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 degrees 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.
Larzul
D
D
Département de Biologie Moléculaire, Institut Henry Beaufour, Les Ulis, France.
Chevrier
D
D
Thiers
V
V
Guesdon
J L
JL
D001322:Autoanalysis; D001483:Base Sequence; D015139:Blotting, Southern; D004279:DNA, Viral / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase; D006509:Hepatitis B / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase
D004279:DNA, Viral; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase