PCR-DIG ELISA with biotinylated primers is unsuitable for use in whole blood samples from patients with brucellosis

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• 作者：Queipo-Ortu&ntilde ; o ; Maria Isabel ; Garcí ; a-Ordo&ntilde ; ez ; Miguel Angel ; Gil ; Ramó ; n ; et. al.
• 刊名：Molecular and Cellular Probes
• 出版年：2004
• 出版时间：August, 2004
• 年：2004
• 卷：18
• 期：4
• 页码：243-250
• 全文大小：399 K

文摘

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.