Polymerase chain reaction for synthesis of digoxigenin-labelled DNA probe: application to parvovirus B19 and to polyomavirus BK.
A one-step polymerase chain reaction (PCR) was used to synthesize a digoxigenin-labelled probe, 176 bp long, for the detection of human polyomavirus BK (BKV). A 104 bp-long digoxigenin-labelled probe was generated by 'nested' PCR for the detection of human parvovirus B19 (virus B19). In both cases the whole viral genome was used as template. Different amounts of template as well as different percentages of dTTP substituted by digoxigenin-dUTP (dig-dUTP) in the reaction mixture were employed in order to determine the optimum conditions for the labelled probe synthesis. The sensitivity and the specificity of these PCR-produced probes, together with the simplicity and the reduced time scale of the procedure, suggest the potential of this technique as an additional method for preparing non-radioactive molecular probes for routine diagnosis of viral infections.
Istituto di Microbiologia, Università di Firenze, Italy.
K
K
Zakrzewska
S
S
Ciappi
A
A
Azzi
D001739:BK Virus / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D015342:DNA Probes / Q000138:chemical synthesis*; D004076:Digoxigenin; D006801:Humans; D008969:Molecular Sequence Data; D010322:Parvoviridae Infections / Q000175:diagnosis / Q000382:microbiology; D016732:Parvovirus B19, Human / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D013997:Time Factors; D014412:Tumor Virus Infections / Q000175:diagnosis / Q000382:microbiology
D015342:DNA Probes; D004076:Digoxigenin