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Aac(6')-Ib-cr genotyping by simultaneous high-resolution melting analyses of an unlabeled probe and full-length amplicon.

ABSTRACT: We have developed a time- and cost-efficient one-step closed-tube assay for genotyping of aac(6')-Ib-cr that is capable of distinguishing between the two genetic aac(6')-Ib-cr variants. Our genotyping assay uses the combined information of simultaneously acquired high-resolution melting data from an unlabeled probe and the full-length amplicon.

SUBMITTER: Bell JM

PROVIDER: S-EPMC2825968 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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aac(6')-Ib-cr genotyping by simultaneous high-resolution melting analyses of an unlabeled probe and full-length amplicon.

Bell Jan M JM Turnidge John D JD Andersson Patiyan P

Antimicrobial agents and chemotherapy 20091214 3

We have developed a time- and cost-efficient one-step closed-tube assay for genotyping of aac(6')-Ib-cr that is capable of distinguishing between the two genetic aac(6')-Ib-cr variants. Our genotyping assay uses the combined information of simultaneously acquired high-resolution melting data from an unlabeled probe and the full-length amplicon. ...[more]

PMID: 20008777

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Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting.

Project description:Amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, or allele-specific polymerase chain reaction. However, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood, amplicon Tm differences of 0.03 to 0.39 degrees C attributable to the extractions were observed. To correct for solution chemistry differences between samples, complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. High- and low-temperature controls bracketing the amplicon melting region decreased the Tm SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included, whereas a 3% error rate was observed without temperature correction. In conclusion, internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments.

| S-EPMC1899416 | biostudies-literature

RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting analysis.

Project description:Single bp mutations in the RET proto-oncogene can cause multiple endocrine neoplasia type 2 syndromes. The conventional approach for genotyping RET mutations is sequencing the exons. A closed-tube RET genotyping assay using a saturating DNA dye, unlabeled probes, and amplicon high-resolution melting analysis was developed. The method required two sequential polymerase chain reaction stages, a primary and secondary assay. The primary assay analyzed RET exons 10, 11, 13, 14, and 16 with a total of seven reactions using eight unlabeled probes. The primary assay genotyped wild-type exons, a common exon 13 polymorphism, and an exon 16 mutation, whereas other RET sequence variation was detected. The primary unlabeled probe data limited the possible genotypes for the detected RET sequence variation, which permitted genotyping in a secondary assay with only two to five reactions. Six probes were designed with the masking technique and masked selected sequence variations to allow unambiguous analysis of other mutations elsewhere under the probe. After this two-stage RET genotyping assay, less than 0.2% of exons tested would require sequencing for genotype. A blinded study generated from five wild type and 29 available RET sequence variation samples was 100% concordant with sequencing. Amplicon high-resolution melting analysis with unlabeled probes and the masking technique is a fast, accurate method for genotyping the >50 RET sequence variations.

| S-EPMC1867443 | biostudies-literature

Mechanistic and structural analysis of aminoglycoside N-acetyltransferase AAC(6')-Ib and its bifunctional, fluoroquinolone-active AAC(6')-Ib-cr variant.

Project description:Enzymatic modification of aminoglycoside antibiotics mediated by regioselective aminoglycoside N-acetyltransferases is the predominant cause of bacterial resistance to aminoglycosides. A recently discovered bifunctional aminoglycoside acetyltransferase (AAC(6')-Ib variant, AAC(6')-Ib-cr) has been shown to catalyze the acetylation of fluoroquinolones as well as aminoglycosides. We have expressed and purified AAC(6')-Ib-wt and its bifunctional variant AAC(6')-Ib-cr in Escherichia coli and characterized their kinetic and chemical mechanism. Initial velocity and dead-end inhibition studies support an ordered sequential mechanism for the enzyme(s). The three-dimensional structure of AAC(6')-Ib-wt was determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 A. Observation of the direct, and optimally positioned, interaction between the 6'-NH 2 and Asp115 suggests that Asp115 acts as a general base to accept a proton in the reaction. The structure of AAC(6')-Ib-wt permits the construction of a molecular model of the interactions of fluoroquinolones with the AAC(6')-Ib-cr variant. The model suggests that a major contribution to the fluoroquinolone acetylation activity comes from the Asp179Tyr mutation, where Tyr179 makes pi-stacking interactions with the quinolone ring facilitating quinolone binding. The model also suggests that fluoroquinolones and aminoglycosides have different binding modes. On the basis of kinetic properties, the pH dependence of the kinetic parameters, and structural information, we propose an acid/base-assisted reaction catalyzed by AAC(6')-Ib-wt and the AAC(6')-Ib-cr variant involving a ternary complex.

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Characterization of aberrant melting peaks in unlabeled probe assays.

Project description:An unlabeled probe assay relies on a double-stranded DNA-binding dye to detect and verify target based on amplicon and probe melting. During the development and application of unlabeled probe assays, aberrant melting peaks are sometimes observed that may interfere with assay interpretation. In this report, we investigated the origin of aberrant melting profiles observed in an unlabeled probe assay for exon 10 of the RET gene. It was determined that incomplete 3' blocking of the unlabeled probe allowed polymerase-mediated probe extension resulting in extension products that generated the aberrant melting profiles. This report further examined the blocking ability of the 3' modifications C3 spacer, amino-modified C6, phosphate, inverted dT, and single 3' nucleotide mismatches in unlabeled probe experiments. Although no 3' blocking modifications in these experiments were 100% effective, the amino-modified C6, inverted dT, and C3 spacer provided the best blocking efficiencies (1% or less unblocked), phosphate was not as effective of a block (up to 2% unblocked), and single nucleotide mismatches should be avoided as a 3' blocking modification.

