A ratio type DNA electrochemical sensor for precise determination of swing type base pair allelotyping

Abstract

This invention discloses a ratiometric DNA electrochemical sensor for genotyping of wobbly base pairs, characterized by the innovative use of a non-specific amplification strategy (ligase chain reaction, LCR) and the higher amplification efficiency and dominant role of Watson base pairs (GC, AT) compared to wobbly base pairs (GT, AC). Using alleles as target genes, and based on the Watson base pair complementarity principle, two LCRs (LCRs) are designed. Fc→G and LCR MB→A Ferrocene (Fc) and methylene blue (MB), electroactive indicators with stable detection performance in clinical samples, were selected as the electrochemical response signals for the two LCRs, and their current ratio (IL) was used. MB / I Fc As the output signal for allele typing, finally, based on I... MB / I Fc Regional division enables precise determination of swing-type base pair allele genotyping (homozygous wild type, homozygous mutant type, heterozygous type).

Description

Technical Field

[0001] This invention relates to a ratiometric DNA electrochemical sensor for precise determination of swing-type base pair allele genotyping, capable of specifically distinguishing genotypes in human whole blood genomic DNA. CYP2C19*2 With three genotypes, this invention provides important design reference value for how to accurately determine the genotyping of swinging base pair alleles, and can also provide new ideas and directions for personalized drug genotyping detection methods. Background Technology

[0002] CYP2C19 is a crucial drug-metabolizing enzyme in the human body. Drugs metabolized by this enzyme exhibit significantly different therapeutic effects and adverse reactions depending on the patient's genotype. This is due to the CYP2C19 gene polymorphism. CYP2C19*2 It is one of the most common mutation sites in the Han Chinese population. Studying its genotype is beneficial to ensuring the safety of patients' medication and to achieving individualized drug administration.

[0003] Alleles are the most common genotypes in the human genome, typically characterized by single nucleotide polymorphisms (SNPs). With new discoveries regarding the roles of alleles in disease pathogenesis, progression, prognosis, and drug treatment response, allele typing has become a key area of ​​cutting-edge research. The ability to distinguish single-base mismatches is a prerequisite for allele typing; however, the intrinsic impact of single nucleotide variations on the overall physicochemical properties of nucleic acid sequences is small, and the frequent G:T mismatch integration errors of DNA polymerases pose significant challenges to polymerase chain reaction (PCR)-based allele typing methods. In particular, the GT and AC swinging base pairs formed by the DNA probes and alleles make swinging allele typing extremely difficult because the structural stability of swinging base pairs is similar to that of the Watson base pairs AT and GC (also known as perfect base pairs). To date, many swinging alleles (such as...) ApoEε2-4 , HLA-B*1502 , CYP2C19*2 This has been shown to be closely related to disease progression and drug treatment. Therefore, there is an urgent need to develop an accurate and universal method for swing-type base pair allele typing.

[0004] Based on the above analysis, we creatively utilized the non-specific amplification of ligase chain reaction (LCR). Taking advantage of the fact that perfect base pairs and rocking base pairs have similar but higher amplification efficiencies, we constructed a ratiometric DNA electrochemical sensor called R-eLCR for precise measurement. CYP2C19*2Allelic genotyping. Currently, researchers have developed many swinging allelic genotyping methods, such as allelic-specific PCR, blocker-displacement amplification (BDA)-PCR, and gold nanoparticle-modified isothermal amplification. These methods focus solely on reducing non-specific amplification, neglecting the swinging base pairs (GT and AC), thus limiting their effectiveness in inhibiting mismatch amplification. Furthermore, current allelic genotyping methods employ a single-signal output mode, with results dependent on absolute values, making them susceptible to inaccurate readings due to factors such as instrumentation, reagents, environmental conditions, and operational biases.

