Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays
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.
- Research Organization:
- Idaho National Lab. (INL), Idaho Falls, ID (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- DE-AC07-99ID-13727
- OSTI ID:
- 911981
- Report Number(s):
- INEEL/JOU-02-01572; AEMIDF; TRN: US200801%%426
- Journal Information:
- Applied and Environmental Microbiology, Vol. 69, Issue 8; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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