiplex PCR was established to detect ISKNV(550 bp),SCRIV(400 bp)and SCRV(780 bp)simultaneously.The sensitivity and specificity of the method were verified by comparing with single PCR using 22 suspected infected ISKNV,SCRIV and SCRV samples.【Result】The triple PCR method was established successfully and it was able to detect ISKNV,SCRIV and SCRV simultaneously with good specificity and no amplification of common fish viruses such as IPNV,GCRV,KHV,SGIV,NNV,TiLV and SVCV.The method had good sensitivity with lower detection limit of 1×10-2 ng/μL for the three viral DNAs.The testing of 22 clinical samples showed that the established method and single PCR methods were 100%identical.The positive rates of ISKNV,SCRIV,and SCRV were 27%,41%and 9%.The positive rate of mixed infection of ISKNV and SCRIV was both 9%,while the positive rate of mixed infection of ISKNV,SCRIV and SCRV was 0.【Conclusion】The established multiple PCR detection method had high specificity and sensitivity.Thus,it can be used for rapid identification and diagnosis of the three viruses.