[0005] This invention utilizes the non-specific amplification of ligase chain reaction (LCR) and the higher amplification efficiency of perfect base pairs compared to rocking base pairs to design two LCRs (LCRs). Fc→G and LCR MB→A Ferrocene (Fc) and methylene blue (MB), electroactive indicators with stable detection performance in clinical sample testing, were selected as the electrochemical response signals. Clinical samples were amplified separately by two LCRs, then mixed in equal volumes, and the electrochemical signals were acquired using square wave voltammetry. The current ratio of the two LCRs (IL) was used as the electrochemical response signal. MB / I Fc As the output signal for allele typing, finally, based on I... MB / I Fc Regional division (I) MB / I Fc <1, 1<I MB / I Fc <2,I MB / I Fc >2) Can be achieved CYP2C19*2 Wobbling base pair allele typing ( CYP2C19*1*1, CYP2C19*1*2, CYP2C19*2*2 The precise measurement of ). Summary of the Invention

[0006] One of the objectives of this invention is to construct a ratiometric DNA electrochemical sensor that can be used for allelic typing of rocking base pairs in clinical samples, taking advantage of the non-specific amplification of ligase chain reaction (LCR) and the higher amplification efficiency of perfect base pairs compared to rocking base pairs.

[0007] The second objective of this invention is to apply this method to human whole blood genomic DNA samples. CYP2C19*2 Allelic typing detection.

[0008] To achieve the purpose of the invention, the present invention adopts the following technical solution:

[0009] A detection method for a ratiometric DNA electrochemical sensor that can be used for accurate determination of swing-type base pair allele typing is characterized by the following steps: (1) constructing a ratiometric DNA electrochemical sensor R-eLCR using ligase chain reaction (LCR); (2) selecting ferrocene (Fc) and methylene blue (MB), electroactive indicators with stable detection performance in clinical samples, as electrochemical response signals of LCR, and experimentally verifying that Fc and MB have similar electrochemical signal responses in R-eLCR; (3) using CYP2C19*2 Two LCRs were designed for the detection target, namely LCR Fc→G and LCR MB→A Based on its current ratio (I) MB / I Fc The region is divided into three current ratio intervals, namely I MB / I Fc <1, 1<I MB / I Fc <2,I MB / I Fc >2, in human whole blood genomic DNA testing, it can specifically distinguish CYP2C19*2 The three genotypes of alleles, namely CYP2C19*1*1 , CYP2C19*2* 2 , CYP2C19*1*2 .

[0010] Furthermore, in designing two LCRs (LCRs) Fc→G and LCR MB→A When probing: For probe LCR MB→A Of the four primer probes designed, probe HT1 and probe HT2 A They are respectively CYP2C19*2 The allele-specific gene fragment A-contained half of the target, while the auxiliary probe AP and the MB-modified signal probe SP... MB With probes HT1 and HT2 respectively A Complementary, the specific sequences and modifying groups of the four primers and probes are shown in Table 1; for probe LCR Fc→G Of the four primer probes designed, probe HT1 and probe HT2 G They are respectively CYP2C19*1 Half of the allele-specific gene fragment G-contained target, while the auxiliary probe AP and the Fc-modified signal probe SP. Fc With probes HT1 and HT2 respectively GThe specific sequences and modification groups of the four primer probes are shown in Table 1. Among them, probe HT1 and probe AP are common probes of the two LCRs. The 5' end of the auxiliary probe AP has a 12 nt sequence that is complementary to the 3' end of CP. The 5' end of CP is modified with a -SH group, so that after the signal probes SP and AP of the two LCRs are connected, the electroactive indicator MB / Fc approaches the electrode surface in an inverted form, undergoes electron transfer, and generates the corresponding electrochemical signal.

[0011] Table 1 shows the primer and probe sequences for R-eLCR.

[0012]

[0013] Furthermore, in the use of ligase chain reaction (LCR): CYP2C19*2 The three genotypes of alleles ( CYP2C19*1*1 , CYP2C19*2*2 , CYP2C19*1*2 These are considered as three detection targets, each amplified by two LCRs and then mixed in equal volumes; based on the two LCRs described in claim 2 Fc→G and LCR MB→A Probe design, when the detection target is CYP2C19* 1*1 At that time, I MB For LCR MB→A Non-specific amplification signal, when the detection target is CYP2C19*2*2 At that time, I Fc For LCR Fc→G Non-specific amplification signal, while when the detection target is CYP2C19*1*2 At that time, its I MB and I Fc Both contain non-specific amplified signals; experiments have verified that Fc and MB have similar electrical signal responses in R-eLCR. Therefore, the current ratio (IC) of the two LCRs is used to determine the current ratio (IC) of the two LCRs. MB / I Fc As CYP2C19*2 The output signal for allele typing can be based on I MB / I Fc Regional division CYP2C19*2 The three allele types are specifically distinguished.

