| Norwalk-like viruses (NLVs) are now the most widely recognized viral agents associated with food and waterborne outbreaks of non-bacterial gastroenteritis world-wide. American CDC's investigation indicated that about 42% outbreaks of gastroenteritis were related with NLVs. Moreover, in outbreaks of viral gastroenteritis caused by polluted food, 90% of pathogen is NLVs. Epidemiology's investigation indicated that mollusk is the most important vehicle of NLVs. As a coastal city, the consumption of shellfish in Qingdao is great. But there are not reports of pollution status of NLVs in shellfish in Qingdao.The identification and detection of the NLVs has been difficult prior to develop- ment of molecular methods because they are non-culturable, they can't grow in food, there is no animal model, and they show great genetic diversity. These factors limit the use of traditional immunology and serotyping assays. With the development of molecular biology, RT-PCR method and Nucleic acid sequence-based amplification (NASBA) have applied to the detection of viruses and pathogens more and more. Det- ection of NLVs in marine shellfish in Qingdao is carried out by RT-PCR and NASBA. And the basic pollution status of NLVs in shellfish is obtained.Experimental oysters are treated with two different methods of virus elution, acid adsorption-elution method and alkaline elution method. The result of RT-PCR indicates that the effect of acid adsorption-elution is one time higher than alkaline elution. In RNA extraction process, different references report different precipitation time which is from 10min to several hours. Thus, the effects of different RNA precipitation time are compared to, and confirm the RNA precipitation time is more than 2h finally. Research on the detection of NLVs in oysters by NASBA is also completed, and the method of NASBA for detection of NLVs in oysters is established. But the detection rate of NASBA is lower than RT-PCR.In order to investigate the pollution states of NLVs in shellfish, 208 oyster specimens, 169 clam specimens, 169 blood specimens and 100 scallop specimens are sampled in primary aquatic market in Qingdao including Shina borough, Shibei borough and Sifang borough from Dec. 2005 to Apr. 2007. Using RT-PCR method to detect NLVs. The results are that all of calm and scallop samples are not detected , 21 of 208 oyster samples are detected that are contaminated with NLVs, the detection rate is 10.3%, and 9 of 169 blood clam samples are detected that are contaminated with NLVs, the detection rate is 5.3%.In winter, oysters are polluted by NLVs seriously and the detection rate is 18.8% (12/64) which is the highest. Autumn follows, and the detection rate is 12.5% (6/48). The detection rate in spring and summer is lower than in autumn, is 4.2% (2/48) and 2.1% (1/48) respectively. Blood clams are similar with oysters. Autumn and winter are the primary seasons in which blood clams are polluted by NLVs. In autumn, the detection rate is 7.7% which is the same in winter. Spring follows, and the detection rate is 5.1%. There are not NLVs positive blood clam samples in summer. PCR results of different tissues in positive oyster specimens show that NLVs can only be found in oysters'alimentary canal, can not be detected in gill and muscle. The results of investigation indicate that the detection rate of oyster specimens which are from Shibei borough is 14.1% which is the highest. Shinan borough follows, which is 10.2%. Sifang borough is the lowest, and its detection rate is 3.8%. The detection rate of blood clam specimens which are from Shinan borough is 7.6% which is the highest. Shibei borough follows, which is 6.1%. There are not positive samples from Sifang borough. Positive shellfish samples are centralized in Tuandao Aquatic Market and Nanshan Aquatic Market.Risk assessment is a scientifically based process. Research on qualitative risk assessment of NLVs in shellfish in Qingdao which is based on overseas case of risk assessment, compare the state of research on epidemiology in Europe and America to ours, and combine the status of the pollution of NLVs in shellfish in Qingdao is done. The result of risk assessment is the risk of people who are infected with NLVs by eating marine shellfish is 25 people/100000 populations. And the risk management is brought forward according to the result of risk assessment.
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