Department of Laboratory Animals, College of Animal Sciences, Jilin University, Changchun, Jilin, China.
State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Front Cell Infect Microbiol. 2024 Oct 16;14:1461448. doi: 10.3389/fcimb.2024.1461448. eCollection 2024.
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.
在猪病的临床诊断中，多种病毒的混合感染常常导致相似的临床症状。仔猪断奶后多系统衰竭综合征（PMWS）可由 TTSuV 或 PCV2 感染引起，而 PCV2、PRV 和 PPV 可引起猪的呼吸和生殖障碍。这些感染的临床和病理特征重叠，需要开发一种快速、特异的方法来区分和检测这四种 DNA 病毒。
本研究设计了针对 TTSuV 保守序列、PCV2 Rep 基因、PRV gE 基因和 PPV VP2 基因的四对引物和 TaqMan 探针。在优化反应条件，包括退火温度、引物浓度和探针浓度后，建立了四重实时 PCR 方法。
该方法可特异性同时检测 TTSuV1、PCV2、PRV 和 PPV，与 ASFV、CSFV、PRRSV、PEDV、PSV 和 TGEV 无交叉反应。每种病毒的最小检测限为 10 拷贝/μl，批内和批间变异系数范围为 0.33%至 1.43%。随后，用 150 份临床样本评估该方法的实际适用性。TTSuV1、PCV2、PRV 和 PPV 的阳性率分别为 8.6%（13/150）、10.67%（16/150）、14%（21/150）和 11.33%（17/150）。
结果表明，建立的四重实时 PCR 方法可用于临床准确、快速诊断 TTSuV1、PCV2、PRV 和 PPV，为这些感染的防控提供有力支持。
