A duplex fluorescent quantitative PCR assay to distinguish the genotype I, II and I/II recombinant strains of African swine fever virus in China

Abstract

African swine fever (ASF) is a severe, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) in both domestic pigs and wild boars. In China, ASFV has been present for over six years, with three genotypes of strains prevalent in field conditions: genotype I, genotype II, and genotype I/II recombinant strains. In order to differentiate among these three ASFV genotypes, a duplex fluorescent quantitative PCR method was established using specific probes and primers designed based on viral genes MGF_110-1L and O61R from ASFV strains reported in the GenBank database. Following optimization of reaction conditions, a duplex fluorescent quantitative PCR method was successfully developed. This method demonstrated no cross-reactivity with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine circovirus 2 (PCV2), porcine circovirus 3 (PCV3), highlighting its specificity. Sensitivity analysis revealed that the limits of detection (LODs) of this method were 2.95 × 10^-1 copies/μL for the MGF_110-1L gene and 2.95 × 10⁰ copies/μL for the O61R gene. The inter- and intra-group coefficients of variation were both <1%, indicating high reproducibility. In summary, the establishment of this duplex fluorescent quantitative PCR method not only addresses the identification of the ASFV recombinant strains but also allows for simultaneous identification of the three epidemic genotype strains.

Keywords: ASFV genotype I; ASFV genotype I/II recombinant strain; ASFV genotype II; duplex fluorescent quantitative PCR; genotype identification.

Figure 1

Location of primer and probe sequences in the genomes of different ASFV strains.

Figure 2

Sensitivity amplification curves and standard curves of the duplex fluorescent quantitative PCR. (A,B) The sensitivity amplification curves of MGF_110-1L gene with FAM channel (A) and O61R gene with VIC channel (B). Number 1–11: 2.95 × 10⁹–2.95 × 10⁻¹ copies/uL. (C) Standard curves of both MGF_110-1L gene and O61R gene.

Figure 3

Specific amplification curve of the duplex fluorescent quantitative PCR. No detection signal was obtained for PEDV, TGEV, PRRSV, CSFV, PRV, PCV2, PCV3 or NC.

Figure 4

Amplification curves of clinical samples (N = 96). (A) Five positive samples of ASFV genotype I strains (No. 1–5). (B) Six positive samples of ASFV genotype II strains (No. 1–6). (C) Six positive samples of ASFV genotype I and II recombinant strains (No. 1–12). Blue lines represent the MGF_110-1L gene, and yellow lines represent the O61R gene.