Detection of the A2058G and A2059G 23S rRNA Gene Point Mutations Associated with Azithromycin Resistance in Treponema pallidum by Use of a TaqMan Real-Time Multiplex PCR Assay
| Autor: | Kai-Hua Chi, John R. Su, Cheng-Yen Chen, Eli Nachamkin, Ronald C. Ballard, Allan Pillay |
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| Rok vydání: | 2013 |
| Předmět: |
Microbiology (medical)
Genotype Microbial Sensitivity Tests Azithromycin Biology Real-Time Polymerase Chain Reaction Drug Resistance Bacterial Multiplex polymerase chain reaction TaqMan Humans Point Mutation Treponema pallidum Treponema Point mutation Genes rRNA Bacteriology biology.organism_classification Virology Molecular biology United States Anti-Bacterial Agents RNA Ribosomal 23S Restriction enzyme Restriction fragment length polymorphism Multiplex Polymerase Chain Reaction Nested polymerase chain reaction |
| Zdroj: | Journal of Clinical Microbiology. 51:908-913 |
| ISSN: | 1098-660X 0095-1137 |
| Popis: | Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum . |
| Databáze: | OpenAIRE |
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