A Rapid Real Time Fluorescence Recombinase-aided Amplification Method for Detecting Porcine Pseudorabies Virus

  Objective  A rapid method applying the recombinase-aided amplification (RAA) method for detecting porcine pseudorabies virus (PRV) was developed.

  Methods   Based on the sequence of gE gene in PRV, specific primers and probes were designed. Amplification conditions were optimized, and assay specificity, sensitivity, and reproducibility scrutinized by a verification trial on clinical samples.

  Results   The newly developed assay successfully amplified the PRV nucleic acids in merely 23m under the constant temperature of 43 ℃ with a detection limit of 111 copies·μL⁻¹. There were no cross reactions with viruses that produced reproductive and respiratory syndromes, epidemic diarrhea, rotavirus, transmissible gastroenteritis, circovirus 2, or circovirus 3 on pigs. The coefficients of variation within a group and between groups on the repeatability test were less than 5%. And, on 40 clinical samples, the positive detection on PRV of the assay was 15% (6/40), which was comparable to that of the conventional PCR.

  Conclusion   A simple, rapid, efficient, and accurate method of fluorescence RAA detection on PRV was established for laboratory testing and epidemiological investigation of the disease.