Ｈ7 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，李金平，尹馨，彭程，刘朔，李阳，于晓慧，王静静，刘华雷

Abstract

为建立灵敏、特异的 H7 亚型禽流感病毒（AIV）微滴式数字 PCR（ddPCR）检测方法，本研究依据 GenBank 和 GISAID 收录的 H7 亚型 AIV 血凝素（HA）基因核苷酸序列，设计并合成 H7 亚型 AIV 特异性引物与探针，优化扩增反应条件，建立 H7 亚型 AIV ddPCR 检测体系，并评价其特异性、敏感性与重复性。结果显示：最佳引物浓度为900 nmol/L、探针浓度为250 nmol/L，最佳退火温度55 ℃，最佳升降温速率2.0 ℃/s，最佳反转录时间20 min。特异性试验表明，该方法仅可特异性检出 H7 亚型 AIV，无法检出 H5 亚型 AIV、H9 亚型 AIV、新城疫病毒、鸡传染性支气管炎病毒及传染性法氏囊病毒。敏感性试验结果显示，该方法对重组质粒标准品的最低检测下限为0.8 copies/μL，灵敏度较荧光定量 PCR（qPCR）高出 10 倍。组内与组间重复性试验变异系数均小于3 %。对 60 份临床样品开展检测，该方法与鸡胚分离法检测符合率达100 %。综上，本研究建立的 H7 亚型 AIV ddPCR 检测方法特异性强、重复性好、灵敏度高，可应用于 H7 亚型 AIV 早期诊断、流行病学调查、检测方法评价、核酸标准物质研制及免疫效果评估，可为 H7 亚型 AIV 科学防控与相关基础研究提供可靠技术支撑。

In order to establish a sensitive and specific droplet digital PCR (ddPCR) method for the detection of H7 subtype avian influenza virus (AIV), this study designed and synthesized primers and probe according to the nucleotide sequence of H7 subtype AIV HA genes in GenBank and GISAID, optimized the reaction conditions to establish a ddPCR method, and determined the specificity, sensitivity and repeatability. The results showed that the optimal primers and probe concentration were 900 nmol/L and 250 nmol/L, respectively; the optimal annealing temperature was 55 ℃; the optimal rising and cooling rate was 2.0 ℃/s; and the optimal time of reverse transcription was 20 min. The specificity test results showed that this method detected only H7 subtype AIV, but not H5 subtype AIV, H9 subtype AIV, Newcastle disease virus, infectious bronchitis virus and infectious bursal disease virus. Sensitivity test results showed that the detection limit of this method was 0.8 copies/μL, which was 10 times more sensitive than qPCR. The coefficients of variation were less than 3 % in both intra-batch and inter-batch reproducibility tests. The results of 60 clinical samples showed that the coincidence rate was 100 % compared with the virus isolation method. Thus, the ddPCR method established in this study has excellent specificity, high sensitivity and good reproducibility, allows for the early diagnosis, epidemiological investigation, diagnostic method evaluation, standard substance development and immune effect evaluation of H7 subtype AIV, and provides strong technical support for the prevention and control of H7 subtype AIV.