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 × 102 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. © 2001 Elsevier Science B.V.

Zerbini M., Gentilomi G., Cricca M., Manaresi E., Bonvicini F., Musiani M. (2001). A system to enhance the sensitivity of digoxigenin-labelled probe: Detection of B19 DNA in serum samples. JOURNAL OF VIROLOGICAL METHODS, 93(1-2), 137-144 [10.1016/S0166-0934(01)00265-8].

A system to enhance the sensitivity of digoxigenin-labelled probe: Detection of B19 DNA in serum samples

Zerbini M.;Gentilomi G.;Cricca M.;Manaresi E.;Bonvicini F.;
2001

Abstract

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 × 102 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. © 2001 Elsevier Science B.V.
2001
Zerbini M., Gentilomi G., Cricca M., Manaresi E., Bonvicini F., Musiani M. (2001). A system to enhance the sensitivity of digoxigenin-labelled probe: Detection of B19 DNA in serum samples. JOURNAL OF VIROLOGICAL METHODS, 93(1-2), 137-144 [10.1016/S0166-0934(01)00265-8].
Zerbini M.; Gentilomi G.; Cricca M.; Manaresi E.; Bonvicini F.; Musiani M.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/964719
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