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

• Published in: Frontiers in cellular and infection microbiology 2024-10, Vol.14, p.1461448
• Main Authors: Quan, Fushi, Geng, Yulu, Wu, Yang, Jiang, Faming, Li, Xuemei, Yu, Changqing
• Format: Article
• Language: English
• Subjects: Animals; Cellular and Infection Microbiology; Circovirus - genetics; Circovirus - isolation & purification; Coinfection - diagnosis; Coinfection - veterinary; Coinfection - virology; DNA Primers - genetics; DNA Virus Infections - diagnosis; DNA Virus Infections - veterinary; DNA Virus Infections - virology; DNA, Viral - genetics; DNA, Viral - isolation & purification; Herpesvirus 1, Suid - genetics; Herpesvirus 1, Suid - isolation & purification; Parvovirus, Porcine - genetics; Parvovirus, Porcine - isolation & purification; PCV2; PMWS; Porcine Postweaning Multisystemic Wasting Syndrome - diagnosis; Porcine Postweaning Multisystemic Wasting Syndrome - virology; PPV; PRV; quadruplex; Real-Time Polymerase Chain Reaction - methods; Sensitivity and Specificity; Swine; Swine Diseases - diagnosis; Swine Diseases - virology; Torque teno virus - genetics; Torque teno virus - isolation & purification; TTSuV 1
• ISSN: 2235-2988; 2235-2988
• DOI: 10.3389/fcimb.2024.1461448

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. 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. 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. 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.