Study On The Methods Of Concentration And Deteciton Of Hepatitis A Virus In Water Samples

The aim of this study was to build up methods of the concentration and detectionhepatitis A virus (HAV) in water samples. Two different kinds of HAV concentrationmethods were established: One of the concentration methods in a lab environment whichwas based on membrane was built and applied in East Lake water samples by comparingthe concentration efficiency of the secondary enrichment and membrane alone toconcentrate HAV.The effects of concentration efficiency were explored by using differentkinds of filter membranes. Another concentration method was built to concentrate HAVwhich was based on immunomagnetic beads. The immunomagnetic beads were preparedfor carboxyl beads and HAV monoclonal antibodies. To ensure the accuracy and efficiencyof the experiment, it was necessary to optimize the quantity of conjugated antibodies of1mg magnetic beads and choose a viral RNA extraction method which was suitable forImmunomagnetic beads separation techniques(IMBS). In addition, the SYBR Greenreal-time PCR method to detect HAV was established by using the HAV plasmid standardmolecular and standard curve.The data showed that the standard curve of SYBR Green real-time PCR had a goodlinear relationship（the reliability was0.999）. The limit of detection was improved as lowas9.27×101copies/μL. The average concentration efficiency of HAV achieved(79.39±12.06)%by using Mixed Cellulose Esters membrane in East Lake water samples.Its average concentration efficiency was higher than the average concentration efficiencyby using the secondary enrichment. The volume of solution was concentrated250-fold. Asfar as using IMBS to concentrate HAV,1mg immunomagnetic beads which coated with75μg HAV antibodies had91.45%of concentration efficiency to capture1.52×106copies/μL HAV in300μL water samples and78.95%of concentration efficiencyto capture1.52×106copies/μL HAV in3mL water samples.The results showed that using the methods of Mixed Cellulose Esters membrane andIMBS to concentrate HAV could achieved a higher concentration efficiency. Both the twoconcentration methods combination with SYBR Green real-time PCR are rapid, sensitive,specific and reliable, which provide a strong technical basis for detection of HAV andevaluation of HAV in water samples.