The development and application of a duplex reverse transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method for Macrobrachium rosenbergii nodavirus and extra small virus isolated in China

Abstract

White tail disease (WTD), a major disease prevailing in the larval stage of Macrobrachium rosenbergii, caused by Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), led to the economic loss of shrimp industry in China. In order to establish a convenient, sensitive and selective molecular diagnostic method to detect MrNV and XSV for the Chinese shrimp (MrNV/XSV-chin), a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay combined with a lateral flow dipstick (LFD) method were developed. A set of four specific primers and a labeled probe were designed according to the six conserved gene sequence regions encoding for the MrNV capsid protein CP43 and the XSV capsid protein CP17. The detection of MrNV and XSV simultaneously by RT-LAMP was performed at 61 °C in a single reaction for 60 min followed by hybridization with an FITC-labeled probe for 5 min and visualized by LFD. The RT-LAMP-LFD assay had a sensitivity of approximately 100-fold higher than conventional PCR. In addition, the assay could detect MrNV/XSV-chin from limited amount of RNA extracts as low as 1.0 pg extracted from Macrobrachium rosenbergii. This assay was simple to use, required little instrumentation, and exhibited excellent specificity for the MrNV/XSV-chin compared with other shrimp viruses. In conclusion, a convenient, sensitive and selective practical molecular diagnostic method was developed with the potential for diagnosis and prevention of WTD.