Aac(6)-Ib-cr genotyping by simultaneous high-resolution melting analysis of an unlabelled probe and full length amplicon
Abstract number: P754
Objectives: There are several variants of the gene encoding aminoglycoside acetyltransferase aac(6')-Ib, and of greatest clinical concern is the aac(6')-Ib-cr variant which extends the enzyme targets to include fluoroquinolones in addition to aminoglycosides. The aac(6')-Ib-cr variant is characterised by amino acid changes at codon 102 (Trp®Arg) and codon 179 (Asp®Tyr). There are two described aac(6')-Ib-cr variants which differ from the wild type aac(6')-Ib (wt) at nucleotide 304 by a T®C (cr-C) or T®A (cr-A) change. Both nucleotide changes result in a Trp®Arg amino acid substitution. We sought to design a rapid and simple assay able to distinguish between the three known alleles found in the aac(6')-Ib gene at codon 102.
Methods: A 58bp fragment was amplified using asymmetrical primer concentrations in the presence of a 25bp unlabelled probe with a perfect match to the wt allele. The combined information from simultaneously acquired high resolution melting data of the full length amplicon and the unlabelled probe, allows unambiguous genotyping, even of discrete T®A nucleotide changes.
Results: Two melting events were observed: one around 6575°C, describing the probe disassociation and one around 7984°C, describing whole amplicon melting (see figure). In brief, the probe and amplicon melting points were: 72.0°C and 81.9°C for wt; 69.7°C and 82.7°C for cr-C; 68.8°C and 81.7°C for cr-A. Heterozygote isolates containing both wt and cr alleles showed a distinct shoulder on the amplicon melting peak, due to the melting of heteroduplex fragments. The probe melting event for heterozygotes showed two distinctive peaks representative of the two individual allele probe peaks.
The assay was validated using 211 isolates with known aac(6')-Ib status and genotype, and further tested on 732 isolates with unknown aac(6')-Ib status and genotype. The assay performed reliably in all tests.
Conclusion: We have developed a one-step closed-tube assay for the detection and genotyping of aac(6')-Ib-cr, capable of differentiating between the two genetic variants responsible for the aac(6')-Ib-cr phenotype, which from an epidemiological perspective is of importance. The assay is rapid at <2 hours, costs less than US $1 per isolate and addresses the need of an efficient detection system for the monitoring of the growing prevalence of clinically significant aac-Ib-cr variants.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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