Detection And Quantitation Of Norovirus Genogroup â…¡ By Real-time Reverse Transcription-pcr Assay And Disinfection Of Human Norovirus Using Chlorine

Objective:Norovirus (NoV) is a major pathogen which can cause the non-bacterial acutegastroenteritis of children and adults all of world, it’s a principal member of thecalicivirus family,and it can cause the acute diarrhea and vomiting, however, becauseof the dehydration or immunodeficiency of some peoples, that may lead to seriouscomplications or death. Since the1990s, the epidemiological survey in world-wideshew that NoV GII.4has gradually became the dominant epidemic strains in a globalfashion and caused four times the global epidemic at least. NoV is mainly transmittedthrough the fecal-oral route, which can spread the virus by person-to-personcontact,having the contaminated food, oysters or water; otherwise, though theparticles of vomit flying got into the respiratory tract may also be a route oftransmission. In the70’s of the last century, NoV was found by electron microscopy,,but because of the lack of cell culture systems for the NoV， the disinfection of NoVcommonly used alternatives just like: mouse norovirus (murine norovirus, MNV),feline calicivirus (feline calicivirus, FeCV) and canine calicivirus (of caninecalicivirus CaCV). Now the research on disinfection of NoV of using chlorine andchlorine dioxide in drinking water didn’t developed in our country. Therefore, theinvestigation of China’s norovirus Prevalence, to establish a sensitive and specificreal-time Reverse Transcription-PCR for detection and Quantitation of NoV G II, ithas hardly significance for public health, we may provide the basis for thedevelopment of appropriate control strategies and measures, about the use ofdisinfectants in sewage, which further ensure the achievement of bacteriology andvirology in the sewage. Therefore, we use the RT-PCR to study the prevalence of thediarrhea due to norovirus in infants and young children of NoVmber to December2009in Tianjin,construction and evaluation real-time Reverse Transcription-PCR fordetection and Quantitation of Norovirus genogroup II, disinfection of norovirus usingchlorine, to develope the reference method and basis about detection of norovirus infecal specin,food and water, the control of disinfection NoV and rapid detection ofpublic health emergencies. Methods:From October to NoVember2009，we collected a total of319stool specimensfrom infants and children with acute non-bacterial diarrhea in Tianjin Children’sHospital,that198cases were male and female121cases, the minimum age for the twodays after birth up to9years old. RT-PCR was used to detect Norovirus RNA andthen the PCR products were cloned and sequenced. The results of sample sequencewas submissed to Genbank and Blast to determine the genotype of the samples.Phylogenetic tree was constructed by Neighbor-joining method in Mega4.1. Statisticalmethods were used to analyze the demographic data and clinical data of the samples.For the strong positive samples of NoV G II, PCR product was cloned andchoose the positive clones by the blue-whiteing screening, and then we constructedplasmids, and transcription of the copy RNA (cRNA)was synthetized as a standardproduct, the establishment and optimization of NoV G II RT-qPCR and the reactionsystem, constructed of the standard curve and evaluated the sensitivity, specificity,reproducibility of the reaction system, and clinical evaluation by detecting stoolsamples.Norovirus, RT-PCR-positive, RT-qPCR detection of viral load samplesartificially contaminated sterile ultrapure water, in the amount of disinfectant chlorineunder the action of the different time points sampling, using RT-PCR method initiallyto determine the point of virus damage, quantitative PCR detection of viral RNAfurther reductions, to research the damage of chlorine and chlorine dioxide NoV andviral RNA reduction rate of the corresponding law, in order to determine thedisinfection effect of these two disinfectants to NoV.Results:1, Detection of Norovirus among children in Tianjin in fall-winter seasons of200960cases were NoV positive in319specimens by RT-PCR,That59cases wereNoV G II,; one cases of NoV G I.24cases of norovirus positive samples, PCR products were purified andsequenced, the sequencing results were submitted