生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (1): 74-.doi: 10.3969/j.issn.2095-1736.2023.01.074

• 研究报告 • 上一篇    下一篇

β冠状病毒通用RT-PCR检测方法建立及昆明地区动物带毒状况调查

邓晏琼1， 李德氾1， 李晓红2， 宋春莲1， 舒相华1， 齐晓朋2， 陈培富1   

  1. 1. 云南农业大学 动物医学院， 昆明 650201； 2. 中国科学院昆明动物研究所， 昆明 650201
  • 出版日期:2023-02-18 发布日期:2023-02-21
  • 通讯作者: 陈培富，博士，教授，硕士生导师，从事动物分子微生物学与免疫学研究，E-mail: cltwins2003@163.com
  • 作者简介:邓晏琼，硕士研究生，研究方向为预防兽医学，E-mail: 1036785302@qq.com；邓晏琼和李德氾为共同第一作者
  • 基金资助:
    昆明市科技计划项目“新型冠状病毒感染肺炎科技防治”专项（2020-1-H-002）； 云南农业大学兽医公共卫生省创新团队建设项目（202105AE160014）

Development of a universal RT-PCR method for detection of β coronaviruses and investigation of the virus carriage in animals in Kunming area

DENG Yanqiong1, LI Defan1, LI Xiaohong2, SONG Chunlian1, SHU Xianghua1,QI Xiaopeng2, CHEN Peifu1   

  1. 1. College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China;
    2. Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650201, China
  • Online:2023-02-18 Published:2023-02-21

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摘要： 为调查昆明地区猪、牛及鼠β冠状病毒携带状况及充当重症急性呼吸综合征冠状病毒（SARS-CoV）-2中间宿主的可能性，建立基于β冠状病毒属RNA依赖性RNA聚合酶（RdRp）的特异性逆转录-聚合酶链式反应（RT-PCR）检测方法，并用于2020—2021年昆明9个县区具有呼吸道或消化道症状的猪（186份）、牛（164份）及随机捕获的鼠（140份）的β冠状病毒核酸检测；对RdRp阳性样品进一步进行SARS-CoV-1和SARS-CoV-2刺突蛋白（S）基因特异性扩增。建立的RT-PCR方法能特异扩增β冠状病毒属各病毒种，对SARS-CoV-2核酸检测的灵敏度为310个质粒拷贝；应用该法检测的490份样品中，猪、牛、鼠的β冠状病毒检出率分别为5.91%、9.76%和8.57%，但RdRp阳性样品未检出SARS-CoVs的S基因同源片段。成功建立可特异检测β冠状病毒属的通用RT-PCR方法，应用该法联合S基因扩增调查昆明地区猪、牛及鼠，未发现其携带与SARS-CoVs的S基因有联系的β冠状病毒。

关键词: 哺乳动物, β冠状病毒, RNA依赖性RNA聚合酶, 逆转录-聚合酶链式反应

Abstract: For investigation of the status of β coronaviruses carriage by swine, cattle and murine in Kunming area and the possibility of these animals acting as intermediate host for severe acute respiratory syndrome coronavirus (SARS-CoV)-2, a reverse transcription-polymerase chain reaction (RT-PCR) detection method based on RNA-dependent RNA polymerase (RdRp) of the β-coronavirus genus was built and then used to examine β-coronavirus nucleic acid in pigs (n=186) and cattle (n=164) that displayed respiratory or digestive symptoms and randomly captured mice (n=140) from nine counties in Kunming area in 2020 to 2021. RdRp-positive samples were further subjected to amplification with the primers specific for the spike protein (S) genes of SARS-CoV-1 and SARS-CoV-2. The developed RT-PCR method could be employed for specific amplification of all species within the β-coronavirus genus but not non-β-coronaviruses as well as other viruses and reached a minimum detection limit of 310 copies of plasmid bearing the RdRp gene of the SARS-CoV-2. Among the 490 samples analyzed using this method, there was a detection rate of 5.91%, 9.76% and 8.57% for β-coronaviruses in pigs, cattle and mice, respectively. However, no nucleic acid fragment homologous to the S genes of the SARS-CoVs was detected when these RdRp-positive samples were further amplified. The results showed that a universal RT-PCR method suitable for specific detection of the β-coronavirus genus had been successfully established in this study and that no coronaviruses related to the S genes of the SARS-CoVs were detected when this method was used in combination with amplification of the S genes to pigs, cattle and mice in Kunming area.

Key words: mammals, β coronaviruses, RdRp, RT-PCR

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