Detection and discrimination of B pertussis and B holmesii by real-time PCR targeting IS481 using a beacon probe and probe-target melting analysis.
A beacon probe was designed to detect one of the two documented single nucleotide changes in IS481 target allele of Bordetella holmesii genome as compared to Bordetella pertussis. PCR amplified product targeting a region of IS481 in presence of the probe was subjected to a post-PCR hybridization and melting cycle. Hybrid of the probe with B. pertussis specific target had a different thermal stability than that with allele having the single nucleotide change in B. holmesii. The melting of B. pertussis-probe hybrid occurred in a single phase; while that of B. holmesii-probe hybrid was biphasic-one for allele identical to that in B. pertussis and the other for that with a single nucleotide change in B. holmesii genome, with a difference in melting temperature (T(m)) of 6.5 degrees C. The characteristic melting profile and T(m) analysis was the basis for discriminatory detection of B. pertussis from B. holmesii. The method was applied in a representative set of clinical isolates of B. pertussis and B. holmesii and the result was in agreement with conventional culture method.
Poddar
S K
SK
Pediatric Pharmacology Research Unit (PPRU), University of California at San Diego (UCSD), La jolla, CA 92093-0808, USA. spoddar@ucsd.edu
D001884:Bordetella / Q000235:genetics / Q000302:isolation & purification*; D001886:Bordetella pertussis / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry; D005453:Fluorescence; D009691:Nucleic Acid Denaturation; D044367:Phase Transition; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D013696:Temperature
D015342:DNA Probes; D004269:DNA, Bacterial