Establishment and Application of a LAMP Method for Visual Detection of Norovirus GⅡ in Aquatic Products

Abstract:

[Objective] This study aims to develop a method for the rapid detection of norovirus GⅡ based on loop-mediated isothermal amplification (LAMP). [Method] Four primers were designed based on the conserved region of norovirus and the reaction system and conditions were optimized. [Result] The optimal reaction system was composed of 0.32 U/μL Bst DNA polymerase, 10 mmol/L MgSO₄, 1.2 mmol/L dNTP, 1.6 μmol/L BIP, 1.6 μmol/L FIP, 0.4 μmol/L F3, and 0.4 μmol/L B3. The optimal reaction conditions were reaction at 63 ℃ for 50 min and then at 85 ℃ for 5 min. The established LAMP method specifically recognized norovirus GⅡ, with the sensitivity of 1×10^-7 ng/μL, which was better than that of conventional PCR. In addition, three visualization dyes were screened. The results showed that SYBR Green I, phenol red, and hydroxynaphthol blue (HNB) demonstrated significant color contrast between positive and negative results, indicating that all the three dyes could be used for visualization. [Conclusion] A fast, specific, and simple method is successfully established for detecting norovirus GⅡ and applied to the detection of aquatic products.