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Abstract
Clopidogrel is an antiplatelet medicine used to prevent blood clots in patients who have had a heart attack, stroke, or other symptoms. Variability in the clinical response to clopidogrel treatment has been attributed to genetic factors. In particular, five SNPs of rs4244285, rs4986893, rs12248560, rs662 and rs1045642 have been associated with resistance to clopidogrel therapy in Chinese population. This work involves the development of a multiplex high-resolution melting (HRM) assay to genotype all five of these loci in 2 tubes. Amplicons corresponding to distinct SNPs in a common tube were designed with the aid of uMelt prediction software to have different melting temperatures Tm by addition of a GC-rich tail to the 5′ end of the certain primers. Two kinds of commercial methods, Digital Fluorescence Molecular Hybridization (DFMH) and Sanger sequencing, were used as a control. Three hundred sixteen DFMH pretested samples from consecutive acute coronary syndrome patients were used for a blinded study of multiplex HRM. The sensitivity of HRM was 100% and the specificity was 99.93% reflecting detection of variants other than the known resistance SNPs. Multiplex HRM is an effective closed-tube, highly accurate, fast, and inexpensive method for genotyping the 5 clopidogrel resistance associated SNPs.
Highlights
Clopidogrel is widely used in antiplatelet therapy in patients undergoing percutaneous coronary intervention (PCI) to reduce ischemic complications[1]
The 5 clopidogrel resistance associated SNPs were amplified in duplex and triplex PCR and genotyped by duplex and triplex High-resolution melting (HRM)
The Tm differences between neighboring amplicon melting transitions in the multiplex HRM were from 2–5 °C
Summary
Clopidogrel is widely used in antiplatelet therapy in patients undergoing percutaneous coronary intervention (PCI) to reduce ischemic complications[1]. PCR- restriction fragment length polymorphism (PCR-RFLP) genotyping uses restriction enzymes to digest the PCR product for analysis by gel electrophoresis This is a less expensive than Sanger sequencing for clopidogrel resistance SNP genotyping, but it involves several steps and some are open tube and susceptible to contamination[11]. SPARTAN assay is a kind of point-of-care testing which is based on portable PCR instrument and Taqman fluorescent probe[17,18] It is closed-tube, fast, flexible used, and only buccal swab www.nature.com/scientificreports/. We have developed multiplex HRM to genotype the five clopidogrel resistance associated SNPs in just two reactions, one triplex and one duplex This method is convenient, fast, closed-tube, cost efficient and reliably accurate
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