Development of a duplex SYBR GreenI based real-time PCR assay for detection of porcine epidemic diarrhea virus and porcine bocavirus3/4/5

Lan Lan Zheng, Jian Tao Cui, Hao Ying Han, Hua Lin Hou, Leyi Wang, Fang Liu, Hong Ying Chen

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Article number101544
JournalMolecular and Cellular Probes
Volume51
DOIs
StatePublished - Jun 2020

Keywords

  • Duplex real-time PCR
  • Porcine bocavirus
  • Porcine epidemic diarrhea virus

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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