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

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.

Development of a SYBR green I-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine epidemic diarrhea virus and porcine circovirus 3.

The development of a rapid, specific, and sensitive SYBR Green I-based duplex real-time quantitative PCR assay is described for the simultaneous detection of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 3 (PCV3). The assay specifically detected PEDV and PCV3, with no fluorescence detected for other non-targeted pig pathogens. The assay showed a good linear relationship, and the limits of detection for this assay were 34.6 copies/µL and 61.2 copies/µL for PEDV and PCV3, respectively. The assay exhibited high repeatability and reproducibility, with intra-assay and inter-assay variation coefficients less than 2.0%. A clinical evaluation using intestinal tissue and fecal samples from piglets suffering from diarrhea at different pig farms in China revealed that the singular infection rates of PEDV and PCV3 were 43.94% (29/66) and 16.67% (11/66), respectively, while the co-infection rate of PCV3 with PEDV was 27.27% (18/66). The results indicate this assay is a rapid and reliable diagnostic tool for PEDV and PCV3 monitoring and surveillance in the field, and provides technical support for the quantitative detection of clinical samples infected or co-infected with PEDV and PCV3.