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. (August 2018)

• Record Type: Journal Article
• Authors: Lin, Feng; Liu, Li; Hao, Gui-jie; Sheng, Peng-chen; Cao, Zheng; Zhou, Yang; Lv, Peng; Xu, Ting; Shen, Jinyu; Chen, KePing
• Is Part Of: Molecular and cellular probes. Volume 40(2018)
• Page Start: 1
• Page End: 7
• Publication Date: 2018-08
• Subjects: Macrobrachium rosenbergii nodavirus -- Extra small virus -- Duplex reverse transcription -- Loop-mediated isothermal amplification -- Lateral flow dipstick; Molecular probes -- Diagnostic use -- Periodicals; Pathology, Cellular -- Technique -- Periodicals; Cell Biology -- Periodicals; Molecular Biology -- Periodicals; Sondes moléculaires -- Utilisation diagnostique -- Périodiques; Cytopathologie -- Technique -- Périodiques; 572
• DOI: 10.1016/j.mcp.2018.05.001
• Languages: English
• ISSNs: 0890-8508

Abstract

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. Highlights: 1 The RT-LAMP-LFD for detecting MrNV and XSV simultaneously was developed by amlification at 61 °C for 60 min. 2 The RT-LAMP-LFD was specific for MrNV and XSV. 3 Sensitivity of RT-LAMP-LFD was about 100 times higher in comparison to PCR. 4 The duplex RT-LAMP-LFD would be a simple, rapid and reliable method for detection of WTD. 5 We applied the RT-LAMP-LFD to detect MrNV and XSV in the larvae.