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. 2024 Oct 16:14:1461448.
doi: 10.3389/fcimb.2024.1461448. eCollection 2024.

Development and application of a quadruplex real-time PCR method for Torque teno sus virus 1, Porcine circovirus type 2, pseudorabies virus, and porcine parvovirus

Fushi Quan  1 , Yulu Geng  1 , Yang Wu  2 , Faming Jiang  3 , Xuemei Li  3 , Changqing Yu  3
Affiliations

Development and application of a quadruplex real-time PCR method for Torque teno sus virus 1, Porcine circovirus type 2, pseudorabies virus, and porcine parvovirus

Fushi Quan et al. Front Cell Infect Microbiol. .

Abstract

Introduction: In clinical diagnosis of porcine diseases, co-infection with multiple viruses often leads to similar clinical symptoms. Postweaning multisystemic wasting syndrome (PMWS) can be caused by infections with TTSuV or PCV2, while PCV2, PRV, and PPV can cause respiratory and reproductive disorders in pigs. The overlapping clinical and pathological features of these infections necessitate the development of a rapid and specific method for differentiating and detecting these four DNA viruses.

Methods: In this study, four pairs of primers and TaqMan probes were designed targeting the conserved sequence of TTSuV, the Rep gene of PCV2, the gE gene of PRV, and the VP2 gene of PPV. After optimizing reaction conditions, including annealing temperature, primer concentration, and probe concentration, a quadruplex real-time PCR method was developed.

Results: This method can specifically detect TTSuV1, PCV2, PRV, and PPV simultaneously, with no cross-reactivity with ASFV, CSFV, PRRSV, PEDV, PSV, and TGEV. The minimum detection limit for each virus was 10 copies/μl, and the inter-assay and intra-assay coefficients of variation ranged from 0.33% to 1.43%. Subsequently, 150 clinical samples were tested to evaluate the practical applicability of this method. The positive rates for TTSuV1, PCV2, PRV, and PPV were 8.6% (13/150), 10.67% (16/150), 14% (21/150), and 11.33% (17/150), respectively.

Discussion: The results indicate that the established quadruplex real-time PCR method can assist in the accurate and rapid diagnosis of TTSuV1, PCV2, PRV, and PPV in clinical settings, providing robust support for the prevention and control of these infections.

Keywords: PCV2; PMWS; PPV; PRV; TTSuV 1; qPCR; quadruplex.

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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
The standard curves of the quadruplex real-time quantitative PCR assay. (A–D) Standard curves of the standard plasmid pMD-TTSuV1 (A), pMD-PCV2 (B), pMD-PRV (C), and pMD-PPV (D) at final reaction concentrations ranging from 1.0 × 10^9 to 1.0 × 10^2 copies/µL.
Figure 2
Figure 2
Specificity analysis of the quadruplex real-time quantitative PCR assay. (A) TTSuV1; (B) PCV2; (C) PRV; (D) PPV; (E-K) ASFV,CSFV, PRRSV, PEDV, PSV and TGEV.
Figure 3
Figure 3
Sensitivity of the quadruplex real-time quantitative PCR assay. The amplification curves were generated by using the standard plasmid pMD-TTSuV1 (A), pMD-PCV2 (B), pMD-PRV (C), and pMD-PPV (D). 1–9: 1.0 × 10^9–1.0 × 10^1 copies/µL (final concentration).
Figure 4
Figure 4
Clinical sample testing.
See this image and copyright information in PMC

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