Ｈ5 亚型高致病性禽流感病毒微滴式数字 PCR 检测方法的建立与应用

蒋文明，刘朔，李金平，彭程，尹馨，于晓慧，李阳，王静静，刘华雷

Abstract

为建立特异、灵敏的 H5 亚型高致病性禽流感病毒（high pathogenicity avian influenza virus, HPAIV）微滴式数字 PCR（droplet digital PCR, ddPCR）检测方法，根据 GISAID 和 GenBank 公布的 H5 亚型 HPAIVs HA 基因的核苷酸序列，设计并合成引物和探针，对反应体系和条件进行优化，建立针对 H5 亚型 HPAIVs 的 ddPCR 检测方法，并对该方法的特异性、重复性以及敏感性进行评估。结果显示，引物探针浓度为900 nmol/L和250 nmol/L、退火温度为55 ℃、升降温速率为2.0 ℃/s时 ddPCR 反应效率最高。该方法可特异性检测 H5 亚型 HPAIVs，对 H9N2、H7N9 等常见禽病病原均不能检出。该方法对标准品最低检测限为1.97 拷贝 /μL，敏感性比 qPCR 方法高 10 倍。组内和组间重复性试验的变异系数均小于5 %。对 80 份临床样品检测结果显示，与金标准方法的符合率为100 %。本研究建立的 ddPCR 方法特异性强、敏感性高、重复性好，为 H5 亚型 HPAIVs 的诊断和检测以及精准防控提供了重要技术支撑。

To establish a specific and sensitive droplet digital PCR (ddPCR) method for detection of H5 subtype high pathogenicity avian influenza virus (HPAIV), primers and probes were designed and synthesized based on the nucleotide sequences of H5 subtype HPAIV HA genes published in GISAID and GenBank. The reaction system and conditions were optimized, and the specificity, repeatability and sensitivity of the method were systematically evaluated. The results showed that the optimal concentrations of primers and probe were 900 nmol/L and 250 nmol/L, respectively. The optimal annealing temperature was 55 ℃, and the optimal heating and cooling rate was 2.0 ℃/s. The method could specifically detect H5 subtype HPAIV, but showed no cross-reactivity with H9N2 and H7N9 subtype AIVs or other common avian pathogens. The limit of detection for the standard substance was 1.97 copies/μL, and the sensitivity of this method was 10 times higher than that of qPCR. The coefficient of variation for both intra- and inter-assay repeatability tests was less than 5 %. The detection results of 80 clinical samples showed that the coincidence rate with the gold standard method was 100 %. The ddPCR method established in this study has strong specificity, high sensitivity and good repeatability, which provides important technical support for the diagnosis, detection and precise prevention and control of H5 subtype HPAIVs.