Cao Lihua, Lv Wenke, Li Anqi, Yang Lulu, Zhou Feng, Wen Feng, Yuan Sheng, Huang Shujian, Li Zhili, Guo Jinyue
College of Life Science and Engineering, Foshan University, Foshan, Guangdong, 528231, China.
BMC Vet Res. 2025 Jan 7;21(1):10. doi: 10.1186/s12917-024-04440-x.
Pseudorabies virus (PRV), porcine parvovirus (PPV) and porcine circovirus 3 (PCV3) are common in swine farms in China. Single infection or co-infection with PRV, PPV and/or PCV3 was difficult to distinguish between their clinical symptoms and pathological changes. Therefore, a quick and accurate detection method is needed for epidemiological surveillance, disease management, import and export control.
In the present study, we established a multiplex real-time PCR assay based on SYBR Green I for the simultaneous detection of PRV, PPV and PCV3 genomes.
PRV, PPV and PCV3 were distinguished in the same sample by their different melting temperatures (Tm), with melting peaks at 90 °C for PRV, 84 °C for PPV and 80 °C for PCV3, respectively, and other non-targeted swine pathogens did not exhibit specific melting peaks. The assay showed a high degree of linearity (R≧0.995), and the detection limits were 4.76 copies/μL for PRV, 3.67 copies/μL for PPV, 3.07 copies/μL for PCV3 and 1.87 × 10 copies/μL for the three mixed plasmids, respectively. In this research, 81 clinical samples from pig farms in nine different regions of Guangdong Province were used to evaluate this new method. The detection rate of the multiplex real-time PCR assay was higher than that of the conventional PCR assay.
This multiplex real-time PCR assay could be used as a diagnostic tool that is rapid, sensitive and reliable for the detection of co-infection of PRV, PPV and PCV3 as well as for molecular epidemiological surveillance.
伪狂犬病病毒（PRV）、猪细小病毒（PPV）和猪圆环病毒3型（PCV3）在中国猪场中普遍存在。PRV、PPV和/或PCV3的单一感染或共同感染，在临床症状和病理变化上难以区分。因此，需要一种快速准确的检测方法用于流行病学监测、疾病管理、进出口控制。
在本研究中，我们建立了一种基于SYBR Green I的多重实时荧光定量PCR检测方法，用于同时检测PRV、PPV和PCV3基因组。
PRV、PPV和PCV3在同一样本中通过不同的熔解温度（Tm）得以区分，PRV的熔解峰在90℃，PPV在84℃，PCV3在80℃，其他非目标猪病原体未出现特异性熔解峰。该检测方法具有高度线性（R≧0.995），PRV的检测限为4.76拷贝/μL，PPV为3.67拷贝/μL，PCV3为3.07拷贝/μL，三种混合质粒为1.87×10拷贝/μL。本研究中，使用来自广东省九个不同地区猪场的81份临床样本对该新方法进行评估。多重实时荧光定量PCR检测方法的检出率高于传统PCR检测方法。
这种多重实时荧光定量PCR检测方法可作为一种诊断工具，用于快速、灵敏且可靠地检测PRV、PPV和PCV3的共同感染以及进行分子流行病学监测。
