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.

中文翻译：

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基于双向SYBRGreenⅠ的实时PCR检测试剂盒的开发，用于检测猪流行性腹泻病毒和猪博卡病毒3/4/5。

建立了基于SYBR Green的双重实时PCR检测试剂盒，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。设计了两对特异性引物，分别靶向PEDV的N基因序列和PBoV3 / 4/5的VP1基因序列。PEDV和PBoV3 / 4/5可以通过在一个样品中的不同熔化温度（Tm）来区分。PEDV的Tm值为83.5°C，PBoV3 / 4/5的Tm值为78.5°C，而其他猪病原体则没有特定的解链峰。PEDV和PBoV3 / 4/5的检测限均为10拷贝/μL。从腹泻的仔猪中收集总共六十三种肠组织样品，并通过实时PCR测定法和常规PCR测定法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的患病率分别为85.7％和46％，两种病毒的共感染率为28.6％。这些结果表明，这种双工实时荧光定量PCR方法是区分PEDV和PBoV3 / 4/5或它们共同感染的灵敏，特异且可重现的方法。