Quantitation of parvovirus B19 DNA sequences by competitive PCR: differential hybridization of the amplicons and immunoenzymatic detection on microplate

Molecular and Cellular Probes
G GallinellaE Manaresi

Abstract

A competitive PCR assay was developed to quantify B19 DNA sequences. Target and internal standard sequences were co-amplified by the same set of primers. The internal standard competitor was constructed by recombinant PCR and differed from the original genome sequence in a 21-bp mutagenized fragment, internal to the region amplified by the same set of primers. The internal standard competitor was cloned in a plasmid vector and the cloned fragment used in all the experiments. Target and internal standard sequences were labelled with digoxigenin during the co-amplification reaction and the different amplicons were detected in two separate hybridization reactions by biotinylated probes specific for the original 21-bp sequence or the mutagenized one. Hybridized amplicons were captured onto streptavidin-oated microtitre wells and detected by anti-digoxigenin antibodies conjugated to peroxidase. The chromogenic reaction for peroxidase was quantitatively evaluated by optical density determination. The titration curve subsequently developed showed a linear relationship over the range 10(2) to 10(5) genome copies, thus obtaining an exact quantitative evaluation over a wide range together with good sensitivity. Nine reference serum sampl...Continue Reading

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Citations

Jul 16, 2002·Pathologie-biologie·M ZerbiniM Musiani
Jul 15, 2003·Journal of Medical Virology·G GallinellaM Musiani
Sep 8, 2004·Journal of Virological Methods·T RenaultC Lipart
May 6, 2004·Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology·F BonviciniM Zerbini
Jun 13, 2006·Clinica Chimica Acta; International Journal of Clinical Chemistry·Dimitri PeterlanaClaudio Lunardi
May 7, 2002·Journal of Medical Virology·Elisabetta ManaresiMonica Musiani
Aug 25, 1999·Journal of Medical Virology·M ZerbiniS Venturoli
Mar 8, 2005·The Pediatric Infectious Disease Journal·Sean A McGheeE Richard Stiehm
Nov 4, 2016·Clinical Microbiology Reviews·Jianming QiuNeal S Young
Jun 2, 2000·Journal of Clinical Microbiology·M Clementi

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