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.