Applied and Environmental Microbiology, 2003-08, Vol.69 (8), p.4753-4759
2003
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- Autor(en) / Beteiligte
- Titel
- Real-time PCR detection of Brucella abortus: a comparative study of SYBR Green I, 5'-exonuclease, and hybridization probe assays
- Ist Teil von
- Applied and Environmental Microbiology, 2003-08, Vol.69 (8), p.4753-4759
- Ort / Verlag
- Washington, DC: American Society for Microbiology
- Erscheinungsjahr
- 2003
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches--SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)--were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 0099-2240eISSN: 1098-5336DOI: 10.1128/AEM.69.8.4753-4759.2003Titel-ID: cdi_highwire_asm_aem_69_8_4753
- Format
- –
- Schlagworte
- AMPLIFICATION, animal pathogenic bacteria, Bacteria, Bacteriological methods and techniques used in bacteriology, Bacteriology, Benzothiazoles, Biological and medical sciences, BRUCELLA, brucella abortus, Brucella abortus - genetics, Brucella abortus - isolation & purification, Brucella melitensis biovar Abortus, brucellosis, DETECTION, Diamines, disease diagnosis, DNA, DNA primers, DNA probes, dyes, exodeoxyribonucleases, exonuclease, FLUORESCENCE, fluorescence resonance energy transfer, Fluorescent Dyes - metabolism, Fundamental and applied biological sciences. Psychology, GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE, GENES, genetic techniques and protocols, HYBRIDIZATION, hybridization probe assay, hybridization probes, Livestock, Methods, microbial detection, Microbiology, MONITORING, Nucleic Acid Hybridization, Organic Chemicals, Pathogens, Phosphodiesterase I, Phosphoric Diester Hydrolases - metabolism, PHOSPHORS, polymerase chain reaction, Polymerase Chain Reaction - methods, PROBES, Quinolines, Real-time PCR, RESONANCE, SENSITIVITY, Sensitivity and Specificity, SPECIFICITY, SYBR Green I, TARGETS