Norovirus Genome Sequence Analysis And Norovirus Gi Genotype-specific Detection System Design

Norovirus is the main pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide,and ranks first among the global causes of foodborne diseases.Current classification of norovirus,at least 10 genogroups(GI～GX)and 48 genotypes,has been made in accordance with the amino acid sequence of major capsid protein,as the mutation of nucleic acid sequence in norovirus is fairly common.The mode of transmission of norovirus varies with genotype.The current detection methods can only detect norovirus specifically at the genogroup level,and the exact genotyping information of norovirus cannot be obtained,which has hindered the traceability of norovirus.Therefore,it is particularly important to establish a norovirus typing system to trace the source of norovirus.In order to be able to detect norovirus quickly and accurately,this study collected stool samples from patients with clinical diarrhea,and used the norovirus GI/GII nucleic acid detection kit to detect norovirus.At the same time,we obtained the genome of norovirus by RNA-Seq,and performed genotyping and evolution analysis on the norovirus genome sequence.Based on the results of norovirus detection,we designed a genotype-specific detection system for the norovirus genogroup with a high detection rate in this study.The accuracy,sensitivity,and specificity are evaluated,and a real-time fluorescent quantitative PCR detection method is established at the same time.A total of 116 stool samples from patients with diarrhea were collected in this study.The main results are as follows:1.15 stool samples were positive for norovirus,of which 12 were positive for norovirus GI,6 were positive for norovirus GII,and 3 were stool samples that were both positive for norovirus GI and GII.Three norovirus genome sequences were successfully obtained through RNA-Seq,namely:Haidian 13,Haidian 46,and Haidian 56.Haidian 13 and Haidian 46 are both norovirus GI.1,and Haidian 56 is norovirus GII.17.The similarity between Haidian 13and Haidian 46 is 99.59%.Haidian 56 and KT589391.1 have the greatest similarity,with a similarity of 98.16%.2.This study designed a genotype-specific detection system for norovirus GI,and designed 14 pairs of norovirus genotype-specific detection primers to detect 9 norovirus GI genotypes.Among 14 pairs of primers,GI.1＿2F/R,GI.6＿1F/R,GI.6＿2F/R and GI.7F/R have been verified,and they have good specificity and can be accurately detected The corresponding norovirus genotypes in the samples,the detection limits were4.598×10~4copies/μL,3.906×10~4copies/μL,4.254×10~3copies/μL,3.585×10~3copies/μL,respectively.In summary,this study successfully constructed a rapid detection method for norovirus GI genotype based on the sequence information of the norovirus genome.In the high incidence period of norovirus,this method can be used to trace the source of norovirus rapidly,comprehensively,and efficiently.