Tyramide signal amplification of biotinylated probe in dot-blot hybridization assay for the detection of parvovirus B19 DNA in serum samples.
Highly sensitive assay systems are necessary for large-scale virological screenings. We evaluated the use of tyramide signal amplification (TSA) for biotinylated probe in dot-blot hybridization assay to detect B19 DNA in serum samples. The probe was constructed by PCR and directly labeled with biotin during amplification reaction. The sensitivity of the dot-blot hybridization assay with TSA detection method was evaluated in comparison with a hybridization assay using the direct detection of biotinylated probe by streptavidin-biotin-alkaline phosphatase substrate. The TSA detection was able to detect 1 pg of B19 DNA and proved to be 10-50 times more sensitive than the hybridization assay with the direct detection of biotinylated probe. The analysis of 720 serum samples by TSA detection of biotinylated probe showed that the assay may be a valid diagnostic tool in routine testing of B19 DNA in serum samples.
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
Cricca
M
M
Gentilomi
G
G
Venturoli
S
S
Gallinella
G
G
Musiani
M
M
D001710:Biotin; D019921:Biotinylation; D015342:DNA Probes; D004279:DNA, Viral / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009693:Nucleic Acid Hybridization; D010322:Parvoviridae Infections / Q000175:diagnosis*; D016732:Parvovirus B19, Human / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral; D001710:Biotin