Rapid Detection of Norovirus in Shellfish by Dual Fluorescence Reverse Transcription-Enzymatic Recombinase Amplification

Abstract

Objective: To establish a dual fluorescence reverse transcription-enzymatic recombinase amplification (RT-ERA) method for the rapid detection of GI and GII noroviruses (NoV) in shellfish using MS2 as the model process control virus. Methods: The target sequences of GI and GII NoV were individually cloned into vectors with a T7 promoter. Then, high-purity RNA was obtained by in vitro transcription. Dual fluorescence RT-ERA was performed using the primers and probes of MS2 phage designed in this study and the primers and probes of GI and GII NoV selected previously. The reaction procedure and system were optimized. The sensitivity of the method was analyzed. Finally, this method was used to detect real samples, and its accuracy and viability were determined by comparing the results with those obtained by the reference method specified in the national standard GB 4789.42-2016. Results: The optimal volumes of primers and probes for the dual fluorescence RT-ERA assay were 4.1 and 1.8 μL, respectively. The method took only about 10 min to perform, and could detect as low as 102 copies of NoV nucleic acid. When the established method was applied to detect 29 real samples, the results were consistent with those from GB 4789.42-2016. Conclusion: The dual fluorescence RT-ERA assay can simultaneously detect MS2, GI and GII NoV with high sensitivity and accuracy, which will lay a foundation for rapid on-site detection of NoV in the future.

Keywords

• norovirus; reverse transcription-enzymatic recombinase amplification; dual fluorescence; rapid detection; shellfish