Detection and discrimination of B pertussis and B holmesii by real-time PCR targeting IS481 using a beacon probe and probe-target melting analysis

Molecular and Cellular Probes
S K Poddar

Abstract

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.

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Citations

Jan 7, 2005·Journal of Clinical Microbiology·G MuyldermansS Lauwers
Mar 25, 2006·BMC Infectious Diseases·Laina KnorrJasmine Ahmed-Bentley
Oct 18, 2011·Emerging Infectious Diseases·Roxanne E StrachanUNKNOWN Australian Research Network in Empyema
Apr 12, 2014·The Lancet Infectious Diseases·Laure F PittetKlara M Posfay-Barbe
Mar 16, 2007·Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases·A MénardB de Barbeyrac
Jun 20, 2007·Journal of Medical Microbiology·Benoit VincartOlivier Denis
May 27, 2016·Microbiology Spectrum·Laure F Pittet, Klara M Posfay-Barbe
Sep 22, 2006·Enfermedades infecciosas y microbiología clínica·Jesús García-MartínezJoaquín R Otero

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