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Characterization of Tn6238 with a new allele of aac(6')-Ib-cr.

Project description:Here, we report that the genetic structure of Tn1331 remained conserved in Argentina from 1989 to 2013 (72 of 73 isolates), with the exception being the plasmid-borne Tn1331-like transposon Tn6238 containing a new aac(6')-Ib-cr allele recovered from a colistin-resistant Klebsiella pneumoniae clinical isolate. A bioinformatic analysis of aac(6')-Ib-like gene cassettes suggests that this new aac(6')-Ib-cr allele emerged through mutation or homologous recombination in the Tn1331 genetic platform. Tn6238 is a novel platform for the dissemination of aminoglycoside and fluoroquinolone resistance determinants.

| S-EPMC4394824 | biostudies-literature

Expression of the aac(6')-Ib-cr Gene in Class 1 Integrons.

Project description:aac(6')-Ib-cr is a plasmid-mediated quinolone resistance gene embedded within a gene cassette, most often within an integron. It confers resistance to quinolones and aminoglycosides. We investigated the role of a 101-bp fragment frequently present upstream of the aac(6')-Ib-cr gene cassette and found that it contributes to the expression of aac(6')-Ib-cr and provides an alternative start codon, confirming the length of the AAC(6')-Ib-cr protein to 199 amino acids.

| S-EPMC5404527 | biostudies-literature

Detection of aac(6')-Ib-cr in avian pathogenic Escherichia coli isolates in Japan.

Project description:We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in avian pathogenic Escherichia coli (APEC) strains in Japan. A total of 117 APEC strains collected between 2004 and 2007 were examined for PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, qepA and oqxAB) by polymerase chain reaction. None of the APEC strains carried qnrA, qnrB, qnrC, qnrD, qnrS, qepA or oqxAB, but one of the isolates was identified as an AAC (6')-Ib-cr producer. Phylogenetic grouping, multi-locus sequence typing and serotyping showed that this isolate belonged to phylogenetic group A, sequence type 167 and untypable serogroup. To our knowledge, this is the first report of the aac (6')-Ib-cr gene in bacteria from food-producing animals in Japan.

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First report of the aac(6')-Ib-cr gene in Providencia stuartii isolates in Brazil.

Project description:IntroductionThe aac(6')-Ib-cr and bla KPC genes are spreading among Enterobacteriaceae species, including Providencia stuartii, in some countries of world.MethodsThese genes were investigated in 28 P. stuartii isolates from a public hospital in Recife, Pernambuco, Brazil, by PCR and sequencing.ResultsThe aac(6')-Ib-cr gene was detected in 16 resistant isolates, and the bla KPC gene was seen in 14.ConclusionsThe presence of these genes in P. stuartii multi- and extensively drug-resistant isolates indicates that the resistance arsenal of this species is increasing, thus limiting the therapeutic options.

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Rapid diagnosis of MEN2B using unlabeled probe melting analysis and the LightCycler 480 instrument.

Project description:Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant, inherited cancer syndrome. MEN2B patients have a high risk of developing medullary thyroid carcinoma, and prophylactic thyroidectomy is recommended by 6 months of age. Genetic testing can identify MEN2B patients before cancer progression. Two RET proto-oncogene mutations, in exon 15 at codon 883 (GCT>TTT) and in exon 16 at codon 918 (ATG>ACG), account for more than 98% of MEN2B cases. An assay using unlabeled probes and the LightCycler 480 instrument was developed to genotype these two common MEN2B RET mutations. Asymmetric polymerase chain reaction was used to increase ssDNA products followed by melting analysis of the unlabeled probe/ssDNA amplicon duplex. The available samples were either patient DNA of known RET genotype or artificial templates. Analysis of the codon 883 heterozygous mutation demonstrated a DeltaT(m) of 5.70 +/- 0.11 degrees C, while the codon 918 heterozygous mutation generated a DeltaT(m) of -5.72 +/- 0.11 degrees C. Samples with the targeted RET mutation genotypes were accurately detected and easily distinguishable from five other reported sequence changes using these probes. Thus, MEN2B diagnosis using unlabeled probes and the LightCycler 480 is a rapid, closed-tube method that is less time consuming and less expensive than sequencing. This assay demonstrates 100% specificity and sensitivity for the identification of RET mutations causative of MEN2B.

| S-EPMC2259465 | biostudies-literature

PMQR genes oqxAB and aac(6')Ib-cr accelerate the development of fluoroquinolone resistance in Salmonella typhimurium.

Project description:Emergence of multidrug-resistant Salmonella typhimurium strains, especially the ACSSuT and nalidixic acid R types, has significantly compromised the effectiveness of current strategies to control Salmonella infections, resulting in increased morbidity and mortality. Clinical S. typhimurium isolates recovered in Hong Kong during the period of 2005-2011 were increasingly resistant to ciprofloxacin (CIP) and antibiotics of the ACSSuT group. Our data revealed that oqxAB and aac(6')Ib-cr were encoded on plasmids of various sizes and the presence of these two elements together with a single gyrA mutation in S. typhimurium were sufficient to mediate resistance to CIP. Acquisition of the oqxAB and aac(6')Ib-cr encoding plasmids by S. typhimurium caused a fourfold increase in CIP minimal inhibitory concentration. Furthermore, the presence of oqxAB and aac(6')Ib-cr in Salmonella dramatically increased the mutation prevention concentration of CIP which may due to mutational changes in the drug target genes. In conclusion, possession of oqxAB and aac(6')Ib-cr encoding plasmid facilitate the selection of CIP resistant S. typhimurium, thereby causing a remarkable increase of CIP resistance among clinical Salmonella strains in Hong Kong.

| S-EPMC4183184 | biostudies-literature

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