鳜弹状病毒与传染性脾肾坏死病毒双重PCR检测方法的建立  

Establishment of duplex PCR assay for detecting Siniperca chuatsi rhabdovirus and infectious spleen and kidney necrosis virus

作　　者：梁红茹[1] 范芷仪 蔡秀珠 付小哲[1] 林强[1] 刘礼辉[1] 黄志斌[1] 牛银杰 林蠡 李宁求[1] LIANG Hongru;FAN Zhiyi;CAI Xiuzhu;FU Xiaozhe;LIN Qiang;LIU Lihui;HUANG Zhibin;NIU Yinjie;LIN Li;LI Ningqiu(Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Key Laboratory of Fishery Drug Development,Ministry of Agriculture and Rural Affairs,Key Laboratory of Aquatic Animal Immune Technology,Guangzhou,Guangdong 510380,China;College of Animal Sciences and Technology,Zhongkai University of Agriculture and Engineering,Guangzhou,Guangdong 570228,China)

机构地区：[1]中国水产科学研究院珠江水产研究所,农业农村部渔用药物创制重点实验室,广东省水产动物免疫技术重点实验室,广东广州510380 [2]仲恺农业工程学院动物科技学院,广东广州570228

出　　处：《西北农林科技大学学报（自然科学版）》2020年第3期39-46,53,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：中国-东盟海上合作基金项目;广东省促进经济发展专项资金(海洋经济发展用途)项目(GDME-20181007);广州市珠江科技新星项目(201710010087)。

摘　　要：【目的】建立鳜弹状病毒(SCRV)和传染性脾肾坏死病毒(ISKNV)的双重PCR检测方法。【方法】分别针对SCRV N基因和ISKNV MCP基因的保守区域设计特异性引物,通过优化退火温度和引物用量(μL)比,建立SCRV和ISKNV的双重PCR检测方法,并对该方法的敏感性和特异性进行检验。应用建立的双重PCR方法对20份临床样品进行单一和双重PCR检测,比较单一和双重PCR的检测结果。【结果】成功建立了SCRV和ISKNV双重PCR方法,该法特异性较强,对草鱼呼肠孤病毒(GCRV)、锦鲤疱疹病毒(KHV)、神经坏死病毒(NNV)、鲤春病毒血症病毒(SVCV)、新加坡石斑鱼虹彩病毒(SGIV)、罗非鱼湖病毒(TiLV)等鱼类常见病毒无扩增;敏感性较好,对2种病毒DNA的检测下限均为0.01 ng/μL。20份临床样品的双重PCR样品检测结果显示,3份为SCRV阳性,11份为ISKNV阳性,2份为SCRV和ISKNV混合感染,结果与单一PCR检测结果一致。【结论】建立的SCRV和ISKNV双重PCR检测方法特异性较强,敏感性较好,可用于鳜鱼SCRV与ISKNV的快速鉴别诊断和分子流行病学调查。【Objective】This study aimed to establish a duplex reverse transcription-polymerase chain reaction(PCR)assay for detecting the the Siniperca chuatsi rhabdovirus(SCRV)and infectious spleen and kidney necrosis virus(ISKNV)in Mandarin fish at the same time.【Method】Two pairs of specific primers were designed from the conserved sequence of N gene in SCRV and MCP gene in ISKNV.By optimizing the reaction conditions and system,a duplex PCR was established to detect the two genotypes simultaneously.After further testing the sensitivity and specificity,the duplex PCR and sequencing methods were used to detect 20 clinical samples.【Result】The duplex PCR for detecting SCRV and ISKNV was established successfully with high sensitivity for SCRV and ISKNV and no amplification for Grass Carp Reovirus(GCRV),Koi herpesvirus(KHV),Nervous necrosis virus(NNV),Spring Viremia of Carp Virus(SVCV),Singapore grouper iridovirus(SGIV)and Tilapia Lake Virus(TiLV).The DNA detection limit was 0.01 ng/μL for both SCRV and ISKNV.Among the 20 samples,3 were SCRV positive,11 were ISKNV positive and 2 were SCRV and ISKNV positive.The results of the duplex PCR were consistent with those of single PCR.【Conclusion】The established duplex PCR method had high sensitivity for SCRV and ISKNV and could be used for rapid diagnosis and epidemiology investigation on SCRV and ISKNV of Mandarin fish.

关 键 词：鳜弹状病毒 传染性脾肾坏死病毒 双重PCR 病毒检测 

分 类 号：S941.41[农业科学—水产养殖]