Detection of hepatitis B virus DNA in serum samples via nested PCR and MALDI-TOF mass spectrometry.
In a blind study, nested polymerase chain reaction (PCR) was performed with control DNA and DNA preparations from serum samples of six patients. The detection limit was determined to be 100 molecules of template in 1 ml of serum. Hepatitis B virus (HBV) related products of nested PCR were purified by ultrafiltration and immobilisation on streptavidin coated magnetic beads. The immobilized PCR products were denatured from the beads and analyzed via matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. The results of MALDI-TOF MS analysis were in agreement with the results obtained by polyacrylamide gel electrophoresis (PAGE) and with the data obtained by serological analysis. The detection strategy introduced here has a high potential for automation and represents a fast and reliable method of detection for HBV DNA in serum without the need for time consuming gel electrophoresis and labeling or hybridization procedures.
Jurinke
C
C
Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University of Hamburg, Germany.
Zöllner
B
B
Feucht
H H
HH
Jacob
A
A
Kirchhübel
J
J
Lüchow
A
A
van den Boom
D
D
Laufs
R
R
Köster
H
H
D004279:DNA, Viral / Q000032:analysis*; D006509:Hepatitis B / Q000097:blood / Q000821:virology*; D006512:Hepatitis B Core Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D004279:DNA, Viral; D006512:Hepatitis B Core Antigens