College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, Henan Province, People's Republic of China.
College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, Henan Province, People's Republic of China; College of Animal Science and Technology, Guangxi University, Nanning, 530004, Guangxi Province, People's Republic of China.
Mol Cell Probes. 2020 Jun;51:101544. doi: 10.1016/j.mcp.2020.101544. Epub 2020 Feb 25.
The duplex real-time PCR assay based on SYBR Green І was developed for detection of porcine epidemic diarrhea virus (PEDV) and porcine bocavirus (PBoV) 3/4/5 genotypes simultaneously. Two pairs of specific primers were designed targeting the N gene sequence of PEDV and VP1 gene sequence of PBoV3/4/5. PEDV and PBoV3/4/5 could be distinguished by their different melting temperatures (Tm) in one sample. The Tm value of PEDV was 83.5 °C, and the Tm value of PBoV3/4/5 was 78.5 °C, while other swine pathogens showed no specific melting peaks. The detection limits of this assay were 10 copies/μL for both PEDV and PBoV3/4/5. A total of sixty-three intestinal tissue samples were collected from piglets suffering from diarrhea, and the viral nucleic acids detected and identified by the real-time PCR assay and conventional PCR assay. The duplex real-time PCR detection results showed that the prevalence of PEDV and PBoV3/4/5 was 85.7% and 46%, respectively, and the co-infection rate of the two viruses was 28.6%. These results indicated that this duplex real-time PCR assay was a sensitive, specific and reproducible method for differentiating PEDV and PBoV3/4/5 or their co-infection.
基于 SYBR Green І 的双重实时 PCR 检测方法被开发用于同时检测猪流行性腹泻病毒（PEDV）和猪博卡病毒（PBoV）3/4/5 基因型。针对 PEDV 的 N 基因序列和 PBoV3/4/5 的 VP1 基因序列设计了两对特异性引物。PEDV 和 PBoV3/4/5 可以通过它们在一个样品中的不同熔解温度（Tm）来区分。PEDV 的 Tm 值为 83.5°C，而 PBoV3/4/5 的 Tm 值为 78.5°C，而其他猪病原体则没有显示出特异性的熔解峰。该检测方法的检测限对于 PEDV 和 PBoV3/4/5 均为 10 拷贝/μL。从患有腹泻的仔猪中收集了 63 个肠组织样本，并用实时 PCR 检测和常规 PCR 检测来检测和鉴定病毒核酸。双重实时 PCR 检测结果表明，PEDV 和 PBoV3/4/5 的流行率分别为 85.7%和 46%，两种病毒的合并感染率为 28.6%。这些结果表明，该双重实时 PCR 检测方法是一种敏感、特异和可重复的方法，可用于区分 PEDV 和 PBoV3/4/5 或它们的合并感染。
