IHHNV假病毒核酸标准物质定值研究  

Research of Quantification for IHHNV Pseudovirus Nucleic Acid Reference Materials

作　　者：陈桂芳 李会杰 高运华[3] 杨佳怡 董莲华[3] CHEN Gui-fang;LI Hui-jie;GAO Yun-hua;YANG Jia-yi;DONG Lianhua(College of Chemistry Engineering,Shenyang University of Chemistry Technology,Shenyang,Liaoning 110142,China;Shenzhen Institute for Technology Innovation,National Institute of Metrology,Shenzhen,Guangdong 518107,China;Center for Advanced Measurement Science,National Institute of Metrology,Beijing 100029,China)

机构地区：[1]沈阳化工大学化学工程学院,辽宁沈阳110142 [2]深圳中国计量科学研究院技术创新研究院,广东深圳518107 [3]中国计量科学研究院前沿计量科学中心,北京100029

出　　处：《计量学报》2023年第3期392-400,共9页Acta Metrologica Sinica

基　　金：国家市场监管总局科技计划项目(2021MK149)。

摘　　要：传染性皮下及造血组织坏死病毒(IHHNV)感染对虾引起急性传染病,对幼虾危害尤其明显。基于Genbank收录的IHHNV基因序列(AF218266.2),制备了含IHHNV全基因组序列的假病毒标准物质(NIM-RM4062)。针对ORF1基因设计特异性引物探针,建立了IHHNV数字PCR检测方法,进行IHHNV假病毒核酸标准物质的均匀性和稳定性检验。标准物质拷贝数浓度的标准值及扩展不确定度为:(1.22±0.15)×10^(3)copies/μL(k=2)。利用多种数字PCR平台对标准物质进行定值验证,测量的相对标准偏差(RSD)小于4%,标准物质量值在不同检测平台复现性较好。采用市售IHHNV核酸检测试剂盒进行验证,标准物质量值与荧光定量PCR试剂盒的检测结果具有较好的一致性。Infectious hypodermal and haematopoietic necrosis virus(IHHNV)infection caused acute infectious diseases,especially to juvenile shrimp.According to the IHHNV gene sequence(AF218266.2)accepted by GenBank,pseudoviruses containing the whole genome sequence of IHHNV have been produced.Referring to GB/T 25878-2010 guidance on IHHNV PCR test,ORF1 gene was aimed to design special primers and probes,digital PCR detection method for IHHNV was established.Then digital PCR method was employed to verify the uniformity and stability of the reference material.The reference values with expanded uncertainty was(1.22±0.15)×10^(3) copies/μL(k=2).Multiple dPCR platforms were recruited to validate the values of the reference materials,and the relative standard deviations value was less than 4%,showing good reproducibility.Commercial IHHNV nucleic acid detection kit was used to detect the reference materials,the results were in good agreement with the reference values.

关 键 词：计量学 传染性皮下及造血组织坏死病毒 假病毒标准物质 数字PCR 对虾病毒 不确定度 

分 类 号：TB99[一般工业技术—计量学]