CN-116559257-B - Ratio type DNA electrochemical sensor for accurate determination of swing type base pair genotyping

Abstract

The invention discloses a ratio type DNA electrochemical sensor for swing type base pair position genotyping, which is characterized by creatively utilizing a nonspecific amplification strategy (ligase chain reaction, LCR) and having the characteristics of higher amplification efficiency of Wo Kesen base pairs (GC, AT) and dominant effect in LCR compared with swing type base pairs (GT, AC). The method is characterized in that alleles are used as target genes, two LCRs (LCR Fc→G and LCR MB→A ) are designed based on Wo Kesen base pair complementation principle, an electroactive indicator ferrocene (Fc) and Methylene Blue (MB) with stable detection performance in clinical samples are selected as electrochemical response signals of the two LCRs, the current ratio (I MB /I Fc ) is used as an output signal of allele typing, and finally, accurate determination of rocking base pair genotyping (homozygous wild type, homozygous mutant type and heterozygous type) can be realized according to I MB /I Fc region division.

Inventors

• LIU ZHOUJIE
• CHEN JINYUAN
• XU YANFANG
• HUANG CHEN
• LIANG YUQI
• YANG LIANGYONG
• LAN RUILONG

Assignees

• 福建医科大学附属第一医院

Dates

Publication Date

20260512

Application Date

20230507

Claims (3)

1. 1. A detection method for precisely typing CYP2C19 alleles is characterized by comprising the following steps of adopting a ratio type DNA electrochemical sensor for detection, (1) utilizing a ligase chain reaction LCR to construct a ratio type DNA electrochemical sensor R-eLCR, (2) selecting an electroactive indicator ferrocene (Fc) and Methylene Blue (MB) as electrochemical response signals of the LCR, experimentally verifying that the Fc and the MB have similar electric signal responses in R-eLCR, (3) taking CYP2C 19X 2 as detection targets, designing two LCRs, namely LCR Fc→G and LCR MB→A , dividing three current ratio intervals, namely I MB /I Fc ＜1,1＜I MB /I Fc ＜2,I MB /I Fc >2 according to a current ratio (I MB /I Fc ) region, and specifically distinguishing three genotypes of the CYP2C19 alleles, namely CYP2C 19X 1*1, in human whole blood genome DNA detection, CYP2C19 x 2, In the design of two LCR (LCR Fc→G and LCR MB→A ) probes, for the probe LCR MB→A , the designed four primer probes are respectively half of CYP2C 19-2 allele-specific gene fragments A-contained target, and auxiliary probes AP and MB-modified signal probes SP MB are respectively complementary to probes HT1 and HT2 A , the specific sequences and modification groups of the four primer probes are shown in Table 1, for the probe LCR Fc→G , the designed four primer probes are half of CYP2C 19-1 allele-specific gene fragments G-contained target, the auxiliary probes AP and Fc-modified signal probes SP Fc are respectively complementary to probes HT1 and HT2 G , the specific sequences and modification groups of the four primer probes are shown in Table 1, wherein the common sequences of probes HT1 and AP are two LCRs, the auxiliary probes CP 5 'ends of the probes are complementary to the respective signal probes, and the complementary ends of the probe SP 3' ends of the probe AP are adjacent to the Fc-35, and the signal ends of the probe CP 38 are in an inverse electrochemical manner, and the electrochemical signal ends of the probe ends are adjacent to the Fc ends of the probe ends; primer probe sequences of tables 1R-eLCR 。
2. 2. A process for preparing the electrochemical sensor of ratio-type DNA for precisely typing CYP2C 19X 2 allele includes such steps as preparing R-eLCR of electrochemical sensor of ratio-type DNA, preparing 100L buffer solution containing 1X AMPLIGASE, thermostable ligase 1.0. 1.0U Ampligase, preparing the electrochemical sensor of ratio-type DNA, and preparing the electrochemical sensor of ratio-type DNA, 120 The reaction solution of the four LCR primers and the target is fully vortexed, and then LCR thermocycling reaction is carried out to obtain a mixed LCR amplification product; R-eLCR is prepared by hybridization at 53deg.C for 2 min, denaturation at 94 deg.C for 1 min, and repeating for 30 cycles; thereafter, equal volumes of LCR MB→A and LCR Fc→G products were mixed and 5 μl of 0.2M phosphate buffer was added to the mixed product; (2) self-assembly of capture probe CP on gold electrode (AuE) by dropping 3. Mu.L of 350 nM CP onto AuE surface, shading 16 h at room temperature to form thiol-DNA self-assembled monolayer, then washing with 10 mM phosphate buffer and immersing in 100. Mu.L of 2 mM mercapto hexanol (MCH) solution, sealing against light 2 h to replace nonspecifically adsorbed DNA and inactivate the remaining AuE region, obtaining modified AuE, (3) dropping 3. Mu.L of mixed LCR amplification product prepared in step (1) onto 1 h of modified AuE surface obtained in step (2) at room temperature, finally performing electrochemical assay by square wave voltammetry, designing four primer probes for LCR MB→A , respectively, probe HT1 and probe HT2A being half of 2C19 allele-specific gene fragment A-56, and complementary probe SP 43 and probe SP 43 being complementary to four primer probe 4332, respectively, designing four primer probes for LCR MB→A , and probe HT1 and probe 53R 53, respectively, the probe HT1 and the probe HT2 G are respectively half of CYP2C19 x 1 allele specific gene segment G-contained target, the auxiliary probe AP and the Fc modified signal probe SP Fc are respectively complementary with the probe HT1 and the probe HT2 G , the specific sequences and the modification groups of the four primer probes are shown in the table 1, wherein the probe HT1 and the probe AP are common probes of two LCRs, a section of 12 nt sequence is complementary with the 3' end of the CP at the 5' end of the auxiliary probe AP, the 5' end of the CP modifies a-SH group, so that the signal probes SP of the two LCRs are connected with the AP and then the electroactive indicator MB/Fc approaches to the electrode surface in an inverted mode, and electron transfer occurs, so that corresponding electrochemical signals are generated; primer probe sequences of tables 1R-eLCR 。
3. 3. The method of claim 2, wherein the method comprises the step of preparing a ratio-based DNA electrochemical sensor for accurate genotyping of CYP2C19 x 2 alleles.

