Establishment and preliminary application of fluorescent RT-RAA diagnostic method for porcine epidemic diarrhea virus

  Objective  To establish a quick, simple, sensitive detection of porcine epidemic diarrhea virus (PEDV) the RT- RAA detection method, to improve the efficiency of porcine epidemic diarrhea virus clinical detection.

  Method  Primers and probes were designed for the conserved region of PEDVS gene fragment, and a standard plasmid PEDV-S was constructed. Through specificity, sensitivity, repeatability and condition optimization, a recombinant enzyme-mediated chain replacement nucleic acid amplification fluorescence assay (RT-RAA) for detection of PEDV was established.

  Result  Under the condition of constant temperature at 42 ℃ for 20 min,The established assay is effective for the detection of porcine transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV) and other porcine viruses were negative, porcine epidemic diarrhea virus (PEDV) was positive.The minimum detection limit was 4.43× 10² copies·μL⁻¹ standard plasmid.The reproducibility results showed that there was little difference between the standard plasmids with the same concentration.The positive rate of 40 swine virus samples was 7.5% (3/40) by RT-RAA method, which was the same as that of real-time fluorescence quantitative PCR.

  Conclusion  The RT-RAA detection method for PEDV established in this study is suitable for the rapid diagnosis of PEDV due to its rapid and simple detection, short time-consuming, high specificity, strong sensitivity and good reproducibility.