AN AUTOMATIC MODIFIED POLYMERASE CHAIN-REACTION PROCEDURE FOR HEPATITIS-B VIRUS-DNA DETECTION

In order to perform an efficient and reproducible diagnostic test for hepatitis B virus (HBV) infection using the polymerase chain reaction (PCR), sixteen primer couples specific for the HBV genome were selected. Primers 15-31 nucleotides in length containing between 31-73% GC permitted amplification of fragments corresponding to the whole HBV genome. The specificity and efficiency of PCR amplification were studied in detail using DNA extracted from either a viral particle preparation or from the liver of a patient with chronic active hepatitis. Three primer couples in the X, C and PreS regions i.e. MD24/MD26, MD27/MD31 and MD19/MD18, respectively, gave satisfactory results and performed efficiently under highly stringent hybridization conditions. A modified PCR procedure was then developed using only two thermal steps with a temperature shift of 16°C. This simple method was as efficient as conventional PCR and permitted detection of a single HBV DNA molecule with the X region specific primer couple. The automatization of this PCR-based procedure permitted 40 amplification cycles in 105 min. © 1990.