Discussion
We developed a method for rapid identification of CYP2C19 * 2/3/17 polymorphisms and named it RPCR-MC. With the continuous progress medical science development, many drugs are reported in the world but later removed from research because of adverse reactions(27). In general, drug efficacy and adverse reactions are related to ethnic and individual differences associated with genetic differences between people(28). Single-nucleotide polymorphism involves a difference in one base(29). There are several kinds of genome sequence differences, with SNPs being the most common, which is of great clinical significance. When patients with the same disease take the same drugs, some may have different reactions, and drugs are divided into significantly effective, effective, and ineffective. In clinical treatment, doctors usually cannot determine which drugs are most effective according to the patient’s symptoms and must rely on average statistical results of clinical research data and personal experience for decision-making. Moreover, patients with the same symptom or disease may have different treatment outcomes, even with the same drug, and this difference can be predicted by SNP detection. An SNP may alter the amino acid sequence of the expressed protein, which may change the target site of a drug, with decreases in or lack of affinity. Therefore, SNP analysis can help to achieve correct diagnosis and effective treatment. As a detection system for CYP2C19 * 2/3/17, an important gene for guiding clopidogrel use, RPCR-MC can accurately distinguish different CYP2C19 * 2, CYP2C19 * 3 and CYP2C19 * 17 types. Based on detection of different sample concentrations, the detection limit of the method was determined to be 1 copy/µl, and the detection limit for the blood genome was 0.1 ng/µl, indicating high detection sensitivity.
Some traditional methods for detecting SNPs, such as dideoxy sequencing, pyrosequencing, and whole-genome sequencing, can reveal important features required for SNP typing, such as the type of SNP and its accurate location. However, this method is cumbersome, expensive, and not suitable for general use. In addition, SSCP occurs when single-stranded DNA forms a secondary structure under neutral conditions, and different secondary structures exhibit different mobilities by electrophoresis. The disadvantage of this method is that the mutation type and specific location cannot be determined. Hybridization sequencing-chip sequencing technology is based on imprinting hybridization or nucleic acid in situ hybridization, in which an oligonucleotide probe hybridizes with the target DNA; the fluorescent group carried by the oligonucleotide probe marks the DNA and identifies the specific sequence. Nevertheless, there are some issues with this kind of technology: as it is difficult to identify nonspecific signals through only a single hybridization, it may lead to sequence misreading due to some nonspecific hybridization. In addition, the chip is expensive, as is the equipment required, which is not conducive to universal application. According to the Tm value analysis system established in this study, which recognizes the target sequence using specific primers and probes for PCR amplification and then analyzes the Tm value through melting curve analysis, our system has high specificity and can accurately identify a single-base mutation in the target gene in a complex mixture. Furthermore, RPCR-MC is easy to operate, and the sample release agent is used for biological samples without extraction, reducing the cumbersome process of extracting sample nucleic acids. The detection time is also short and completed within 1 h, and it has high-throughput multiple detection capabilities. Indeed, RPCR-MC can detect 9 genotypes of 3 sites in a single tube.
Recent studies have shown that the determination of SNPs between different population groups can effectively identify the target molecules of new drugs. Due to SNP differences between patients, such findings can be used to reduce adverse reactions to known drugs and clinical trial drugs as well as improve their efficacy. In addition, beneficial drugs that have been eliminated due to adverse reactions may be revisited; as long as the associated SNP is found, such effective drugs can be used in clinical practice. Therefore, quickly and accurately identifying a single-base mutation is particularly important. In view of the rapid and convenient, high sensitivity and good specificity of the RPCR-MC detection system, this method will improve detection efficiency by medical staff, reduce workloads, and play an important role in the early recovery of patient health. Regardless of the time cost or test reagent cost, our detection system is the best choice.
In conclusion, we have developed a method for identifying the CYP2C19 * 2/3/17 locus, which is helpful for guiding the use of clopidogrel.