A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum.
The detection of a little as 0.2 pg (60,000 molecules) of hepatitis B viral (HBV) DNA in human serum samples in 4 h has been demonstrated using a solution-hybridization and bead-capture method. An amplification method based on chemically crosslinked oligodeoxyribonucleotides was coupled with a horseradish peroxidase-labeling scheme for the ultimate detection of the analyte. Two sets of HBV complementary synthetic oligodeoxyribonucleotide probes containing one of two types of single-stranded (ss) overhangs were employed. These ss overhangs were used to capture the probe-analyte complex onto a bead and subsequently to label it. Detection was achieved with either a chemiluminescent or colorimetric output substrate for the enzyme. Only in the presence of the virus was label specifically bound to the support. The assay was relatively unaffected by either sample composition or by the presence of heterologous nucleic acids.
Urdea
M S
MS
Chiron Corporation, Emeryville, CA 94608.
Running
J A
JA
Horn
T
T
Clyne
J
J
Ku
L L
LL
Warner
B D
BD
D001483:Base Sequence; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics*; D006509:Hepatitis B / Q000097:blood / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D009693:Nucleic Acid Hybridization
D004274:DNA, Recombinant; D004279:DNA, Viral