Affinity capture-polymerase chain reaction for quantitation of hepatitis B virus DNA

Although the adhesion of virus molecules to plastic tubes has been previously mentioned, the basis and dynamics of this adhesion have not been studied in detail. Most laboratories have simply used antigen-capture with an antibody-coated capture tube. Reports concerning viral detection using the principle without any intermediate ligand are not citable. The stable adhesion of virus molecules to the polypropylene tube was repeatedly observed in our laboratory during our attempts to develop an accurate methods of quantitation for HBV-DNA. For a diagnostic-routine laboratory, normally, due to the time-consuming nature of virus extraction and its subsequent PCR, the serum samples are not immediately assayed after arrival. Therefore, when we made an attempt to quantitate the infectious viral molecules using this simple method, it was necessary to ask how many viral molecules has been lost by being bound to the plastic (after removal of the samples for extraction and PCR). We were surprised to find that a very large amount of remaining virus was still detectable by PCR even after an intensive washing procedure. From this observation, we proposed that such a kind of adhesion was probably due to interactions among the viral surface molecules, extracellular glycosylated matrix, and the polypropylene molecules. Some form of hydrophobic interaction with the plastic of the PCR tube might also have been occurring in this situation. Using this affinity nature, we report here our development of a fast, simple and practical method for HBV-DNA quantitation in serum samples. Concisely put, the short method presented here is based upon the affinity-capture of hepatitis B virus with a polypropylene tube, the digoxigenin-dUTP labelling of PCR products and the subsequent, solid-support quantitation of the biotinylated PCR products on a 96-well, plastic microplate. The reproducible and linear results suggest that it could be available for routine diagnostics.