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Applied and Environmental Microbiology, August 2003, p. 4753-4759, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4753-4759.2003

Real-Time PCR Detection of Brucella abortus: a Comparative Study of SYBR Green I, 5'-Exonuclease, and Hybridization Probe Assays

D. T. Newby,1* T. L. Hadfield,2 and F. F. Roberto1

Biotechnology Department, Idaho National Engineering and Environmental Laboratory, Idaho Falls, Idaho 83415,1 Armed Forces Institute of Pathology, Washington, D.C. 203062

Received 11 December 2002/ Accepted 8 May 2003

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.

* Corresponding author. Mailing address: Idaho National Engineering and Environmental Laboratory, Biotechnology Department, P.O. Box 1625, Idaho Falls, ID 83415-2203. Phone: (208) 526-7779. Fax: (208) 526-0828. E-mail: newbdt{at}inel.gov.

Applied and Environmental Microbiology, August 2003, p. 4753-4759, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4753-4759.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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