| Objective:The multiple qRT-PCR was established to inspect concurrently and fleetly Nor ovirus(NOV),A group of Rotavirus(RV),Sapovirus(SaV),Human Adenovirus(HAd V)and Human Astrovirus(HAstV)from the stools of acute gastroenteritis patients The epidemiologial status of these viruses and clinic characteristics of these patient s were investigated in some areas of Zhenjiang during 2018.11-2019.11.The distrib uted characteristics of NOV ? Genotype were investigated in the city.The predom inant and epidemiologial virulence strains and their evolution were researchedMethods:1.The recombinant plasmids of pACYC184-NOV-G?,pACYC184-NOV-G? and p ACYC184-RV were construct by utilization of RNA of NOV GI,NOV G? and A group RV.The plasmids of pBluescript ? SK-SaV,pBluescript ? SK-HAstV and pBluescript?SK-HAdV were chemically produced.The standard preparation of R NA and DNA of these viruses were made2.The standard curves of two steps singleplex qRT-PCR were established with the templates of the standard preparation of RNA and DNA so that the sensitivity an d specificity of their systems were evaluted.The standard preparation of DNA wer e done as thetemplates by qRT-PCR to evalute the repeatability of their systems3.On the basis of the singleplex qRT-PCR,the primers and probes of these virus es were divided into three groups.The feasibility array of two kinds of primers an d probes in one group were done.The standard curves of multiple qRT-PCR were established with the templates of the standard preparation of RNA and DNA to e valute the sensitivity and specificity of their systems.The standard preparation of DNA were done as the templates by qRT-PCR to evalute the repeatability of their systems.4.We selected Stool samples from 121 patients,which were clinically diagnosed a s the patients of acute gastroenteritis in the partial areas of Zhenjiang during Nove mber 2018 to November 2019.We extracted nucleinic acid of these samples and e stablished multiple qRT-PCR to inspect these samples to evalute the positive rates of acute gastroenteritis virus(AGV)of adult and infant in the areas,the distributio n of the age and the sex of the patients,and the season in which patients got thes e diseases and clinical characteristics,and to analyze the molecular epidemiological characteristics of acute gastroenteritis virus.5.To select of the postive samples of NOV G?,semi-nest RT-PCR amplified the RdRp and capsid VP1 region of NOV and sequenced the DNA fragments.The genetic sequences were retrieved at GenBank with the similarity searching.The phylogenetic tree was constructed using Mega 7.0 software Neighbor-joining(N-J)based on nucleotide to carry out the system and developed analysis.Results:1.There were good linear relations between the Ct of the standard preparation of RNA or DNA of NOV G?,NOV G?,A group RV,SaV,HAdV or HAstV and t he different dilution of the copy numbes of RNA or DNA by singleplex qRT-PCR;The singleplex qRT-PCR established hasn't cross reaction with other enteroviruses s o that it has a good specificity.As the limits of detection of NOV G?,NOV G?,A group of RV,HAdV,SaV and HAstV by simple qRT-PCR were 102,102,101,102,102,102 and 102,respectively,the results showed that the inspections were g ood sensitivity.The most coefficient of variation of detection of NOV G?,NOV G?,A group of RV,HAdV,SaV and HAstV by singleplex qRT-PCR were 1.56%,2.50%,1.75%,1.56%,2.35%and 2.39%,respectively,which of all were less than 10%,so that the detection system established has a good stability and repeatabilit y.2.The standard curve of detecting at the same time NOV G?/NOV G?,ARV/HA dV,SaV/HAstV by multiple qRT-PCR were established.All of R2,which represent ed the relative coefficient between their Ct value and copy number of templates,were more than 0.99.Their amplification efficiency were among 90%?110%.Thei r primers and probes have no cross reaction with other AGE viruses.Their sensiti vity were the same as the singleplex qRT-PCR.The coefficients of variation in gr oup were less than 10%.The results showed that the detection system established has a good specificity,sensitivity and repeatability.3.From Nov 2018 to Nov 2019,a total collection of 121 feces samples,the positive rate of NOV was 10.74%(13/121),in which the rate of GI group was 4.13%(5/121),and the one of G? group was 6.61%(8/121).The positive rate of group A of RV was 15.70%(19/121).The positive rate of SaV was 2.48%(3/121).The positive rate of HAdV was 0.83%(1/121).There were no significant differences in the rates detected of the AGE viruses between these male and female patients infected(P>0.05),among every age groups of patients infected by NOV GI or SaV(P>0.05).The positive rate of G? group patients among children aged 0 to 10 years old was the highest up to 16.00%,which was more significantly different than other age group patients(P<0.05).The infection of group A of RV mainly happened among young people aged from 0 to 20 years old.The positive rate of group A of RV was the highest among people aged 10 to 20 years old,up to 31.30%,in which there was the significant difference with other age groups(P<0.05)The infection of HAdV mainly happened the old people after 60 years old,and there were no significant differences among other age groups(P>0.05).In the distribution of the season of these morbidity,the peak of the infection of NOV GI was concentrated in February,which was up to 22.22%.The rate of NOV G? was mainly in March,up to 25.00%.The rate of group A of RV was mainly in November,up to 30.00%.The rate of HAdV was 0.83%,which had only been detected in April.The rate of SAV was mainly in January,up to 20%4.The multiple qRT-PCR inspected 8 positive samples of NOV,which was coinc idence with the results of their sequences.All of them were NOV G?,which acc ounted for 75%(6/8)of all NOV.There was a significant difference between NO V G? and other genotypes(P<0.05).The other NOV genotypes were G?.6 and G?.17.NOV G?.4 genotype was at the same branch of the world epidemic strain NOV G?.4/2006(CQ845367)on the same branch,in high homology with 96%.N OV G?.17/2019 was at the same branch of reference strain KU663020,and NOV G?.6/2019 was at the same branch of reference strain AJ277620,and all of them were in high homology with 99%Conclusion:1.A multiple qRT-PCR for the rapid detection of NOV G?,NOV G?,group A RV,SaV,HAdV and HAstV at the same time were established to find 36 AGV from a total collection of 121 stool samples of the patients of acute gastroenteritis2.The prevalence of NOV in the area was mainly NOV G? genogroup,which had the apparent distribution characteristics of seasons and population.The NOV GI infection was only characteristics of seasons.The A group RV had two the distribution characteristics.3.The incidence rate of AGV in nonage group was significantly highter that that one in adult group.There was no significant difference in the incidence rate of AGV between male and female.The common symptoms of the patients of all with AGV were diarrhea,nausea and vomiting4.There were three genotypes G? 4,G? 6,G? 17 of NOV G? in partial areas of Zhenjiang region.The main prevalent genotypes was G?.4,which G?.4 genotype has 96%homology with world epidemic strain G?.4/2006(CQ845367). |
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