Description

Ratio type DNA electrochemical sensor for accurate determination of swing type base pair genotyping Technical Field The invention relates to a ratio type DNA electrochemical sensor for accurate determination of swing type base pair position genotyping, which can specifically distinguish three genotypes of CYP2C19 x 2 in human whole blood genome DNA, the invention has important design reference value for accurately determining the swinging type base pair position genotyping, and can provide a new thought and a new direction for the personalized medicine genotyping detection method. Background CYP2C19 is an important drug metabolizing enzyme in human body, the therapeutic effect and adverse reaction of drugs metabolized by the enzyme are obviously different along with the different genotypes of patients, the gene polymorphism of the CYP2C19 is the cause, CYP2C19 x 2 is one of the most common mutation sites in Han population, and researching the genotypes of the drugs is beneficial to guaranteeing the medication safety of the patients and realizing personalized administration. Alleles are the most common genotypes in the human genome and are generally characterized by single nucleotide polymorphisms. With the new discovery of the role of alleles in disease onset, progression, prognosis, and drug therapy response, allelic typing has become an important area of leading-edge research. The ability to distinguish single base mismatches is a prerequisite for allelic typing, however, single nucleotide changes have little intrinsic impact on the overall physicochemical properties of the nucleic acid sequence, and the frequent occurrence of G: T mismatch integration errors by DNA polymerase, makes Polymerase Chain Reaction (PCR) based allelic typing methods a great challenge. In particular, the GT and AC wobble base pairs formed by the DNA probes employed and the alleles make wobble allele typing extremely difficult, because the structural stability of wobble base pairs is similar to Wo Kesen base pairs AT and GC (also known as perfect base pairs). To date, many wobble alleles (e.g., apoE epsilon 2-4, hla-B1502, cyp2c 19) have been shown to be closely related to disease progression and drug treatment. Therefore, there is an urgent need to develop an accurate and versatile wobble base pair genotyping method. Based on the above analysis, we creatively utilized the nonspecific amplification of Ligase Chain Reaction (LCR), and constructed a ratio-type DNA electrochemical sensor named R-eLCR for accurate determination of CYP2C19 x2 allele typing based on the characteristic that the amplification efficiency of perfect base pairs and wobble base pairs is close but higher. Currently, researchers have developed a number of methods for wobble-type allele typing, such as allele-specific PCR, blocker Displacement Amplification (BDA) -PCR, gold nanoparticle modified isothermal amplification, and the like. These methods focus only on how to reduce non-specific amplification, while wobble base pairs (GT and AC) are not of interest, limiting the inhibitory effect on mismatch amplification. In addition, the current allele typing method adopts a single signal output mode, the detection result depends on an absolute value, and is easily influenced by factors such as instruments, reagents, environmental conditions, operation deviation and the like, so that the reading is inaccurate. According to the invention, by utilizing the characteristics of nonspecific amplification of Ligase Chain Reaction (LCR) and higher amplification efficiency of perfect base pairs compared with wobble base pairs, two LCRs (LCR Fc→G and LCR MB→A) are designed, and electroactive indicators ferrocene (Fc) and Methylene Blue (MB) with stable detection performance in clinical sample detection are selected as electrochemical response signals. After the clinical samples are amplified by two LCRs respectively, the electrochemical signals are mixed in equal volume, a square wave voltammetry is used for collecting the electrochemical signals, the current ratio (I MB/IFc) of the two LCRs is used as the output signals of allele typing, and finally, accurate determination of CYP2C 19-2 rocking base pair genotyping (CYP 2C 19-1*1, CYP2C 19-1*2 and CYP2C 19-2) can be realized according to I MB/IFc region division (I MB/IFc＜1,1＜IMB/IFc＜2,IMB/IFc > 2). Disclosure of Invention One of the purposes of the invention is to construct a ratio type DNA electrochemical sensor for clinical sample rocking base pair genotyping by utilizing the characteristics of nonspecific amplification of Ligase Chain Reaction (LCR) and higher amplification efficiency of perfect base pairs compared with rocking base pairs. The second object of the present invention is to apply the method to the detection of CYP2C19 x 2 allele genotyping of human whole blood genomic DNA samples. In order to achieve the aim of the invention, the invention adopts the following technical scheme: A detection method of a ratio