Research On Colloidal Gold Test Strip For Norovirus And Sapovirus

Norovirus and sapovirus are the major causative agents of acute non-bacterial gastroenteritis in outbreaks and in sporadic in public,thus they also be called human caliciviruses.They have high infectivity,especially norovirus.They can be transmitted by the ingestion of contaminated food,water,vegetable or other contaminated environmental surfaces and fomites,and they can cause epidemic diarrhea,even a large diarrhea outbreak.Nowadays,human caliciviruses is the most second uppermost pathogen after rotavirus that can bring out acute diarrhea.Therefore,many developed countries and regions are strengthening the power of research,prevention and control for the virus.So it is time to develop a more effective,quick and easy method for the detection of the viruses.In this study,noroviruse and sapoviruse were taken as the research objects.Starting with the construction of multi-epitope antigen,preparation of antibody,then using of colloidal gold immune chromatography technology,a rapid detection method for noroviruse and sapoviruse was developed.The main results were as follows:1.Preparation and purification of antibody against norovirus.After Inducting BL21/ pET32a/HuNoV strains conducting prokaryotic expressing by IPTG,a 34 kDa recombinant fusion protein was purified using affinity chromatography.Using recombinant fusion protein to immune BALB/c mice to get mice serum,after 5 times immune,the titer of serum was more than 10000.Later,through cell fusion,cloning,screening,4 strains of cell lines producing McAb was obtained.Choosing two strains of cell lines to further prepare ascites for producing and purifing antibody with the greater titer(> 100000)was obtained.2.According to the principles of competitive inhibition and double antibody sandwich method respectively to establish colloidal gold immune chromatography assay to detect the norovirus.Using sodium citrate reduction method to prepare colloidal gold with particle diameter of 30 nm and the colloidal gold labeled antibody,and then assembling the colloidal gold strips.The test strips of competitive inhibition and double antibody sandwich method for testing rHuNoV were built by using two different PcAbs of rabbit IgG(rIgG)and mouse IgG(mIgG),and the test strips of double antibody sandwich method for testing rHuNoV were also built by using two different sub-types mice McAbs IgG of mIgG1 and mIgG2 b.The results of simulated sample tests revealed that the sensitivity of test strips of competitive inhibition and of double antibody sandwich method of PcAbs and McAbs were 10?g/mL,10?g/mL and 1?g/mL order of magnitudes,respectively.3.Preparation and purification of antibody against sapovirus.Several epitopes from capsid protein coding genes for sapoviruses were analyzed within bioinformatics and predicted by DNAstar software,following organized and become a multi-epitope sequence via linkers called sapovirus multi-epitope antigen with the length of 348 bp.DNA sequence of the sapovirus multi-epitope antigen synthesised were digested and linked by enzymes,which become a part of the recombinant vector of the pET-32a/HuSaV.Obtaining some transformants after transforming the recombinant vectors to BL21(DE3)cells.Recombinant fusion protein with the molecular weight of 36 KDa was gotten when target transformants were induced and expessed.SDS-PAGE showed most fusion proteins are inclusion body.Rabbit serum against sapovirus was prepared by use of purified target proteins with a way of SDS-PAGE & recycling after 5 times the immune.PcAb were clarificated by octanoic acid-ammonium sulfate salting out method,with a titer of 25600 with indirect ELISA.4.Application of competition law principle to establish colloidal gold immune chromatography assay for detecting sapovirus.The concentration of rHuSaV for line T on the NC membrane is 1mg/mL,line C adsorbed the secend rabbit IgG,colloidal gold labeled rabbit IgG assembling the test strips,and the sensitivity of the test strips was 10?g/mL order of magnitudes by testing simulated samples.5.Establishment of ddPCR detection method for norovirus GII.Digital PCR technology was through counting individual molecules so as to realize the quantitative of DNA copy number.Designing norovirus GII type-specific primers and probes and building reaction system.Obtaining the optimized PCR annealing temperature for 57?.We preliminarily established a ddPCR detection method for norovirus GII.