Detection for norovirus genotype II with RT-LAMP

Objective To establish a rapid detection method, RT-LAMP, for norovirus detection at the grassroots level, and apply it to detect clinical stool samples of adults diagnosed with viral diarrhea, in order to provide the scientific data for understanding the prevalence of norovirus-induced viral diarrhea in Nanjing. Methods The RT-LAMP method was established with the norovirus positive samples conserved in the lab. Fifty-seven stool samples from patients diagnosed with viral diarrhea in a hospital of Nanjing in the autumn and winter of 2011 were detected and analyzed for norovirus genotypeⅡ, including quantitative PCR method and reverse transcription loop-mediated isothermal amplification(RT-LAMP) method. And the positive PCR samples were sequenced.Results Two methods, RT-LAMP and fluorescence quantitative assay were established and they both could detect 10 000 dilution of the positive samples with norovirus virus genotype Ⅱ reserved in the lab, which indicated that two methods had the same sensitivity.Meanwhile, there was no amplification for the norovirus type I, hepatitis A virus and hepatitis B virus with these two methods,which indicated that they had specificity of norovirus II-amplification. A total of 57 cases of fecal samples were detected with quantitative PCR method and LAMP method. Six cases were detected positive with Norovirus genotype Ⅱ by both methods, including five cases with norovirus subtype GⅡ.4 and one case with subtype GⅡ.6. Sequences blast results showed that these six cases came from four original clones and three countries or regions, including Brazil, Russia and Beijing. Conclusion The majority subtype of norovirus of viral diarrhea patients in Nanjing is GⅡ.4 and the minority subtype is GⅡ.6. The results indicate the wide spread of the virus and the difficulty of prevention and control of the disease. Both quantitative PCR and RT-LAMP method can quickly detect pathogens, but the RT-LAMP method is more suitable at grassroots level because of its simplicity, requiring no special equipment,and direct visual observation of the results.