HYBRIDIZATION ANALYSIS FOR SERUM HEPATITIS-B VIRUS-DNA

A study was carried out to determine optimal assay conditions for an i n-house hybridisation assay for detection of hepatitis B virus (HBV) g enome in serum samples. Pre-treatment of samples, blot treatment and h ybridisation conditions mere found to affect assay performance. The in direct serum blot procedure was more robust and reliable than direct s erum blotting. In the former, viral particles were isolated from the s ample, lysed and then extracted. In comparison, no approaches to the d irect serum spot method performed adequately. Sensitivity studies show ed that labelling of the nucleic acid probe with dCTP was more efficie nt than with dATP. Using probes labelled to a specific activity of > 1 x10(8) and an autoradiography period of about 48 h we could achieve a detection limit of <1 pg. Specificity was achieved by use of a highly purified probe and moderately stringent hybridisation and wash conditi ons. Background binding was minimal and there was no non-specific bind ing of probe to negative control samples. Factors affecting speed of t he assay were studied to identify steps that could be modified to shor ten assay time without sacrificing performance. A shorter centrifugati on step and the use of a high specific-activity probe permitted comple tion of an assay within four days.