Journal of Biology ›› 2023, Vol. 40 ›› Issue (1): 74-.doi: 10.3969/j.issn.2095-1736.2023.01.074

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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

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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