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.
Newby
D T
DT
Biotechnology Department, Idaho National Engineering and Environmental Laboratory, Idaho Falls, Idaho 83415. Armed Forces Institute of Pathology, Washington, D.C. 20306, USA. newbdt@inel.gov
Hadfield
T L
TL
Roberto
F F
FF
D052160:Benzothiazoles; D002003:Brucella abortus / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D009930:Organic Chemicals; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I