Resistance of Bordetella pertussis to erythromycin has been increasingly reported. We have developed an allele specific PCR method for rapid detection of erythromycin-resistant B. pertussis directly from nasopharyngeal (NP) swabs submitted for diagnostic PCR. Based on the proven association of erythromycin resistance with the A2047G mutation in 23S rRNA of B. pertussis, four primers, two of which were designed to be specific for either wild-type or the mutant allele, were used in two different versions of the allele specific PCR method. The methods were verified according to the results obtained by PCR-based sequencing of 16 recent B. pertussis isolates and 100 NP swabs submitted for diagnostic PCR. The detection limits of the two PCR assays ranged from 10 to 100 fg per reaction for both erythromycin susceptible and resistant B. pertussis. Two amplified fragments of each PCR with the respective sizes of 286-bp and 112-bp were obtained from mutant allele of the isolates and/or NP swabs containing B. pertussis DNAs. For the wild type, only a fragment of 286-bp was visible when the allele specific PCR 1 was performed. No amplification could be found when a number of non-Bordetella bacterial pathogens and NP swabs that did not contain DNAs of B. pertussis were examined. This assay can serve as an alternative for PCR based sequencing, especially for local laboratories in resource-poor countries.
Authors:Wang Z1, Han R2, Liu Y2, Du Q2, Liu J2, Ma C2, Li H2, He Q3, Yan Y4.
Journal:J Clin Microbiol. 2015