白斑综合征病毒和对虾内参基因双重荧光定量PCR检测方法的建立  

Development of a duplex real-time fluorescent quantitative PCR assay for simultaneous detection of white spot syndrome virus and shrimp reference gene

作　　者：李海平 葛辉[2] 温凭[2] 吴丽云 李慧耀 巫旗生[2] 张哲[2] 杨求华[2] 何丽斌[2] 徐春燕[2] LI Haiping;GE Hui;WEN Ping;WU Liyun;LI Huiyao;WU Qisheng;ZHANG Zhe;YANG Qiuhua;HE Libin;XU Chunyan(Fujian Institute of Oceanography,Xiamen 361013,China;Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province,Fisheries Research Institute of Fujian,Xiamen 361013,China)

机构地区：[1]福建海洋研究所,福建厦门361013 [2]福建省水产研究所,福建省海洋生物增养殖与高值化利用重点实验室,福建厦门361013

出　　处：《渔业研究》2024年第6期572-579,共8页Journal of Fisheries Research

基　　金：福建省属公益类科研院所基本科研专项(2022R1007003、2022R1013007);2023年度“揭榜挂帅”科技项目(2023F05);2023年度福建省促进海洋与渔业产业高质量发展专项资金项目(FJHYF-L-2023-5)。

摘　　要：【背景】白斑综合征病毒(WSSV)是一种高度传染性和致命性的病原体,对全球虾类养殖业构成重大威胁。WSSV可通过水平传播和垂直传播迅速扩散,虾在感染WSSV 3~10 d内累积死亡率最高可达到100%。由于缺乏有效的预防和治疗措施,开发快速、准确和敏感的WSSV检测技术对及时诊断和实施控制措施至关重要。【目的】建立一种WSSV和对虾内参基因双重荧光定量PCR检测方法,提高WSSV检测的灵敏度和特异性。【方法】首先,基于WSSV基因组保守序列设计合成特异性引物和TaqMan探针,建立WSSV单重荧光定量PCR检测体系,经特异性和重复性试验证实,该方法对WSSV具有良好的特异性和重复性。在此基础上,设计合成凡纳滨对虾(Litopenaeus vannamei)内参基因引物和探针,并对引物碱基进行优化替换,建立WSSV-对虾内参基因双重荧光定量PCR检测体系。【结果】敏感性试验结果显示,该双重检测体系对WSSV的检测下限可达15拷贝·μL^(-1),在6.6~6.6×10^(5)拷贝·μL^(-1)范围内,WSSV质粒标准品的起始模板量对数值与循环阈值(Ct值)呈良好的线性关系(R^(2)=0.9930)。同时,内参基因的引入可有效地区分样本中由核酸提取或操作问题引起的假阴性结果。【结论】综上所述,本研究建立的WSSV-对虾内参基因双重荧光定量PCR检测方法灵敏度高、特异性强、重复性好,可为WSSV的快速诊断及流行病学调查提供可靠的技术手段,对控制WSSV的暴发流行、保障对虾养殖业的可持续发展具有重要意义。[Introduction]The white spot syndrome virus(WSSV)is a highly infectious and lethal pathogen that poses a significant threat to the global shrimp farming industry.WSSV can spread rapidly through both horizontal and vertical transmission,leading to mortality rates of up to 100%within 3^(-1)0 days post-infection.Due to the absence of effective prevention and treatment measures,developing rapid,accurate,and sensitive detection technologies for WSSV is crucial for timely diagnosis and implementation of control measures to prevent disease spread.[Objective]This study aimes to establish a sensitive and specific duplex real-time fluorescent quantitative PCR(qPCR)method for the simultaneous detection of WSSV and an endogenous control gene in shrimp.[Methods]First,specific primers and TaqMan probes were designed based on the conserved sequences of the WSSV genome,establishing a single WSSV qPCR detection system.Specificity and repeatability tests confirmed that the method had excellent specificity for WSSV without cross-reactivity,with the coefficient of variation for different gradients of plasmid standards being less than 1.5%,indicating high repeatability.Subsequently,primers and probes for the endogenous control gene of Litopenaeus vannamei were designed and synthesized,and the primer sequences were optimized to establish a duplex qPCR detection system.[Results]Sensitivity tests revealed that the detection limit of the duplex system for WSSV was 15 copies·μL^(-1),with a strong linear relationship between the logarithmic value of the initial template amount and the cycle threshold(Ct)value of WSSV plasmid standards within the range of 6.6 to 6.6×10^(5)copies·μL^(-1)(R^(2)=0.998).Furthermore,the incorporation of the endogenous control gene effectively distinguished false-negative results caused by nucleic acid extraction or operational issues in the samples.[Conclusion]In conclusion,the established duplex qPCR detection method for WSSV and the shrimp endogenous control gene exhibits high sensitivity,strong specifici

关 键 词：白斑综合征病毒(WSSV) 荧光定量PCR TAQMAN探针 病毒检测 水产养殖 

分 类 号：S917.4[农业科学—水产科学]