A SYBR-Green Ⅰ RT-qPCR Assay for Detecting Enzootic Nasal Tumor Virus in Goats

  Objective  A rapid, sensitive detection method for early diagnosis and epidemiological survey on enzootic nasal tumor (ENT) in goats was established.

  Method  Bioinformatics methods were employed for sequence alignment of ENTV-2 with ENTV-1, ERVs, and JSRV in search for the conserved sequence of the virus. Primers for qPCR were designed to establish a SYBR-Green I RT-qPCR methodology for its detection. Reaction conditions were optimized, and a standard positive plasmid used to determine the specificity, sensitivity, and reproducibility of the newly developed assay.

  Result  A linear standard curve was found for the detection with a R²=0.992. The method specifically detected only ENTV-2, not the highly homologous ERVs, nor amplified ORFV, MO, or Mmc. It showed a detection sensitivity of up to 7.51×10² copies·μL⁻¹, which was 100 times greater than the conventional PCR can deliver, a repeatability with a coefficient variations (CV) of less than 1% on intra- and inter-batch tests, and a positive detection rate of 17.3% on 81 clinical samples.

  Conclusion  The newly established SYBR-Green I RT-qPCR was specific, sensitive, repeatable, and considered adequate for early and rapid detection of ENTV-2 in goats.