Key result
The multiplex qRT-PCR assay simultaneously and specifically detected ASFV, CSFV, and PRRSV with a detection limit of 1.78 copies/μL and showed >98% agreement with conventional mRT-PCR in 1,143 clinical samples.
Why the study?
Co-infections of ASFV, CSFV, and PRRSV make distinguishing clinical symptoms and pathological changes difficult, requiring a rapid and specific differential detection method.
The developed multiplex qRT-PCR assay provides a rapid, sensitive, and specific tool for the simultaneous detection and differentiation of ASFV, CSFV, and PRRSV in clinical samples.
May aid differential swine virus detection in co-infected herds; leaves open routine adoption pending multicenter validation.
BACKGROUND: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. OBJECTIVES: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. METHODS: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. RESULTS: copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. CONCLUSIONS: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.
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Chen et al. (2021) studied African swine fever virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus (n=1,143). Multiplex qRT-PCR assay vs. Conventional mRT-PCR was evaluated on Positive detection rate and agreement with conventional mRT-PCR. The multiplex qRT-PCR assay simultaneously and specifically detected ASFV, CSFV, and PRRSV with a detection limit of 1.78 copies/μL and showed >98% agreement with conventional mRT-PCR in 1,143 clinical samples.
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