The sensitive detection of fluorescently labelled PCR products using an automated detection system. 1995

M Maher, and D Dowdall, and M Glennon, and S Walshe, and M Cormican, and P Wiesner, and F Gannon, and T Smith
National Diagnostics Centre, BioResearch Ireland, University College Galway, Ireland.

The polymerase chain reaction plays a central role in many detection assays and methods to improve the sensitivity and specificity of these detection systems are constantly being explored. In this study we investigated the use of an automated laser fluorescent system (ALF) in the context of DNA-based diagnostics for pathogenic bacteria. PCR products were generated using species-specific primer sets, one of which was labelled with a 5' fluorescein. PCR products with a fluorescent label were detected on line with an ALF DNA sequencer and the sensitivity of detection was found to be comparable to that for DNA probe hybridization with a radioactive probe. The technology was successfully applied to the detection of Mycobacterium tuberculosis supplemented into sputum samples and to the detection of listeria in paraffin-embedded tissue samples.

UI MeSH Term Description Entries
D008087 Listeria A genus of bacteria which may be found in the feces of animals and man, on vegetation, and in silage. Its species are parasitic on cold-blooded and warm-blooded animals, including man.
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009169 Mycobacterium tuberculosis A species of gram-positive, aerobic bacteria that produces TUBERCULOSIS in humans, other primates, CATTLE; DOGS; and some other animals which have contact with humans. Growth tends to be in serpentine, cordlike masses in which the bacilli show a parallel orientation. Mycobacterium tuberculosis H37Rv
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D013183 Sputum Material coughed up from the lungs and expectorated via the mouth. It contains MUCUS, cellular debris, and microorganisms. It may also contain blood or pus. Sputum, Induced,Induced Sputum,Induced Sputums,Sputums,Sputums, Induced
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain

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