Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Sep 23:9:998874.
doi: 10.3389/fvets.2022.998874. eCollection 2022.

A duplex fluorescent quantitative PCR assay to distinguish the genotype I and II strains of African swine fever virus in Chinese epidemic strains

Shinuo Cao  1 , Huipeng Lu  1 , Zhi Wu  1 , Shanyuan Zhu  1
Affiliations

A duplex fluorescent quantitative PCR assay to distinguish the genotype I and II strains of African swine fever virus in Chinese epidemic strains

Shinuo Cao et al. Front Vet Sci. .

Abstract

African swine fever (ASF) is a highly contagious hemorrhagic disease that affects domestic and wild pigs. A recent study reported that both ASF virus (ASFV) genotypes I and II have invaded farm-raised pigs in China, causing chronic infection and morbidity. To develop a duplex fluorescent quantitative PCR method to distinguish the ASFV genotypes I and II in Chinese epidemic strains, the probes and primers were designed based on the B646L sequences of genotypes I and II listed in the GenBank database. After optimizing the system, a duplex fluorescent quantitative PCR method for simultaneous detection of ASFV genotypes I and II B646L genes was successfully established. This method had no cross-reaction with Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), or Porcine Parvovirus (PPV), indicating that it has strong specificity. The sensitivity results indicated that the minimum detection limit of ASFV genotypes I and II B646L was 10 copies/Rxn. The inter- and intra-group coefficients of variation were both <3%, indicating that the method was highly reproducible. Therefore, the established duplex fluorescent quantitative PCR assay is important for the differential detection and epidemiological investigation of ASFV.

Keywords: African swine fever virus genotype I; African swine fever virus genotype II; TaqMan probe; diagnosis; fluorescent quantitative PCR.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Nucleotide sequence alignment of B646L genes of African swine fever virus (ASFV) genotypes I and II with indicated position primers and TaqMan probes: ASFV-F1 primer, ASFV-S1 primer, FAM-ASFV-T1 probe and VIC-ASFV-T2 probe.
Figure 2
Figure 2
The standard curves of the dual fluorescent quantitative PCR for ASFV genotype I (A) and II (B) in Chinese epidemic strains. Based on the mean of Ct values against log10 of 10-fold serial dilutions of the synthesized plasmids pUC57-ASFV-B646L-T1 and pUC57-ASFV-B646L-T2.
Figure 3
Figure 3
Specificity of the dual fluorescent quantitative PCR. No detection signal was obtained for Porcine circovirus type 2 (PCV2), Pseudorabies virus (PRV), or Porcine Parvovirus (PPV).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wardley RC, de M Andrade C, Black DN, de Castro Portugal FL, Enjuanes L, Hess WR, et al. . African swine fever virus brief review. Arch Virol. (1983) 76:73–90. 10.1007/BF01311692 - DOI - PubMed
    1. Karger A, Pérez-Núñez D, Urquiza J, Hinojar P, Alonso C, Freitas FB, et al. . An update on African swine fever virology. Viruses. (2019) 11:864. 10.3390/v11090864 - DOI - PMC - PubMed
    1. Wang Y, Kang W, Yang W, Zhang J, Li D, Zheng H. Structure of African swine fever virus and associated molecular mechanisms underlying infection and immunosuppression: a review. Front Immunol. (2021) 12:715582. 10.3389/fimmu.2021.715582 - DOI - PMC - PubMed
    1. Galindo I, Alonso C. African swine fever virus: a review. Viruses. (2017) 9:103. 10.3390/v9050103 - DOI - PMC - PubMed
    1. Blome S, Franzke K, Beer M. African swine fever - a review of current knowledge. Virus Res. (2020) 287:198099. 10.1016/j.virusres.2020.198099 - DOI - PubMed

LinkOut - more resources