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  

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

作　　者：邓晏琼 李德氾 李晓红 宋春莲 舒相华 齐晓朋 陈培富 DENG Yanqiong;LI Defan;LI Xiaohong;SONG Chunlian;SHU Xianghua;QI Xiaopeng;CHEN Peifu(College of Veterinary Medicine Yunnan Agricultural University,Kunming 650201 China;Kunming Institute of Zoology Chinese Academy of Sciences,Kunming 650201 China)

机构地区：[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　　处：《生物学杂志》2023年第1期74-78,共5页Journal of Biology

基　　金：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)。

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。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.

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 

分 类 号：Q939.47[生物学—微生物学]