Yu Hai-Qiong, Cai Xian-Quan, Lin Zhi-Xiong, Li Xiang-Li, Yue Qiao-Yun, Li Rong, Zhu Xing-Quan
Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou, Guangdong Province, 510630, PR China.
Technical Center, Zhongshan Entry-Exit Inspection and Quarantine Bureau, Zhongshan, Guangdong Province, 528403, PR China.
BMC Vet Res. 2015 Feb 28;11:46. doi: 10.1186/s12917-015-0364-2.
Porcine parvovirus (PPV) is the important causative agent for infectious infertility, which is a fairly tough virus that multiplies normally in the intestine of pigs without causing clinical signs in the world.
We developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the detection of PPV. Primers targeting the VP gene were highly specific, as evidenced by the negative amplification of closely related viruses, such as porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The performance of unlabeled real time PCR was compared to TaqMan real time PCR, and the detection limits of the two methods were nearly equal. Moreover, there was good correlation between Cp and diluted genomic DNA when tested with the two methods. The assay has the accuracy of 100% in reference to labeled real time PCR, when it was tested on 45 clinical samples.
The present study demonstrated that the established assay integrating real-time PCR and HRM is relatively cost-effective and more stable, which provides an alternative tool for rapid, simple, specific and sensitive detection of PPV.
猪细小病毒（PPV）是传染性不育症的重要病原体，它是一种相当顽固的病毒，在世界范围内通常在猪的肠道中繁殖而不引起临床症状。
我们开发了一种整合实时荧光定量PCR和高分辨率熔解曲线分析（HRM）的检测方法来检测PPV。靶向VP基因的引物具有高度特异性，如猪圆环病毒2型（PCV2）、猪繁殖与呼吸综合征病毒（PRRSV）、伪狂犬病病毒（PRV）、猪瘟病毒（CSFV）或日本脑炎病毒（JEV）等密切相关病毒的扩增均为阴性，证明了这一点。将未标记的实时荧光定量PCR的性能与TaqMan实时荧光定量PCR进行了比较，两种方法的检测限几乎相等。此外，用这两种方法检测时，Cp值与稀释的基因组DNA之间具有良好的相关性。当对45份临床样本进行检测时，该检测方法相对于标记实时荧光定量PCR的准确率为100%。
本研究表明，所建立的整合实时荧光定量PCR和HRM的检测方法相对具有成本效益且更稳定，为PPV的快速、简单、特异和灵敏检测提供了一种替代工具。
