Introduction
Clopidogrel is an antiplatelet drug widely used in patients with acute coronary syndrome (ACS), including those with non-ST-segment elevation ACS (unstable angina UA or non-Q wave myocardial infarction) and ST-segment elevation myocardial infarction (NSTEMI)(1-3). Specifically, non-ST-segment elevation ACS includes patients with stent implantation after percutaneous coronary intervention, patients with peripheral arterial disease, and patients with recent myocardial infarction or recent ischemic stroke(4-6). Clopidogrel, a prodrug, has no pharmacological activity(7, 8), but active metabolites are mainly produced by the cation of the CYP2C19 enzyme(9-11). The active metabolites produced irreversibly bind to the P2Y12 receptor on the surface of platelets, inhibiting their aggregation and interfering with ADP-mediated platelet activation for an overall antiplatelet effect(12, 13). Genetic variation of the CYP2C19 gene leads to individual differences in CYP2C19 enzyme activity, resulting in four phenotypes: ultrafast metabolizer (UM), fast metabolizer (EM), intermediate metabolizer (IM) and slow metabolizer (PM)(14). CYP2C19 UM patients treated with conventional doses of clopidogrel generate increased levels of active metabolites, with increased platelet inhibition, enhanced antiplatelet function, and increased risk of bleeding(15). Treatment of CYP2C19 PM patients with conventional doses of clopidogrel results in decreased inhibition of platelets, decreased antiplatelet function, and increased risk of thrombosis(16). CYP2C19 * 2 (rs4244285, c.681G > A) and CYP2C19 * 3 (rs4986893, c.636G > A) are two major alleles in the Chinese population(17) with frequencies of 23.1% -35% and 2% -7%, respectively. The CYP2C19 * 17 (rs12248560, c.-806C > T)-encoded CYP2C19 enzyme displays increased activity, and the frequency of occurrence in the Chinese population is approximately 0.5%-4%(18). In general, methods that detect and distinguish CYP2C19 genotypes should be reliable and rapid, especially when the purpose is for clinical medication guidance, which is related to safety and treatment costs.
Several methods have been developed to detect CYP2C19 gene polymorphisms. However, these methods are almost all performed by highly skilled technicians in well-equipped referral hospitals, and the process is complex and time-consuming. This approach may exclude those who need the medication, which is not helpful for rapid guidance clinically.
Sanger sequencing (referred to as polymerase chain reaction-Sanger sequencing) of PCR-amplified fragments from patient samples is the current ’gold standard’ method for detecting CYP2C19 genotypes(19, 20). It facilitates accurate detection of all nucleotide mutations, including new mutations not previously reported. Nonetheless, Sanger sequencing instrumentation is often unaffordable for most local hospitals. In addition, the sequencing process is complex and time-consuming, requiring very careful operation to avoid contamination by PCR amplification. This is also true for other sequencing-based analyses, such as pyrosequencing and next-generation sequencing, as well as analyses based on high-end instruments, such as DNA microchips and mass spectrometry(21). Several simpler and more cost-effective methods have been developed to detect CYP2C19 genotypes(22). Real-time fluorescent quantitative polymerase chain reaction is a good platform for clinical diagnosis because its closed detection format makes it easy to use and fast and reduces the contamination of amplification products(23, 24), and a real-time amplification polymerase chain reaction (PCR) method to detect CYP2C19 genotype polymorphisms has been established(25, 26). However, due to the limited coverage of multiple detection of mutation sites, it is far from practical.
In this study, we developed a detection strategy based on PCR amplification combined with melting curve analysis that uses a combination of adjacent probes and TaqMan probes for amplification. This strategy can detect 9 genotypes of CYP2C19 in a single tube. Here, we describe a new rapid PCR combined with melting curve analysis method for rapid (within 1 h) detection of CYP2C19 * 2/3/17 sites in one tube. We named this method Rapid PCR Melting Curve Method (RPCR-MC). We systematically evaluated its analytical performance, including mutation detection accuracy, analytical sensitivity, specificity and detection, in clinical samples. We tested the RPCR-MC method by analyzing samples from 93 patients with a high risk of thrombosis after percutaneous coronary intervention(PCI) and compared the results with those of Sanger sequencing.