PCR-DIG ELISA with biotinylated primers is unsuitable for use in whole blood samples from patients with brucellosis.
In an attempt to avoid some of the inconveniences associated with conventional PCR, such as electrophoresis in ethidium bromide, we developed and analyzed the yield of a digoxigenin-based enzyme-linked immunosorbent PCR assay (PCR-DIG ELISA) for the detection of specific Brucella target DNA. During the DNA amplification process in healthy subjects and controls (Brucella abortus B-19) non-specific amplification of fragments was formed between genomic DNA and specific biotin-labeled primers. The labeled non-specific fragments bound to streptavidin-coated wells, saturating the solid phase streptavidin by biotin-streptavidin interaction. The formation of these non-specific PCR products was demonstrated by reduction in absorbance with hemin, a Taq polymerase inhibitor, and identified by use of a silver stained method which improves the sensitivity of nucleic acid visualization.
Queipo-Ortuño
Maria Isabel
MI
Department of Biochemistry and Molecular Biology, Facultad de Medicina, Universidad de Málaga, Málaga 29071, Spain.
García-Ordoñez
Miguel Angel
MA
Gil
Ramón
R
Rojas
José
J
de Dios Colmenero
Juan
J
Morata
Pilar
P
D001710:Biotin; D002003:Brucella abortus / Q000235:genetics*; D002006:Brucellosis / Q000097:blood / Q000175:diagnosis*; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D016622:Silver Staining; D019914:Taq Polymerase / Q000737:chemistry
D001710:Biotin; D019914:Taq Polymerase