to Genbank BLAST alignmentfound that1cases is NoV G I type, the remaining23cases were NoV G II.Multi-sequence homology was found that the homology was71.7to99.3%among the sequenced strain reference strain17, the Netherlands Nijmegen115,Lincoln House, reference strains, Beijing151strains and Terneuzen70strains ofsequence, the homology of reference strains with Lincoln House is up to98.0to99.3%.13in15cases are G II-42006b mutant;2cases are NoV G II-3. The nalysis of phylogenetic tree showed that NoV G II/2006b virus strains was the majorepidemic strain for diarrhea norovirus in2009in Tianjin, which is similar to thecurrent study or other places in China, which indicates that the popular predominantstrain is still NoV G II/2006b strain in our country.The distribution analysis of NoV positive cases in different age groups showedthat there were35cases in the0-1age group of children infected with norovirus in60cases, a2-year-old children with16cases of norovirus infection. With the increasingof age, children with diarrhea reduced, fewer children infected with norovirus. Infantpromise such as virus infection is mainly concentrated in the0to2-year-oldpopulation, other studies show that infant serum antibody was lower, with thegrowing, the serum antibody was increasing to resist to diseases.In this study, outpatient diarrhea norovirus virus-positive rate is26.75%(41/157),while the inpatient department of diarrhea in children with norovirus virus-positiverate is11.11%(18/162), the statistical analysis of the difference between the twostatistically is significant, indicating that the disease incidence of acute self-limiting,quick recovery, most patients are outpatient.2, the establishment and evaluation of NoV G II by RT-qPCRNoV G II RT-qPCR amplification using the primers ordinary RT-PCR reaction,using2%agarose gel electrophoresis showed a specific band of98bp and no othernon-specific amplification. The primers and probe fluorescence quantitative RT-PCR,fluorescence amplification curve was typical of the smooth S-shaped curve.The purity of the plasmid DNA, high concentrations of purified cRNA was afterin vitro transcription, cRNA was the OD value at between1.7and2.0middle purity,concentration mean321.83μg/ml. The standard curve slope is-3.41, the intercept is49.79, and the correlation coefficient (R2) is0.998.The sensitivity testing about the method, the results showed good sensitivity, theminimum can be detected in the number of102copies/μl of RNA standard of knownconcentration samples.This method can specifically detection of NoV G II, no cross-reaction with NoVG I, and the Coxsackie virus group B, polio virus, enterovirus, astrovirus, hepatitis Avirus, ECHO virus and rotavirus was no cross reaction.For the standard batch experiment, the Ct value of the coefficient of variation(CV) were1.60%,0.70%, and inter-trial coefficient of variation (CV) were0.40%,0.40%,all were below5%, indicating that the system stability, good reproducibility. 3. Disinfection of norovirus using chlorine in waterFirst of all, RT-PCR results showed that, NV3R the/NV3F primers amplifiedbands were the first to disappear, indicating that the chlorine priority injury NoVnucleic acid3’end; Secondly, with the chlorine disinfectant volume increases,COG2F/G2-SKR primer pairs band disappeared amplified nucleic acid damagepoint of the ORF1and ORF2binding region; Finally, when the chlorine disinfectantfrom5mg/L to10mg/L, NV5R/NV5F primers amplified band disappeared,indicate that nucleic acid may be damaged the5’end of the no-cap structure, the areawith the disappearance of NoV infections.Conclusion:1, That NoV was existed in the Otc and NoV of2009, Tianjin diarrhea NoVvirus-induced diarrhea and NoV is the major pathogens that cause diarrhea; Tianjin2009autumn and winter diarrhea in children with different genotypes of norovirusinfection, NoV G II-42006b mutant is a major epidemic predominant strain.2, we have established and optimized fluorescent quantitative RT-PCR detection ofNoV G II, the method is stable, good sensitivity, specificity and reproducibility can beused for the rapid detection of public health emergencies.3, the chlorine priority damage the NoV nucleic3’end, followed by the bindingregion of ORF1and ORF2, with the chlorine concentration increases, the damageregion maybe is5’ end.