| Noroviruses (NoVs) are commonly occurring pathogens that causegastroenteritis. NoVs can be isolated from water, vegetable, fruit andseafood samples, especially in shellfish samples frequently. It usually causesacute gastroenteritis associated with the consumption of raw or undercookedseafood. On rare occasion, infection can lead to severe dehydration and deathmay also occur subsequently. The annual production of shellfish was nearly upto10,000,000tons in China. For shellfish is delicious, the coastal residents arefond of it. Foodborne outbreaks caused by NoVs associated with theconsumption of shellfish were constantly reported in the coastal area of China.More attention should be paid to food safety issuses arised by NoVs.To investigate the prevalence of NoVs in shellfish, the quantitative detectionmethods were optimized. The level of NoVs in shellfish of seven cities aroundYellow Sea and Bohai Sea in China was investigated by real-time RT-PCR andthen the quantitative assessment was carried out. The recent studies showedthat NoVs recognize histo-blood group antigens (HBGAs) as receptors inoyster.To elucidate the mechanism of NoVs accumulated in oysters, firstly, the ELISA and PCR method was optimized to detect A-type HBGAs and keygenes of HBGAs in oyster. Second, total length cDNA of one key genes FUT3was got and the protein was expressed.Temperature effect on expression levelof FUT2was also studied.The main contents were as follows:1. The detection methods of NoVs in shellfishAimed to identify a simple, rapid and highly efficient recovery method forreal-time RT-PCR detection of NoVs. Four methods were compared forrecovering GI.3and GII.4NoVs from spiked digestive tissues of oysters andclams, respectively. The method based on the application of proteinase K-PEG8000was found most efficient, with9.3%and13.1%of GI.3and GII.4NoVsrecovered from oysters and9.6%and12.3%of GI.3and GII.4NoVsrecovered from clams, respectively.2. Presence of Genogroup II Norovirus in Retail Shellfish from Yellow seasand Bohai seas in ChinaOur objective was to evaluate the presence and contamination levels of NoVin shellfish sold at seafood markets in China. We tested840shellfish samples(Crassostrea gigas, Mytilus edulis, Azumapecten farreri, SinoNoVaculaconstricta, Scapharca subcrenata, Ruditapes philippinarum) that werecollected from seven cities around the Yellow and Bohai Seas in Chinabetween December2009and November2011. We used real-time RT-PCR todetect NoV in purified concentrates from the stomachand digestive diverticula of these shellfish. NoV was detected in19.35%(N=155),16.67%(N=114),5.70%(N=158),8.82%(N=136),13.74%(N=131), and16.44%(N=146) ofoyster, mussel, scallop, razor clam, ark shell, and clam samples, respectively.The average detection rate was13.33%(112/840). Nucleotide sequencing ofthe NoV RT-PCR products demonstrated that all strains belonged to NoVgenotype GII.12, except two that belonged to GI.3. More than102copies ofthe NoV genome were detected in69of112positive shellfish samples. Ourresults suggest that~13%of shellfish harbor NoV, and GII.12NoV is theprimary strain in shellfish purchased at markets in seven coastal cities inChina.3. Risk assessment of NoVs in shellfishRisk assessments of NoVs in shellfish were carried out based on the data ofinvestigation of NoVs presented in shellfish and shellfish diet survey. NoVsgastroenteritis infection probability of consumers along the coast areas ofChina by intake marine shellfish is4.08×10-7per person a day. The annualnumber of desease cases caused by consumption of polluted NoVs shellfish isabout229,17458and651among ages0-4,5-64and above65. Uncertaintyanalysis was also done in the study, which provides suggestion for NoVsassessment in shellfish or other food.4. HBGAs detection in pacific oyster The ELISA method based on was established for detection of A-type ofHBGAs in shellfish by optimized the concentration of heat treated oysterdigestive tisseues.36oyster samples were collected from Weihai, Qingdao andRizhao in2011. A-type of HBGAs and NoVs detection was analysed. Thedetection rate was100%and16.7%respectively. The results showed thatA-type of HBGAs changed along seasonal variation in fluctuating way.Interestingly, NoVs was detected at the time A-type of HBGAs showed peakvalue.5. Prokaryotic expression and cloning of genes related to HBGAs in pacificoysterPresent on the oyster HBGAs synthesis of key gene has not been reported.With reference to the study of the synthetic pathway of human HBGAs, wedetermine the FUT genes for research purposes. By Clustal X and Mega andother analysis software, analysis of human, chimps, mice, dogs and otherspecies of the FUT2sequence, primers were designed based on conservedsequences of “FUT2”; The Pacific oyster (Crassostrea gigas) genomic DNAas the template for PCR amplification, cloning and sequencing the amplifiedproducts confirmed the existence in the oyster genome fragments from FUT2gene. In2012, with the completion of genome sequence of pacific oyster, the1081bp sequence we got was confirmed as FUT3. Prokaryotic expression ofFUT3was studied and We got the expected size of the protein. 6. Temperature effect on the expression regulation of FUT2in differenttissues of oysterReal-time PCR detection method of FUT2genes in pacific oyster wasestablished with β-actin as the reference genes. FUT2genes expression inpacific oyster exposure to high temperature (28℃) and low temperature (4℃)was investigated by real-time quantitative PCR. The tissues analysed byquantitative real-time PCR were mantle, adductor, gill and digestive gland.Theexperiments were performed to investigate the expression level changes withinand between tissues at48h and72h.The results indicated that level of most FUT2gene expression wassignificant higher than control no matter exposure to high temperature or lowtemperature conditions. When exposure to high temperature stress, level ofFUT2.1and FUT2.3genes expresstion have raised in4different tissues ofoyster organizations are raised, while under the stress of low temperature,FUT2.4expressed in mantle and gill tissue raised. |
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