[0014] The present invention describes a method for preparing a ratiometric DNA electrochemical sensor for accurate determination of wobbling base pair allele genotyping, characterized by the following steps: 1) 100 µL of a reaction solution containing 1×Ampligase buffer, 1.0 U Ampligase thermostable ligase, 120 nM of four LCR primers and a target is thoroughly vortexed, followed by an LCR thermal cycling reaction to obtain a mixed LCR amplification product; the LCR process is as follows: hybridization ligation at 53 °C for 2 min, denaturation at 94 °C for 1 min, repeated for 30 cycles; thereafter, an equal volume of LCR... MB→A and LCR Fc→G 1) Mix the products and add 5 µL of 0.2 M phosphate buffer to the mixture; 2) Self-assembly of the capture probe CP on the gold electrode (AuE): 3 µL of 350 nMCP was dropped onto the AuE surface and kept in the dark at room temperature for 16 h to form a thiol-DNA self-assembled monolayer; then, it was rinsed with 10 mM PB and immersed in 100 µL of 2 mM mercaptohexanol (MCH) solution and kept in the dark for 2 h to replace the non-specifically adsorbed DNA and passivate the remaining AuE region to obtain the modified AuE; 3) 3 µL of the mixed LCR amplification product obtained in step 1) was dropped onto the modified AuE surface obtained in step 2) at room temperature for 1 h, and finally electrochemically measured by square wave voltammetry.

[0015] The above-described preparation method of the present invention yields a ratiometric DNA electrochemical sensor that can be used for accurate determination of swing-type base pair allele typing.

[0016] Specifically, the present invention adopts the following technical solution:

[0017] Constructing a DNA self-assembled monolayer on a gold electrode surface includes the following steps:

[0018] Gold electrode (AuE) pretreatment: The AuE was sonicated with Piranha solution (30% H2O2 and concentrated H2SO4, mixed in a volume ratio of 1:3) for 10 min, then sonicated twice with deionized water for 5 min each time. It was then polished to a mirror finish with mixtures of 0.3 μm and 0.05 μm Al2O3 and water, respectively, followed by sonication with ethanol and distilled water for 2 min each. The sonicated electrode was then placed in 0.5 M H2SO4 and cyclically scanned within the potential range of 0–1.6 V until stable. It was then rinsed with double-distilled water, dried with N2, and ready for use.

[0019] Self-assembly of CP on AuE: Pretreated AuE was immobilized on a horizontal surface. CP was reduced using TE+TCEP at room temperature in the dark (to remove disulfide bonds). 3 µL of a 350 nM capture probe (CP) was then drop-coated onto the AuE surface and incubated at room temperature in the dark for 16 h to form a thiol-DNA self-assembled monolayer (SAM). The surface was then rinsed with 10 mM PB and immersed in 100 µL of 2 mM MCH solution, sealed in the dark for 2 h to replace non-specifically adsorbed DNA and passivate the remaining AuE regions. After blocking, the surface was rinsed with 10 mM PB, dried with N2, and ready for use.

[0020] Construction of R-eLCR: 100 µL of reaction solution containing 1×Ampligase buffer, 1.0 U Ampligase thermostable ligase, 120 nM of four LCR primers and the target was thoroughly vortexed and then thermally cycled in a 2720 thermal cycler (Applied Biosystems). The optimized LCR protocol was established as follows: ligation at 53 °C for 2 min, denaturation at 94 °C for 1 min, repeated for 30 cycles. Subsequently, an equal volume (50 µL) of the LCR solution was... MB→A and LCR Fc→G The products were mixed, and 5 µL of 0.2 M PB was added to the mixture to hybridize with CP on AuE. The electrical signal was collected using square wave voltammetry.

