H3亚型禽流感病毒荧光定量RT-PCR检测方法的建立与应用

doi:10.11843/j.issn.0366-6964.2024.03.026

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (3): 1137-1146.doi: 10.11843/j.issn.0366-6964.2024.03.026

H3亚型禽流感病毒荧光定量RT-PCR检测方法的建立与应用

毛秋艳¹, 周淑宁², 刘朔³, 彭程³, 尹馨³, 张雅馨⁴, 周婉婷¹, 李金平³, 侯广宇³, 蒋文明^3*, 宋厚辉^1*, 刘华雷^1,3*

1. 浙江农林大学动物科技学院·动物医学院, 杭州 311300;
2. 青岛农业大学动物医学院, 青岛 266109;
3. 中国动物卫生与流行病学中心, 青岛 266032;
4. 内蒙古农业大学兽医学院, 呼和浩特 010018

收稿日期:2023-06-15 出版日期:2024-03-23 发布日期:2024-03-27
通讯作者: 蒋文明,主要从事畜禽重要疫病防控,E-mail:jiangwenming@cahec.cn;宋厚辉,主要从事动物疫病诊断研究,E-mail:songhh@zafu.edu.cn;刘华雷,主要从事重大动物疫病防控技术研究,E-mail:liuhualei@cahec.cn
作者简介:毛秋艳(1999-),女,浙江丽水人,硕士生,主要从事动物传染病的诊断与防治研究,E-mail:2021120022017@stu.zafu.edu.cn
基金资助:
“十四五”国家重点研发计划项目（2021YFD1800201）；山东省重点研发计划项目（2022CXGC010606）

Establishment and Application of Fluorescent Quantitative RT-PCR for Detection of H3 Subtype Avian Influenza Virus

MAO Qiuyan¹, ZHOU Shuning², LIU Shuo³, PENG Cheng³, YIN Xin³, ZHANG Yaxin⁴, ZHOU Wanting¹, LI Jinping³, HOU Guangyu³, JIANG Wenming^3*, SONG Houhui^1*, LIU Hualei^1,3*

1. College of Animal Science and Technology · College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China;
2. College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China;
3. China Animal Health and Epidemiology Center, Qingdao 266032, China;
4. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China

Received:2023-06-15 Online:2024-03-23 Published:2024-03-27

摘要/Abstract

摘要： H3亚型禽流感病毒（avian influenza virus，AIV）感染宿主广泛，具备跨物种传播的能力，不但造成了家禽的发病，还导致了多起人感染病例，具有重要的公共卫生意义。因此，有必要建立一种快速灵敏的H3亚型AIV的检测方法。本研究参考了GenBank近年来公开的H3亚型AIV HA基因序列，在保守区域设计一对特异性引物和TaqMan探针。通过对反应条件的优化，分别以cRNA阳性标准品和病毒核酸标准品为模板绘制标准曲线，建立了针对H3亚型AIV的荧光定量RT-PCR检测方法，并对其特异性、灵敏性、重复性进行评估。进一步使用实验室攻毒鸡组织样品和临床拭子样品对该方法进行了验证。结果表明，该方法特异性强，与其他亚型AIV和常见禽类病原体均无交叉反应；检测下限分别为1.0×10² copies·μL^-1和10² EID₅₀·0.1 mL^-1，灵敏度与常规RT-PCR相比提高了10倍；组内和组间变异系数均小于1.5%，重复性较好。动物攻毒临床试验样品和临床拭子样品检测结果显示，该检测方法敏感性高于普通RT-PCR方法，与病毒分离鉴定结果一致，符合率为100%，可用于临床检测。综上，本研究建立的H3亚型AIV荧光定量RT-PCR检测方法具备特异、快速、灵敏等特点，为H3亚型AIV的快速诊断和监测及防控提供一定的技术支持。

关键词: 禽流感病毒, H3亚型, 荧光定量, 监测

Abstract: The avian influenza virus (AIV) of subtype H3 infects a wide range of hosts and is capable of cross-species transmission. In addition to causing disease in poultry, it also causes many cases of human infection, which is of great public health importance. Therefore, it is necessary to establish a rapid and sensitive method for the detection of H3 subtype AIV. In this study, the H3 subtype AIV HA gene sequence published by GenBank in recent years was used to design a pair of specific primers and TaqMan probes in the conserved region. By optimising the reaction conditions and drawing the standard curve using cRNA standard and viral nucleic acid standard as templates, a fluorescence quantitative RT-PCR detection method for H3 subtype AIV was established and its specificity, sensitivity and repeatability were evaluated. The method was further validated using laboratory-challenged chicken tissue samples and clinical swab samples. The results showed that the method was highly specific and did not cross-react with other subtypes of AIV and common avian pathogens. The lower detection limits of the method were 1.0×10² copies·μL^-1 and 10² EID₅₀·0.1 mL^-1, respectively, and the sensitivity was 10-fold higher than that of conventional RT-PCR. The coefficient of variation within and between groups was less than 1.5% and the repeatability was good. The results of the animal challenge clinical test and clinical swab samples showed that the sensitivity of the method was higher than that of the conventional RT-PCR method, and the concordance rate was 100%, which could be used for clinical detection. In conclusion, the fluorescent quantitative RT-PCR detection method for H3 subtype AIV established in this study has the characteristics of specific, rapid and sensitive, which provides certain technical support for the rapid diagnosis, monitoring, prevention and control of H3 subtype AIV.

Key words: avian influenza virus, H3 subtype, fluorescence quantification, surveillance

中图分类号:

S852.659.5

毛秋艳, 周淑宁, 刘朔, 彭程, 尹馨, 张雅馨, 周婉婷, 李金平, 侯广宇, 蒋文明, 宋厚辉, 刘华雷. H3亚型禽流感病毒荧光定量RT-PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2024, 55(3): 1137-1146.

MAO Qiuyan, ZHOU Shuning, LIU Shuo, PENG Cheng, YIN Xin, ZHANG Yaxin, ZHOU Wanting, LI Jinping, HOU Guangyu, JIANG Wenming, SONG Houhui, LIU Hualei. Establishment and Application of Fluorescent Quantitative RT-PCR for Detection of H3 Subtype Avian Influenza Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1137-1146.

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H3亚型禽流感病毒荧光定量RT-PCR检测方法的建立与应用

Establishment and Application of Fluorescent Quantitative RT-PCR for Detection of H3 Subtype Avian Influenza Virus

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