A method using non-radioactive material alkaline phosphatase to label HBV DNA as probe has been studied and used in clinical experiments to detect the HBV DNA in hepatitis serum. Alkaline phosphatase coupled with polyethyleneimine (PEI) using P-benzoquine as cross-linking reagent. The modified phosphatase was covalently linked to single strand DNA using glutraldehyde. Such single strand DNA enzyme complexes have been tested for blot hybridization, after hybridization and incubation with a substrate solution, sequences complementary to the probe can be visualized directly in 1 h. The minimum amount about 10 pg of target DNA has been detected in this way, 32P labeled probes are autoradiography 1 h after hybridization can only detect 10 ng, so the enzyme labeled probe is more sensitive than isotope labeled probe in 1 h fast test. Comparing the enzyme-labeled HBV DNA probe with 32P labeled the same one, positive proportion of detecting the HBV DNA in hepatitis patients was about 95.7%. Because the positive patient's serum detected by 32P labeled probe were selected through 1- week radiation, Alkaline Phosphatase labeled probes are color developed for only 1 h. Our experiment certified that it is a sensitive, specific, easy, rapid, safe and economical probe labeling and clinical virus DNA detection method.
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