[0021] CYP2C19*2 Clinical sample testing for allele typing includes the following steps:

[0022] Clinical Sample Collection and Extraction: Department of Pharmacy, First Affiliated Hospital of Fujian Medical University CYP2C19*2 Human whole blood samples for testing (approved by the hospital's medical ethics committee,

[2019] 071), fully automated nucleic acid extractor and matching total nucleic acid extraction kit (human whole blood), stored at -20 ℃;

[0023] PCR amplification of human whole blood genomic DNA: Searching for... CYP2C19*2 The whole genome sequence was obtained, and a gene fragment containing the mutation site was selected, totaling 1170 nt. The PCR primers are shown in Table 2, and the PCR reaction system and reaction steps are shown in Tables 3 and 4.

[0024] Table 2 shows the PCR primer design.

[0025]

[0026] Table 3 shows the PCR reaction system.

[0027]

[0028] Table 4 shows the PCR reaction steps.

[0029]

[0030] Electrochemical detection: MB / Fc electrical signals were collected using a three-electrode system and a CHI 760E dual potentiostat via square wave voltammetry (SWV). The hybridized AuE cells were gently rinsed along the edges with electrolyte before being immersed in the electrolyte for detection. SWV: Amplitude: 25 mV, Frequency: 10–100 Hz, Scan potential: -0.5 V–0.6 V.

[0031] Advantages of the detection method of this invention:

[0032] Achieving precise determination of wobbly base pair allele genotyping: Utilizing the non-specific amplification of LCR and the higher amplification efficiency of perfect base pairs compared to wobbly base pairs, a ratiometric DNA electrochemical sensor called R-eLCR was constructed for use in... CYP2C19*2 Allelic genotyping: This invention provides important design reference value for how to detect swing base pair allelic genotyping, which will promote the application of electrochemical detection methods in clinical samples and is of great significance.

[0033] Achieve clinical sample CYP2C19*2 Three genotype differentiation tests: based on the ratio of two LCR currents I MB / I Fc Regional division, precise measurement CYP2C19*2 The three allele typing methods can provide new ideas and directions for personalized drug genotyping detection methods. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the design principle of the detection method of the present invention;

[0035] Figure 2 This invention compares the electrochemical responses of the electroactive indicators MB and Fc in the detection method and the LCR amplification efficiency of perfect base pairs and rocking base pairs. Figure 2 In the figure, A represents the result of polyacrylamide gel electrophoresis; Figure 2 B in the diagram shows current values ​​similar to MB and Fc; Figure 2 The C in the figure indicates that the specific amplification signal caused by perfect base pairs (AT, GC) is significantly greater than the non-specific amplification signal caused by rocking base pairs (GT, AC);

[0036] Figure 3The electrochemical responses of the electroactive indicators MB and Fc in the detection method of this invention were compared, as well as the LCR amplification efficiencies of perfect base pairs and rocking base pairs. The results showed that the two LCRs had similar amplification efficiencies.

[0037] Figure 4 This is a graph showing the optimized relationship between the concentration of the capture probe CP and the peak values ​​of MB and Fc in the detection method of this invention.

[0038] Figure 5 Optimization of the conditions for the detection method of the present invention; Figure 5 Figures A and B show the optimization of R-eLCR primer concentrations. Increasing the primer concentration can increase the enzyme digestion product (AP-SP). MB AP-SP Fc Increase the number of CPs to improve hybridization efficiency; Figure 5 C and D in the figure represent the optimization of the amount of ligase;

[0039] Figure 6 This is a graph showing the detection performance of the detection method of the present invention and the linear relationship between clinical sample concentration and electrical signal; Figure 6 In the sample, A represents the three alleles (AA: CYP2C19*2*2 GG: CYP2C19*1*1 GA: CYP2C19*1*2 )I MB / I Fc Value region division; Figure 6 B in the text stands for LCR. MB→A The current value (I) MB The concentrations of the three allele samples showed a good linear relationship with the concentrations of the three alleles in the range of 100 fM-100 pM.

