Comparison Of Different Concentration Methods For The Detection Of Hepatitis A Virus From Simulated Water Samples

Hepatitis A virus (HAV) is an important cause of acute hepatitis in humans worldwide. Symptoms of infection include fever, nausea, fatigue, anorexia, dark urine jaundice etc. Thus far, only one serotype and six genotypes have been identified for HAV in humans. Hepatitis A is mainly transmitted via fecal-oral route.The presence of viruses in most environmental water samples is at low concentration levels, it is difficult to detect the viruses directly. HAV grows slowly in cultured cells, and the virus titer is low after the culture. Therefore, choosing a suitable physical method to concentrate HAV from water samples is very important.There are two parts for the paper:Part Ⅰ:Comparison of Hepatitis A Virus Concentration Methods from Simulated Water Samples Using Two Ions MembranesPositively charged membrane from Sartorius and negatively charged membrane from Millipore (as primary concentration method), together with polyethylene glycol (PEG) precipitation (as the secondary concentration method) were used to concentrate HAV from simulated water samples. Then TaqMan real-time RT-PCR was applied to detect the virus RNA. Live attenuated hepatitis A vaccine was added to the ultra-pure water or mineral water samples, while the water samples without adding the virus was treated as negative control. The t-test method was used to compare the concentrating effects of two membranes with SPSS21.0 statistical software. Statistical analysis showed that there was no significant difference (Ct value comparison P = 0.374> 0.05, Viral load comparison P = 0.307> 0.05).Sartorius membrane combined with PEG precipitation was used to compare different elution methods, which includes shaker cleaning, vortex cleaning,20 min ultrasonic cleaning,40 min ultrasonic cleaning and direct lysis method.. TaqMan real-time RT-PCR was applied to detect the virus RNA. The Analysis of Variance(one way ANOVA) was used to compare the different elution methods with SPSS21.0 statistical software. The comparison of Ct value and recovery rate showed that there were significant differences in different treatment methods (P< 0.05). Bonferron pairwise comparison showed that the direct pyrolysis was the best one, and its average recovery rate was 68.17+16.79%, which was the highest among these methods.Two filtration membranes were selected and direct pyrolysis were choosen to concentrate simulate water samples.0.2 TCID50/ml,1 TCID50/ml and 5 TCID50/ml, HepA-L was added into ultrapure water or mineral water samples. The Analysis of Variance (one way ANOVA) was used to compare the different recovery efficiency with SPSS21.0 statistical software.Results showed that the recovery efficiency of the two filtration membranes can reach more than 70% in low virus concentration (0.2 TCID50/ml); the Ct value and viral load of the simulated mineral water and ultra pure water samples had no statistic difference (P = 0.950>0.05), and the average recovery rate indicated the three kind of water samples had similar concentration effect.Summary:For Millipore and Sartorius filtration membranes, the comparison of Ct value and recovery rate shows that there are no significant differences in the concentration of a certain amount of HAV, different elution methods have distinct concentration effect, and direct lysis is the best method.; results indicate the Ct value and viral load of the simulated mineral water and ultra pure water samples have no statistic difference, and the average recovery rate indicate the three kind of water samples have similar concentration effect.The results provide certain experimental basis for further research about HAV concentration from water samples.Part II:Comparison of Hepatitis A Virus Concentration Methods from Simulated Water Samples Using Ultrafiltration and PEG PrecipitationTo compare concentration methods of ultrafiltration and PEG precipitation for hepatitis A virus from simulated water samples. Quantitative hepatitis A virus was inoculated in the simulated water. 100KD and 50KD aperture microporousultrafiltration centrifuge tubes from Millipore, Sartorius, and the final concentration of 10% polyethylene glycol (PEG)-1M sodium chloride (NaCl) precipitation method were selected to concentrate hepatitis A virus. The concentration of virus was detected by fluorescence quantitative RT-PCR method. The data were statistically analyzed with SPSS21.0 The comparison of Ct value and recovery rate showd that there were significant differences between different treatment methods (P< 0.0001, P< 0.0001). There were no statistically significant between the same aperture ultrafiltration centrifuge tubes which are produced by different manufacture (P = 0.25, P=0.532),50KD group had a higher recovery rate (94.57± 16.26%) than 100KD group (52.37± 14.75%), and the 10%PEG-1.0 MNaCl (63.5 ± 8.45%) was in middle of them.Both methods of ultrafiltration and PEG precipitation can be used for HAV concentration from water samples, the ultrafiltration centrifuge tube has the advantages of simple operation, less time-consuming, and selecting the appropriate aperture can improve the HAV recovery rate. PEG is more economical and practical method, and it can process a large volume sample as well.Summary:For Millipore and Sartorius filtration membranes, the comparison of Ct value and recovery rate shows that there are no significant differences in the concentration of a certain amount of HAV, while the direct lysis method is the best one and the three kinds of simulated water samples used in this study have similar concentration effect.Both methods of ultrafiltration and PEG precipitation can be used for HAV concentration from water samples, the ultrafiltration centrifuge tube has the advantages of simple operation, less time-consuming, and selecting the appropriate aperture can improve the HAV recovery rate. PEG is more economical and practical method, and it can process a large volume sample as well.