Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification.
In vitro enzymatic amplification was applied to detect hepatitis B virus (HBV) DNA sequences in serum. This technique, known as the polymerase chain reaction (PCR) was used to amplify a 128 bp DNA fragment including a 112 nucleotide long sequence complementary to a region in the S gene of the HBV genome. Amplified samples were subjected to spot-test hybridization and scintillation counting using a 32P-labeled oligonucleotide probe. A kinetic study, performed for 4 to 32 PCR cycles with a viral particle preparation, showed a time-limited exponential accumulation of the specific amplified DNA fragment. Amplification yield after 32 cycles was at least 4 X 10(6) with a detection limit equal to 3 X 10(2) viral particles per ml of serum. As the reliability of the PCR technique was greatest for 24 PCR cycles, these conditions were used to develop a quantitative test with a detection limit of 4 X 10(4) viral particles per ml of serum. Results of this test were perfectly correlated with those obtained from the classical spot test without amplification. Ethidium bromide stained agarose gel and Southern blot analysis confirmed the specific amplification of the 128 bp HBV DNA fragment.
Larzul
D
D
Laboratoire des Sondes Froides, Institut Pasteur, Paris, France.
Guigue
F
F
Sninsky
J J
JJ
Mack
D H
DH
Bréchot
C
C
Guesdon
J L
JL
D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D004279:DNA, Viral / Q000032:analysis* / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006515:Hepatitis B virus / Q000302:isolation & purification*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides
D004279:DNA, Viral; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase