Differential detection of B. pertussis from B. parapertussis using a polymerase chain reaction (PCR) in presence of SYBR green1 and amplicon melting analysis.
Nucleic acid (DNA) from control stock strains of B. pertussis and B. parapertussis (B. pertussis strain # 9797 and B. parapertussis strain # 15234 from ATCC) was amplified by polymerase chain reaction (PCR) targeting pertussis toxin (PT) promotor region, in presence of SYBR green1 a dye that fluoresces on binding specifically to double stranded DNA; and fluorescent melting profile of amplicon (amplified DNA) was studied. Amplicon of B. pertussis and B. parapertussis generated distinctly different melting bands with melting temperature (Tm) at 89.8 and 91.7 degrees C, respectively. Melting profile and Tm of each randomly selected isolates of B. pertussis and B. parapertussis was identical to that of respective control strains. Distinct difference in Tm between B. pertussis and B. parapertussis specific amplicons allowed differential detection of the two Bordetella species based on a single PCR product. The amplified product of serial diluted control stock of bacteria was analyzed by both agarose gel electrophoresis and melting profile analysis. The analytical sensitivity of detection (1-10 CFU equivalent in the tested volume) by melting profile and Tm analysis was in agreement with that obtained by agarose gel analysis.
University of California at San Diego (UCSD), Pediatric Pharmacology Research Unit (PPRU), La Jolla, CA 92093-0808, USA. spoddar@ucsd.edu
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D015373:Bacterial Typing Techniques / Q000379:methods*; D052160:Benzothiazoles; D042483:Bordetella parapertussis / Q000145:classification / Q000302:isolation & purification*; D001886:Bordetella pertussis / Q000145:classification / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D003959:Diamines; D005456:Fluorescent Dyes; D009690:Nucleic Acid Conformation; D009930:Organic Chemicals; D044367:Phase Transition; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011804:Quinolines; D013696:Temperature
D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I