A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification

Zhang, Fanfan; Ye, Yu; Song, Deping; Guo, Nannan; Peng, Qi; Li, Anqi; Zhou, Xingrong; Chen, Yanjun; Zhang, Min; Huang, Dongyan; Tang, Yuxin

To identify PDCoV in diarrheal samples of piglets, a N-gene-based conventional RT-PCR assay previously established in our lab was employed [10] . The first-strand cDNA was synthesized with reverse primers PDCoV-NR, followed by PCR with primer pairs of PDCoV-NF and PDCoV-NR under the following conditions: denaturation at 94 °C for 5 min, 38 cycles of 94 °C 30 s, 54 °C 30 s, 72 °C 40 s, and consequently with a final extension at 72 °C for 10 min. Expected PCR products of 329 bp in size were purified, cloned and sequenced. The nested RT-PCR method for detection of PDCoV was performed as reported previously [6] . Finally, the amplicons were subjected to electrophoresis on 2% agarose gel, and the target bands were visualized under UV light by staining with ethidium bromide.