[0040] Figure 7 The detection method of the present invention is applied to CYP2C19*2 Clinical sample test results (Figure A) and gene sequencing results (Figure B). Detailed Implementation

[0041] To make the technical problems, technical solutions and effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0042] like Figure 1As shown, the electrochemical method design principle diagram of a ratiometric DNA electrochemical sensor for accurate determination of swing-type base pair allele typing described in this invention is as follows: This invention innovatively utilizes a non-specific amplification strategy (ligase chain reaction, LCR) to construct a ratiometric DNA electrochemical sensor, which is named R-eLCR; (2) Ferrocene (Fc) and methylene blue (MB), electroactive indicators with stable detection performance in clinical samples, are selected as electrochemical response signals of LCR, and our experiments have verified that Fc and MB have similar electrochemical signal responses in R-eLCR; (3) Using CYP2C19*2 For example, two LCRs (LCRs) were designed. Fc→G and LCR MB→A ), based on its current ratio (I MB / I Fc ) Regional division (I MB / I Fc <1, 1<I MB / I Fc <2,I MB / I Fc >2), In human whole blood genomic DNA testing, it can specifically distinguish CYP2C19*2 The three genotypes of alleles ( CYP2C19*1*1, CYP2C19*2*2, CYP2C19*1*2 ). In LCR MB→A Of the four primers, probes HT1 and HT2... A They are respectively CYP2C19*2 The allele-specific gene fragment A-contained half of the target, while the auxiliary probe AP and the MB-modified signal probe SP... MB With probes HT1 and HT2 respectively A Complementary, SP MB The 5' end of the primer is modified with MB, and the remaining primers (HT1, HT2) A AP) 5' end modified with -PO4; for LCR Fc→G probe HT1 and probe HT2 G They are respectively CYP2C19*1 Half of the allele-specific gene fragment G-contained target, while the auxiliary probe AP and the Fc-modified signal probe SP. Fc With probes HT1 and HT2 respectively G Complementarity. Furthermore, the 3' end of the capture probe CP is modified with -SH, and its 5' end has a 12 nt sequence that is completely complementary to the 3' end sequence of AP. Therefore, when SP is connected to AP, MB / Fc can be carried to the surface of AuE in an inverted form, undergoing electron transfer and generating an electrochemical signal. The specific sequences of each probe are shown in Table 1.

[0043] Each clinical sample was subjected to LCR separately.MB→A and LCR Fc→G After amplification, the sample is mixed in an equal volume and hybridized with the CP on AuE, then detected using square wave voltammetry. When the clinical sample contains A-contained target, the LCR is [value missing]. MB→A The amplification products are more than those of LCR. Fc→G The amplification products, because in LCR MB→A A-contained target and SP containing T MB The amplification efficiency of perfect base pairs between them is higher than that of LCR. Fc→G A-contained target and SP containing C Fc The amplification efficiency of the swing base pairs between them; while when the clinical sample contains G-contained target, LCR Fc→G The amplification products of [a specific amplification product] are dominant for the same reason. Therefore, given that the electrochemical responses of MB and Fc are similar, when the clinical sample is [a specific amplification product]... CYP2C19*2*2 At a potential of ~-0.25 V, the current obtained from methylene blue (MB) is higher than the current obtained from ferrocene (Fc) at a potential of ~0.3 V. MB / I Fc The current ratio is much higher than 1; while when the clinical sample is CYP2C19*1*1 At that time, I MB / I Fc The current ratio is much lower than 1; while when the clinical sample is CYP2C19*1*2 At that time, I MB / I Fc The current ratio is close to 1. Therefore, CYP2C19*2 Three allele typing can be done through I MB / I Fc Specific differentiation.

[0044] Example 1:

[0045] Preparation of primer solutions and reagents:

[0046] Phosphate inhibitor (0.2 M PB): Mix 1.4822 g NaH2PO4·2H2O and 14.5046 g Na2HPO4·10H2O into a 500 mL blue cap bottle, and dilute to 250 mL with double distilled water to prepare a 0.2 M phosphate inhibitor (PB) with pH 7.4.

[0047] Phosphate flushing solution (10 mM PB): Take 20 mL of the prepared 0.2 M PB into a 500 mL blue cap bottle, and dilute to 400 mL with double distilled water to prepare a 10.0 mM phosphate flushing solution with a pH of 7.4.

