[Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Assay for the Detection of Genogroup Ⅱ...

To establish a rapid testing method for genogroup Ⅱ (GⅡ) norovirus based on reverse transcription loop-mediated isothermal amplification (RT-LAMP).

The conserved region of norovirus was screened using the genomic data from the National Center for Biotechnology Information (NCBI) database, and specific primers were designed using the online software Primer Explorer V5. The testing system was established through optimization of experimental conditions, including reaction temperature, MgSO₄ concentration, dNTP concentration, etc. The sensitivity and specificity of the method were evaluated. The optimized testing method was used to analyze clinical stool samples, and the results were compared with those of a commercially available kit.

The established testing method exhibited high specificity, showing no cross-reactivity with adenovirus, human enterovirus, or rotavirus. The testing method also exhibited high sensitivity, with a detection limit of 51 copies/μL. The turnaround time was 45 min. For 34 stool samples from patients infected with norovirus, the testing results were highly consistent with those of the commercial kit, with a sensitivity of 100%, a specificity of 83.33%, and an area under the receiver operating characteristic (ROC) curve of 0.97 (95% CI: 0.92-1.00), indicating the high accuracy and reliability of the testing method.

In this study, we successfully developed an RT-LAMP-based testing method for GⅡ norovirus, which exhibits excellent sensitivity, specificity, and practical performance in clinical sample testing, making it suitable for use in primary care laboratories and for rapid testing in field settings.