Development of a TB green II-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine circovirus 2 and 3.

Autor: Zhao Y; Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, Henan Province, People's Republic of China., Han HY; Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, Henan Province, People's Republic of China., Fan L; Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, Henan Province, People's Republic of China., Tian RB; Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, Henan Province, People's Republic of China., Cui JT; Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, Henan Province, People's Republic of China., Li JY; Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, Henan Province, People's Republic of China., Chen HY; Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, Henan Province, People's Republic of China. Electronic address: chhy927@163.com., Yang MF; Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, Henan Province, People's Republic of China. Electronic address: yangmingfan123@163.com., Zheng LL; Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengdong New District Longzi Lake 15#, Zhengzhou 450046, Henan Province, People's Republic of China.
Jazyk: angličtina
Zdroj: Molecular and cellular probes [Mol Cell Probes] 2019 Jun; Vol. 45, pp. 31-36. Date of Electronic Publication: 2019 Apr 10.
DOI: 10.1016/j.mcp.2019.04.001
Abstrakt: Porcine circovirus 3 (PCV3), as a newly emerged circovirus, is widely distributed in pig populations worldwide. Co-infection of PCV2 and PCV3 has been reported frequently in clinical samples. In the present study, a TB Green II-based duplex real-time polymerase chain reaction (qPCR) was developed to rapidly and differentially detect PCV2 and PCV3. The assay specifically detected PCV2 and PCV3, with no fluorescence signals being detected for other non-targeted pig pathogens. The duplex qPCR showed a high degree of linearity (R 2  > 0.998), and its limits of detection were 10 and 78 copies/μL for PCV2 and PCV3, respectively. The duplex qPCR could detect and differentiate PCV2 (melting peaks at 85.5 °C) and PCV3 (melting peaks at 82.5 °C), and showed high repeatability and reproducibility, with intra- and inter-assay coefficients of variation of less than 2.0%. Fifty-six tissue samples from 18 pig farms were used to evaluate the duplex qPCR method. The results revealed infection rates of 66.07% (37/56) and 39.28% (22/56) for PCV2 and PCV3, respectively. The PCV2 + PCV3 co-infection rate was 39.28% (22/56). The developed method could be used as an efficient molecular biology tool for epidemiological investigations of PCV2 and PCV3.
(Copyright © 2019. Published by Elsevier Ltd.)
Databáze: MEDLINE
Externí odkaz: