Development of a SYBR green I-based duplex real-time PCR assay for detection of pseudorabies virus and porcine circovirus 3.

Autor: 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., Jin 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., Xu T; 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., 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., Wang ZY; Key Laboratory of 'Runliang' Antiviral Medicines Research and Development, Institute of Drug Discovery & Development, Zhengzhou University, Zhengzhou, 450001, Henan Province, People's Republic of China. Electronic address: zhenyawang@zzu.edu.cn., 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.
Jazyk: angličtina
Zdroj: Molecular and cellular probes [Mol Cell Probes] 2020 Oct; Vol. 53, pp. 101593. Date of Electronic Publication: 2020 May 05.
DOI: 10.1016/j.mcp.2020.101593
Abstrakt: In the present study, a specific and reliable duplex SYBR green I-based quantitative real-time polymerase chain reaction assay was established to detect pseudorabies virus (PRV) and porcine circovirus 3 (PCV3) simultaneously. Viral genomes of PRV and PCV3 in one specimen were identified by their different melting temperatures with melting peaks at 87 °C and 81 °C for PRV and PCV3 respectively, whilst other non-targeted swine pathogens exhibited no fluorescent signals. The assay displayed a high degree of linearity (R 2  > 0.997), and the limits of detection were 37.8 copies/μL, 30.6 copies/μL and 60 copies/μL for PRV, PCV3 and the mixture of two recombinant plasmids, respectively. It had good repeatability and reproducibility, and the coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. In this research, the duplex assay was further evaluated using 117 clinical tissue specimens from diseased pigs in the field. The results revealed the infection rates of PRV and PCV3 were 23.08% (27/117) and 55.56% (65/117) respectively, and PRV and PCV3 co-infection rate was 14.53% (17/117). The assay could be utilized as a diagnostic tool with specificity, sensitivity, and reliability for molecular epidemiological surveillance of PRV and PCV3.
(Copyright © 2020 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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