Fluorometric Detection of B. pertussis by Polymerse Chain Reaction (PCR) and Molecular Beacon Probe

摘要

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 (1S481) of the genome of B. pertussis was amplified in presence of the probe. The probe sequence was 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 allowed the detection of a B. pertussis target, requiring no additional steps. Presence of B. pertussis in a sample was also examined by agarose gel electrophoresis following PCR. A serial diluted stock of ATCC strain number 9797 of B. pertussis 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. 0 1- 0.1 CFU in the tested volume of the sample and it was equal to or better than that detected by agarose gel analysis.