A non-radioactive diagnostic test for the detection of HBV DNA sequences in serum at the single molecule level.
A non-radioactive diagnostic test, using an acetylaminofluorene-labelled DNA probe, was developed to detect HBV DNA sequences in serum. In vitro enzymatic amplification was employed to increase the amount of HBV DNA sequences, and an amplification rate up to 1.5 x 10(7) was observed. When a dot-blot was performed after amplification with the Klenow fragment for 32 cycles, the detection limit was 3-30 particles. Sera from 20 blood donors and 10 HBs-Ag carriers were screened simultaneously, with the non-radioactive test performed after 28 amplification cycles, and with the classical radioactive test without amplification. An acceptable correlation was obtained between these two techniques. In Southern blot analysis of samples amplified with the thermoresistant DNA polymerase (Taq polymerase) for 40 cycles, a single DNA molecule was detected. Thermal treatment at 115 degrees C efficiently disrupted purified viral particles and allowed the detection of a single viral particle. Applied to crude serum, a kinetic study showed that this treatment was optimal after an incubation time of up to 10 min. Under these conditions, the detection limit was approximately 2 x 10(5) viral particles, after 40 amplification cycles performed with the Taq polymerase.
Larzul
D
D
Laboratoire des Sondes Froides, Institute Pasteur Paris, France.
Chevrier
D
D
Guesdon
J L
JL
D001483:Base Sequence; D015139:Blotting, Southern; D004279:DNA, Viral / Q000032:analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005784:Gene Amplification; D006509:Hepatitis B / Q000097:blood / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000235:genetics*; D015345:Oligonucleotide Probes; D013824:Thermus / Q000201:enzymology
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D004259:DNA-Directed DNA Polymerase