The Research About Using Mussel As The Indicator Of Water Pollution By Human Enterovirus

Objective: Detect the human enterovirus in shellfish with real-time quantitative polymerase chain reaction, explore the relationship of viral load between mussel and the water.Method: Concentrate the enterovirus in shellfish with PEG precipitation, inoculate sediment to Vero cells, observe if there is CPE. There are two methods to elution the virus,traditional and new, which are glycine elution and glycine-threonine elution, we detect the recovery of them with plaque assay. The water samples collected in the same period (filter method) through the beef extract elution, and then PEG precipitation. Shellfish and water samples are used TRIzol extraction method and RNA extraction kit for enterovirus RNA, according to OD260/280 value and the absorbance scan results, analyze the quality of RNA about both of the extraction methods. We design the human enterovirus universal primers in the PCR. We use T-A cloning to make the Specific plasmid(contain the fragment of enterovirus PCR products) for the standard curve in real-time quantitative PCR. Screening and extract pure plasmid with the Bacterium PCR and plasmid PCR, detect DNA concentration for calculate the number of the copies. Observe melting curve , According to the formation of primer-dimers, choose the appropriate concentration of primers (a group: 300 nM / 300 nM, b group: 200 nM / 200 nM, c group: 100 nM / 100 nM, d group: 50 nM / 50 nM). After the 10-fold serial dilution, observe the amplification curve of the plasmid to select the appropriate dilution range. Once there are all ready, begin the real-time quantitative PCR for detect the enterovirus in shellfish and water samples.Results: The vero cells inoculated sediment, observed CPE response. Comparison of two methods for the elution, The recovery of glycine elution (full shell) is 19.6%, and the result of glycine-threonine elution (shellfish gut) is 13.1%, consistent with the reported recovery. Choose Glycine elution for subsequent selection experiment. The difference of OD260/280 value between TRIzol extraction method and RNA extraction kit is significantly (t= 8.53, P<0.05), full scan showed a small Miscellaneousl absorption peak in TRIzol method, which might be molecular salts (OD = 230nm), while the RNA extraction kit have an only nucleic acid absorption peak (OD = 260nm), the RNA purity of the kit is better. Shellfish and water samples by ordinary PCR reaction, whose gel electrophoresis show clear target band fragment, indicate the PEG precipitation of enteroviruses is successful. The Bacterium PCR and plasmid PCR are both show obvious target band, and OD 260/280 value is greater than 1.8, the plasmid DNA extracted is pure. Optimized primer concentration of 100 nM / 100 nM, select the dilution range of the plasmid about 103 to 107 for standard curve in real-time quantitative PCR. The result of real-time quantitative PCR shows that, the amount of enterovirus copies about shellfish samples is 1.12×10~3 , while the water samples collected over the same period is 2.35×10~2. we convert it into the original water sample volume (original volume of 136L), then we find shellfish concentrated enterovirus in water environment up to 3.1×10~4 times.Conclusion: The PEG precipitation of enteroviruses in shellfish is successful, virus recovery compared with the report is better. High purity plasmid is stable, which is suitable for real-time quantitative PCR. shellfish can concentrate enteroviruses in water environment, through the real-time quantitative PCR about shellfish, we can preliminarily estimate enteroviruses in water.