Bordetella pertussis detection by spectrofluorometry using polymerase chain reaction (PCR) and a molecular beacon probe

Bordetella pertussis was detected by spectrofluorometry following PCR incor porating a molecular beacon probe in the reaction. A DNA fragment from the tandem repeat sequence region (IS481) of the genome of B. pertussis was amp lified in presence of the probe complementary to an internal segment of the amplified DNA fragment. Fluorescein (FAM) and DABCYL were used as the fluo rophore and quencher in the probe. The probe was characterized for its sign al to noise ratio by homogeneous solution hybridization with a complementar y 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 e xamined by agarose gel electrophoresis of the PCR product, h serial diluted stock of B. pertussis (ATCC strain #9797) and fourteen clinical isolates o f B. pertussis were examined. The sensitivity of detection by fluorescent m easurement was found to be at least in the range of 0.01-0.1 CFU per 10 mul of the sample and was equal to or better than that detected by agarose gel analysis. (C) 2001 Academic Press.