蒋文明;李金平;尹馨;彭程;刘朔;李阳;于晓慧;王静静;刘华雷;
JIANG Wen-ming;LI Jin-ping;YIN Xin;PENG Cheng;LIU Shuo;LI Yang;YU Xiao-hui;WANG Jing-jing;LIU Hua-lei;China Animal Health and Epidemiology Center,Key Laboratory of Animal BiosafetyRisk Prevention and Control (South);

• 1:中国动物卫生与流行病学中心农业农村部动物生物安全风险预警及防控重点实验室(南方)

摘要(Abstract)：

为了建立灵敏、特异的H7亚型禽流感病毒(AIV)微滴式数字PCR(ddPCR)检测方法，本研究根据GenBank和GISAID中H7亚型AIV血凝素(HA)基因的核苷酸序列，设计合成H7亚型AIV的引物和探针，对反应条件进行优化，从而建立检测H7亚型AIV的ddPCR方法，并测定其特异性、敏感性和重复性。结果显示，最佳引物浓度和探针浓度分别为900 nmol/L和250 nmol/L,最佳退火温度为55℃,最佳升降温速率为2.0℃/s,最佳反转录时间为20 min。特异性试验结果显示，该方法仅对H7亚型AIV进行特异性检测，对H5亚型AIV、H9亚型AIV、新城疫病毒、鸡传染性支气管炎病毒、传染性法氏囊病毒均不能检出。敏感性试验结果显示，该方法对重组质粒标准品检测下限为0.8 copies/μL,比荧光定量PCR(qPCR)方法敏感性高10倍。组内和组间重复性试验结果显示，变异系数均小于3%。对60份临床样品检测显示，该方法与鸡胚分离方法的符合率为100%。结果表明，本研究建立的H7亚型AIV ddPCR检测方法具有较好的特异性、重复性和敏感性，可用于H7亚型AIV的早期诊断、流行病学调查、诊断方法评估、标准物质研制、免疫效果评估，为H7亚型AIV的科学防控和研究提供了有力的技术储备。
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 a strong data support for the control and prevention of H7 subtype AIV.

关键词(KeyWords)： 禽流感病毒;H7亚型;数字PCR
avian influenza virus;H7 subtype;digital PCR

Abstract:

Keywords:

基金项目(Foundation): “十四五”国家重点研发计划(2021YFD1800201);; 山东省重点研发计划(2022CXGC010606)

作者(Authors): 蒋文明;李金平;尹馨;彭程;刘朔;李阳;于晓慧;王静静;刘华雷;
JIANG Wen-ming;LI Jin-ping;YIN Xin;PENG Cheng;LIU Shuo;LI Yang;YU Xiao-hui;WANG Jing-jing;LIU Hua-lei;China Animal Health and Epidemiology Center,Key Laboratory of Animal BiosafetyRisk Prevention and Control (South);

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