Rapid detection of Bordetella pertussis by real-time PCR using SYBR green I and a LightCycler instrument.
A polymerase chain reaction (PCR) assay in real-time for detection of B. pertussis using SYBR green I as the reporter fluorophore and LightCycler instrument (a thermocycler coupled to a fluorescence detection device) was established and evaluated. The amplified amplicon using series diluted control prototype strain (ATCC strain #9797) of B. pertussis was analyzed for the fluorescent melting profile, and melting temperature (Tm) was determined. When examined, amplicons using a representative set of clinical isolates of B. pertussis were found to have the same Tm value (86 +/- 0.5 degrees C, the specificity parameter of detection) as the control prototype strain as expected. Amplified product was also analyzed and detected by agarose gel electrophoresis. The detection limit by fluorescent profile and Tm analysis was 10-fold better than that detected by agarose gel analysis.
Poddar
S K
SK
Pediatric Pharmacology Research Unit, University of California at San Diego, La Jolla 92093-0808, USA. spoddar@ucsd.edu
D052160:Benzothiazoles; D001886:Bordetella pertussis / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics; D003959:Diamines; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; C098022:SYBR Green I