A system to enhance the sensitivity of digoxigenin-labelled probe: detection of B19 DNA in serum samples.
A highly sensitive dot-blot hybridisation assay for the routine screening of numerous samples is described, using parvovirus B19 as a model. Digoxigenin-labelled B19 DNA probe was constructed by PCR, hybrids were detected by an anti-digoxigenin monoclonal antibody followed by a second step, using anti-mouse antibodies conjugated to an alkaline phosphatase-dextran complex (EnVision, Dako) was carried out. The sensitivity of the assay was evaluated using both colourimetric and chemiluminescent substrates for the alkaline phosphatase and was compared with a dot-blot hybridisation assay using the digoxigenin-labelled probe and a standard detection system. With the colourimetric substrate, the EnVision system was able to detect 10 fg of B19 DNA, while with the chemiluminescent substrate the sensitivity increased by up to 2 fg (6 x 10(2) genome copies). This detection system was shown to increase the sensitivity of the assay compared to the standard colourimetric visualisation for the digoxigenin-labelled probe, which could detect 0.1 pg. On account of its sensitivity and specificity the dot-blot hybridisation assay together with the chemiluminescent substrate for the EnVision detection system was used to analyse 760 serum samples; the same sera were tested for B19 DNA with the standard colourimetric visualisation for the digoxigenin-labelled probe used routinely in the diagnostic laboratory.
Zerbini
M
M
Department of Clinical and Experimental Medicine, Division of Microbiology, Osp. S. Orsola, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy. mzerbini@med.unibo.it
Gentilomi
G
G
Cricca
M
M
Manaresi
E
E
Bonvicini
F
F
Musiani
M
M
D015342:DNA Probes; D004279:DNA, Viral / Q000097:blood*; D004076:Digoxigenin; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010322:Parvoviridae Infections / Q000097:blood; D017992:Parvovirus / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral; D004076:Digoxigenin