PMID: 11311352Apr 20, 2001Paper

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

Journal of Virological Methods
M ZerbiniMonica Musiani

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 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; t...Continue Reading

References

Jun 1, 1990·Molecular and Cellular Probes·J S Sevall
Dec 1, 1994·Journal of Virological Methods·E C KimL J Anderson
Feb 1, 1993·Journal of Virological Methods·G GallinellaM La Placa
Nov 24, 1999·Journal of Clinical Pathology·T RichterM Werner
Nov 14, 2000·Clinica Chimica Acta; International Journal of Clinical Chemistry·M ZerbiniM Musiani

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Citations

Jul 16, 2002·Pathologie-biologie·M ZerbiniM Musiani
May 31, 2002·Journal of Clinical Microbiology·P DalyS Doyle
Jul 15, 2003·Journal of Medical Virology·G GallinellaM Musiani

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