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Rapid screening for targeted genetic variants via high-resolution melting curve analysis.

ABSTRACT: Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5.Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (Tm). A standardized software algorithm was used to assign amplicons as 'reference' or 'variant' as compared to duplicate reference sequence DNA controls for each SNP.Intra-assay (n=5) precision of Tms for all SNPs was ?0.19%, while inter-assay (n=20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/?L.The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of 'reference' or 'variant' available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.

SUBMITTER: Chambliss AB

PROVIDER: S-EPMC5550898 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Rapid screening for targeted genetic variants via high-resolution melting curve analysis.

Chambliss Allison B AB Resnick Molly M Petrides Athena K AK Clarke William A WA Marzinke Mark A MA

Clinical chemistry and laboratory medicine 20170301 4

<h4>Background</h4>Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5.<h4>Methods</h4> ...[more]

PMID: 27732553

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Project description:The emergence of Mycobacterium abscessus complex (MABC) infection is the most noteworthy health care problem. Clarithromycin (CLA) and amikacin (AMK) constitute the cornerstone of treatment for patients infected with MABC; thus, early detection of resistance to these two drugs is essential for formulating effective therapeutic regimens. In the present study, we aimed to validate the use of MeltPro MAB assay, a melting curve analysis with dually labeled probes, on a set of clinical isolates to detect CLA and AMK resistance. A total of 103 clinical MABC strains were collected in our analysis, including 76 strains of M. abscessus subsp. Abscessus (MAA) and 27 strains of M. abscessus subsp. Massiliense (MAM). In vitro susceptibility testing revealed that two isolates exhibited intrinsic CLA resistance by harboring A2270T mutation in rrl, and inducible resistance was noted in 42 isolates. Additionally, two MAA isolates with erm(41)T28 genotype were susceptible to CLA. Notably, we found three out of 44 isolates had two melting curve peaks, representing the simultaneous presence of mutant and the wild type in these specimens. In contrast, no known mutations were identified in six AMK-resistant isolates. Further analysis revealed that MeltPro yielded 100% and 96.67% sensitivity and specificity for detecting CLA resistance. In summary, this study firstly demonstrates that MeltPro is a promising diagnostic for early detection of CLA resistance for MABC isolates, which significantly improves the turnaround time within 2 h. Approximate two fifths of MABC isolates are resistant to CLA by 23S rRNA mutation or its methylation, emphasizing the urgent need for early detection of CLA resistance prior to empirical treatment of MABC infections. IMPORTANCE Mycobacterium abscessus complex (MABC) has attracted increasing attention due to the numerous cases of infection. This pathogen is notorious for its intrinsic drug resistance, which complicates clinical management of patients with MABC infections. Clarithromycin (CLA) and amikacin (AMK) are the cornerstone of treatment regimens for MABC. Herein, our data firstly demonstrates that MeltPro is a promising diagnostic for early detection of CLA resistance for MABC isolates. The high frequency of CLA-resistant MABC isolates in China emphasizes the urgent need for early detection of CLA resistance prior to empirical treatment of MABC infections.

Dataset Information

Rapid screening for targeted genetic variants via high-resolution melting curve analysis.

Publications

Rapid screening for targeted genetic variants via high-resolution melting curve analysis.

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