Quantitation of parvovirus B19 DNA sequences by competitive PCR: differential hybridization of the amplicons and immunoenzymatic detection on microplate.
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 samples positive for B19 DNA and eight negative serum samples were tested by the competitive PCR assay for the quantitation of B19 DNA sequences.
Gallinella
G
G
Department of Clinical and Experimental Medicine, University of Bologna, Italy.
Zerbini
M
M
Musiani
M
M
Venturoli
S
S
Gentilomi
G
G
Manaresi
E
E
D003001:Cloning, Molecular; D017931:DNA Primers; D015342:DNA Probes; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D007124:Immunoenzyme Techniques; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D016732:Parvovirus B19, Human / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D004274:DNA, Recombinant; D004279:DNA, Viral