Synthesis of a non-radioactive hepatitis B virus DNA probe from human serum by the polymerase chain reaction.
A method for synthesizing probes for detecting hepatitis B virus DNA in serum was developed. It uses DNA extracted from the serum of an hepatitis B virus carrier as target, and digoxigenin-11-dUTP incorporated in DNA sequences during the polymerase chain reaction as tracer. Using a spot hybridization assay, the sensitivity and specificity of the digoxigenin-labelled DNA probe were compared with two standard hepatitis B virus DNA probes, synthesized with cloned hepatitis B virus DNA and labelled either with digoxigenin or 32P by random priming. Data obtained from the three methods showed an excellent correlation. Thus, hepatitis B virus DNA extracted from human serum and labelled by polymerase chain reaction may be considered a suitable alternative to cloned DNA. It provides reliable probes and eliminates the need for facilities and personnel dedicated to the manipulation of clones. These advantages will allow a wider application of hepatitis B virus DNA testing in clinical practice.
Rodríguez-Frías
F
F
Department of Biochemistry, Hospital General Vall d'Hebron, Barcelona, Spain.
Arranz
J A
JA
Buti
M
M
Esteban
R
R
Jardí
R
R
D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes / Q000032:analysis / Q000138:chemical synthesis*; D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D004587:Electrophoresis, Agar Gel; D006509:Hepatitis B / Q000097:blood* / Q000382:microbiology; D006510:Hepatitis B Antibodies / Q000097:blood; D006513:Hepatitis B e Antigens / Q000097:blood; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; D006510:Hepatitis B Antibodies; D006513:Hepatitis B e Antigens; D007202:Indicators and Reagents; C070202:digoxigenin-11-deoxyuridine triphosphate; D004076:Digoxigenin