Key result
New multiplex qRT-PCR assay simultaneously detects ASFV, CSFV, and APPV with a ~25 copies/μL limit.
Why the study?
Co-infections of ASFV, CSFV, and APPV occur in pig herds, making accurate and differential field detection of these pathogens necessary.
A newly developed one-step multiplex qRT-PCR assay provides a highly specific and sensitive tool for the simultaneous detection of ASFV, CSFV, and APPV in swine.
Facilitates rapid differential diagnosis of co-infections in swine herds; leaves open large-scale field validation before routine adoption.
Background: African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused great economic losses to the swine industry in China. Since there exist co-infections of ASFV, CSFV and APPV in certain pig herds, it is necessary to accurately and differentially detect these pathogens in the fields. In this study, a one-step multiplex real-time quantitative reverse transcription-polymerase chain reaction (multiplex qRT-PCR) was developed for simultaneous and differential detection of ASFV, CSFV and APPV.Results: The developed one-step multiplex qRT-PCR was able to specifically detect ASFV, CSFV and APPV, but could not amplify other viruses, including porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), porcine parvovirus (PPV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PRoV), porcine deltacoronavirus (PDCoV), border disease virus (BDV), bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2 and so on. The detection limit of the assay was 2.52×101 copies/μL for ASFV, CSFV and APPV. Repeatability test using standard plasmids showed that the coefficients of variation of the intra- and inter-assay were less than 2 %. The detection of 509 clinical samples collected in Guangxi Province, Southern China from October 2018 to December 2020 showed that the positive rates of ASFV, CSFV and APPV were 45.58 %, 12.57 % and 3.54 %, respectively, while the co-infection rates of ASFV and CSFV, ASFV and APPV, CSFV and APPV were 4.91 %, 1.38 %, 0.98 %, respectively. Phylogenetic analysis based on the nucleotide sequences of partial ASFV p72 gene showed that all ASFV strains from Guangxi Province, Southern China belonged to genotype I and II.Conclusion: A one-step multiplex qRT-PCR with high specificity, sensitivity, accuracy and repeatability was successfully developed for simultaneous and differential detection of ASFV, CSFV and APPV.
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Liu et al. (2021) studied African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), and Atypical Porcine Pestivirus (APPV) infection in pigs (n=509). One-step multiplex qRT-PCR assay was evaluated on Detection limit and positive rates of ASFV, CSFV, and APPV. A newly developed one-step multiplex qRT-PCR assay demonstrated high specificity and a detection limit of 2.52×10^1 copies/μL for ASFV, CSFV, and APPV, identifying positive rates of 45.58%, 12.57%, and 3.54% respectively in 509 clinical samples.
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