Bordetella pertussis detection by spectrofluorometry using polymerase chain reaction (PCR) and a molecular beacon probe.
Bordetella pertussis was detected by spectrofluorometry following PCR incorporating a molecular beacon probe in the reaction. A DNA fragment from the tandem repeat sequence region (IS 481) of the genome of B. pertussis was amplified in presence of the probe complementary to an internal segment of the amplified DNA fragment. Fluorescein (FAM) and DABCYL were used as the fluorophore and quencher in the probe. The probe was characterized for its signal to noise ratio by homogeneous solution hybridization with a complementary oligonucleotide. Measurement of fluorescent signal at the emission maxima of FAM, immediately after a PCR was used to detect the B. pertussis target, with no additional steps. Presence of B. pertussis in a sample was also examined by agarose gel electrophoresis of the PCR product. A serial diluted stock of B. pertussis (ATCC strain #9797) and fourteen clinical isolates of B. pertussis were examined. The sensitivity of detection by fluorescent measurement was found to be at least in the range of 0.01-0.1 CFU per 10 microl of the sample and was equal to or better than that detected by agarose gel analysis.
Poddar
S K
SK
DoD Center for Deployment Health Research, Naval Health Research Center, San Diego, CA 92186-5122, USA. poddar@nhrc.navy.mil
Le
C T
CT
D001886:Bordetella pertussis / Q000235:genetics* / Q000472:pathogenicity*; D004587:Electrophoresis, Agar Gel; D019793:Fluorescein / Q000378:metabolism; D006801:Humans; D015335:Molecular Probes; D015345:Oligonucleotide Probes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D015335:Molecular Probes; D015345:Oligonucleotide Probes; D019793:Fluorescein