IHHNV假病毒核酸标准物质定值研究

陈桂芳 ¹，李会杰 ²，高运华 ³，杨佳怡 ³，董莲华 ³

Abstract

传染性皮下及造血组织坏死病毒（IHHNV）感染对虾引发急性传染病，对幼虾危害尤为显著。依据 Genbank 收录的 IHHNV 基因序列（AF218266.2），制备含 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，构建 IHHNV 数字 PCR 检测方法，完成假病毒核酸标准物质的均匀性与稳定性考察。该标准物质拷贝数浓度标准值及扩展不确定度：(1.22 ± 0.15) × 10³ copies/μL (k=2)。多平台数字 PCR 定值验证显示，相对标准偏差小于4 %，量值跨平台复现性良好。经商用核酸检测试剂盒比对，其量值与荧光定量 PCR 检测结果一致性佳。

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) infection causes acute infectious diseases, which severely endangers juvenile shrimp. According to the IHHNV gene sequence (AF218266.2) deposited in GenBank, pseudovirus reference material carrying the full-length IHHNV genome was prepared. Specific primers and probes targeting the ORF1 gene were designed to establish a digital PCR detection assay. The uniformity and stability of the reference material were verified via digital PCR. The certified value and expanded uncertainty were (1.22 ± 0.15) × 10³ copies/μL (k=2). Validation across multiple dPCR platforms presented relative standard deviations lower than 4 %, indicating favorable reproducibility. Detection results obtained by commercial IHHNV nucleic acid kits showed great consistency with the certified values.