Application of nested PCR and mass spectrometry for DNA-based virus detection: HBV-DNA detected in the majority of isolated anti-HBc positive sera.
DNA preparations from three different groups of serum samples were examined for HBV-DNA via a nested polymerase chain reaction assay (lower detection limit: 10 viral genomes in 100 microliters serum): Group I consisted of 11 uninfected control sera, group II consisted of sera obtained from 11 HBV infected patients and group III consisted of 21 isolated anti-HBc positive samples. The 21 samples from group III were HBV-DNA negative according to a conventional non-nested PCR assay and hybridization with a 32P-labelled probe. Using nested PCR and mass spectrometry, HBV-DNA was detected in none of group I and in all of group II samples. In 11 out of 21 (52%) of the isolated anti-HBc positive sera from group III, HBV-DNA was detected. No correlation was observed between HBV-DNA positivity and anti-HBc titers. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided a fast, sensitive and non-radioactive assay for the detection of PCR products without the need for gel electrophoresis or hybridization with labelled probes.
Jurinke
C
C
Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University of Hamburg, Germany.
Zöllner
B
B
Feucht
H H
HH
van den Boom
D
D
Jacob
A
A
Polywka
S
S
Laufs
R
R
Köster
H
H
D004279:DNA, Viral / Q000097:blood*; D006509:Hepatitis B / Q000821:virology; D006510:Hepatitis B Antibodies / Q000097:blood*; D006512:Hepatitis B Core Antigens / Q000276:immunology; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D004279:DNA, Viral; D006510:Hepatitis B Antibodies; D006512:Hepatitis B Core Antigens