Rapid and visual detection of novel astroviruses causing fatal gout in goslings using one-step reverse transcription loop-mediated isothermal amplification
Jun Ji, Qinxi Chen, Chaoge Sui, Wen Hu, Zhengli Yu, Zhibin Zhang, Xinhao Mu, Xin Xu, Lunguang Yao, Yunchao Kan, Qingmei Xie
Abstract
To visually and rapidly detect a novel goose astrovirus (N-GoAstV) causing fatal gout in goslings, an isothermal detection method based on one-step reverse transcription loop-mediated isothermal amplification (one-step RT-LAMP) was established. The one-step RT-LAMP assay for N-GoAstV detection, using Bst 3.0 DNA polymerase with strong reverse transcription activity and primer sets targeting the opening reading frame 1b (ORF1b) of N-GoAstV, could be completed in 30 min using a water bath at 61°C; the detection results could be visually observed by adding a pH-sensitive dye containing phenol red and cresol red. The detection limit of the one-step RT-LAMP assay was 57.8 copies, which was similar to that of reverse transcription-quantitative polymerase chain reaction. The assay specifically detected N-GoAstV without any cross-reaction with other reference viruses, and this was further confirmed using enzyme digestion. These results indicated that the newly established RT-LAMP assay could accomplish reverse transcription, amplification, and visual result determination in one step, and the results obtained via this rapid and cost-effective method could be used to support disease control on farms in terms of N-GoAstV infection.