Rapid and visual detection of the emerging novel duck reovirus by using a specific and sensitive reverse transcription recombinase polymerase amplification method

Abstract

Duck spleen necrosis disease (DSND) caused by Novel Duck Reovirus (NDRV), is an emerging infectious disease that causes severely threaten to duck industry. Currently, the popular conventional RT-PCR technique for detecting NDRV is time consuming. So, it is essential to develop a rapid and accurate molecular diagnosis techniques of the pathogen for the purpose to effective control of the disease. In our study, a simple, rapid and reliable detection method was developed by an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA). The RT-RPA primers were designed targeting the S3 gene of NDRV, and its specificity was verified by testing a series of other waterfowl pathogens. A total of 20 field and experimental samples from infected ducklings were tested by the RT-RPA and compared with the results of the conventional RT-PCR and the quantitative RT-PCR simultaneously. The RT-RPA method could detect as little as 4.14 × 10² copies/μl of the target gene in the sensitivity analysis, which was 10×higher sensitive than the conventional RT-PCR. The major advantage of the RT-RPA method is that it could be performed as an isothermal reaction at 37 ℃ and completed within 20 min. In addition, no cross-reactivity was detected with other waterfowl-origin viruses. Also, the amplified products could be visualized faster, without the gel electrophoresis, by adding the SYBR Green I and observing them under an ultraviolet light. The newly developed RT-RPA method offers a simple, rapid and accurate for rapid detection of NDRV, which especially useful in on-site facilities and resource-limited areas.

Keywords: Duck spleen necrosis disease; Isothermal gene amplification; Novel duck reovirus; RT-RPA; Rapid and visual detection.