Development a multiplex RT-PCR assay for simultaneous detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus

Huixin Liu; Kaichuang Shi; Wenchao Sun; Jing Zhao; Yanwen Yin; Hongbin Si; Sujie Qu; Wenjun Lu

doi:10.1016/j.jviromet.2020.114006

Development a multiplex RT-PCR assay for simultaneous detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus

J Virol Methods. 2021 Jan:287:114006. doi: 10.1016/j.jviromet.2020.114006. Epub 2020 Oct 27.

Authors

Huixin Liu¹, Kaichuang Shi², Wenchao Sun³, Jing Zhao¹, Yanwen Yin⁴, Hongbin Si¹, Sujie Qu⁴, Wenjun Lu⁴

Affiliations

¹ College of Animal Science and Technology, Guangxi University, Nanning, 530005, China.
² Guangxi Center for Animal Disease Control and Prevention, Nanning, 530001, China. Electronic address: shikaichuang@126.com.
³ Institute of Virology, Wenzhou University, Wenzhou, 325035, China.
⁴ Guangxi Center for Animal Disease Control and Prevention, Nanning, 530001, China.

PMID: 33127443
DOI: 10.1016/j.jviromet.2020.114006

Abstract

African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused considerable financial losses to the pig industry worldwide, and it is critical to achieve early and accurate diagnosis of these viruses to control the diseases induced by them. In this study, three pairs of specific primers were designed based on the highly conserved genome regions of these viruses, and a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay for ASFV, CSFV and APPV was established after various reaction conditions were optimized. The mRT-PCR assay consisted of two steps, that is, reverse transcription (RT) and mPCR. The assay was highly specific, sensitive, and reproducible for ASFV, CSFV and APPV without cross-reaction with other swine pathogens. The sensitivity of this assay, which used purified plasmid constructs containing specific viral target fragments as templates, was 6.34 × 10² copies/μL for ASFV and 6.34 × 10¹ copies/μL for both CSFV and APPV. A total of 384 clinical samples from piglets suspected to be infected in Guangxi Province, Southern China, during 2018-2019 were analyzed by the established mRT-PCR method. The results showed that the positive rates of ASFV, CSFV and APPV were 43.75 %, 13.28 % and 4.17 %, respectively, and the coinfection rates of ASFV/CSFV, ASFV/APPV and CSFV/APPV were 5.47 %, 1.83 % and 1.30 %, respectively. To understand the epidemiological characteristics of APPV, the newly discovered virus, in Guangxi Province, the clinical samples from APPV-positive animals were selected randomly for amplification and sequencing, and the complete genomic sequences of four APPV strains were obtained. Phylogenetic analysis demonstrated that APPV strains from Guangxi Province had a high degree of genetic diversity. This study provides an important tool for rapid detection and accurate diagnosis of ASFV, CSFV and APPV.

Keywords: African swine fever virus (ASFV); Atypical porcine pestivirus (APPV); Classical swine fever virus (CSFV); Multiplex RT-PCR (mRT-PCR).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

African Swine Fever Virus* / genetics
Animals
China
Classical Swine Fever Virus* / genetics
Classical Swine Fever* / diagnosis
Pestivirus* / genetics
Phylogeny
Reverse Transcriptase Polymerase Chain Reaction
Reverse Transcription
Sensitivity and Specificity
Swine
Swine Diseases* / diagnosis