[0048] Tris-HCl buffer: Place 0.302825 g of Tris powder in a 500 mL blue-capped bottle, and dilute to 250 mL with double-distilled water to prepare a 10.0 mM Tris solution. Then adjust the pH to 8.0 with a small amount of concentrated hydrochloric acid.

[0049] TE buffer: Weigh 2.922 g NaCl and 0.0186 g EDTA, and adjust the volume to 50 mL with the 10 mM Tris-HCl prepared in step (4) to prepare a pH 8.0 TE buffer (containing 1.0 mM EDTA and 0.1 M NaCl).

[0050] TE+TCEP reducing solution: Weigh 0.0286 g of tris(2-carbonylethyl)phosphohydrochloride (TCEP), and dilute to 10 mL with the TE buffer prepared in step (4) to prepare a TE+TCEP reducing solution containing 1 μM TCEP.

[0051] DNA stock solution: The synthesized DNA is a very light dry film attached to the wall of the container tube. It is very easy to lose when opened. Therefore, before dissolving, centrifuge the centrifuge tube containing DNA at 3000 rpm for 10 min, slowly open the cap, and add a certain volume of the TE prepared in step (4) according to the instructions to prepare a 100 µM DNA stock solution. In order to avoid repeated freeze-thaw cycles, the stock solution can be aliquoted into 1 μL per tube and stored at -20 ℃.

[0052] Mercaptohexanol blocking agent (MCH): Add 4 μL of MCH stock solution to 15 mL of double-distilled water to prepare 2 mM MCH blocking agent, and store at 4 °C protected from light.

[0053] Electrolyte: Weigh 23.376 g NaCl and place it in a 500 mL blue cap bottle. Add 20 mL of 0.2 M PB prepared in step (1) and 380 mL of double-distilled water to make up to 400 mL to prepare an electrolyte containing 10 mM PB and 1 mM NaCl (referred to as electrolyte).

[0054] Target probe, primer probe (HT1, HT2) G HT2 A AP, SP MB SP Fc ): Take 1 μL of the corresponding target probe and primer probe stock solution prepared in step (6), add 99 μL of Tris-HCl buffer prepared in step (3), mix evenly in a mixer, first prepare a 1 μM solution, and then prepare different concentrations with Tris-HCl buffer according to experimental requirements, and store at 4℃.

[0055] Capture probe (CP): Take 1 μL of the corresponding capture probe stock solution prepared in step (6), add 99 μL of TE+TCEP reducing solution prepared in step (5), mix evenly in a mixer, first prepare a 1 μM solution, let it stand at room temperature in the dark to reduce, and then prepare different concentrations with TE+TCEP reducing solution according to experimental requirements, and store at 4 ℃.

[0056] 10×Ampligase buffer: 200 mM Tris-HCl, 250 mM potassium chloride, 100 mM magnesium chloride, 5 mM nicotinamide adenine dinucleotide (NAD), 0.1 wt% polyethylene glycol octylphenyl ether (Triton X-100), pH 8.3, prepared as in step (3). Both the thermostable Ampligase ligase and the 10×Ampligase buffer were provided by Epicentre Technologies, Inc., Madison, USA.

[0057] Example 2:

[0058] Comparison of electrochemical responses of electroactive indicators MB and Fc, and comparison of LCR amplification efficiency between perfect base pairs and rocking base pairs.

[0059] To achieve accurate determination of rocking allele genotyping using the ratiometric DNA electrochemical sensor R-eLCR, it is necessary to determine that electroactive indicators MB and Fc have similar electrochemical responses in R-eLCR and that perfect base pairs have higher LCR amplification efficiency compared to rocking base pairs. Therefore, we designed experiments to verify this, such as... Figure 2 The results of polyacrylamide gel electrophoresis in LCR are shown in Figure A. MB→A and LCR Fc→G Under identical amplification conditions (primer concentration 1 nM, target concentration 0.1 μM, ligase concentration 5 U), the LCR amplification efficiency induced by perfect base pairs was higher than that induced by nonspecific amplification by wobbly base pairs (lane 4 vs. lane 5, lane 7 vs. lane 8). Gray-scale analysis was performed on the amplification products of lane 4 and lane 7. Figure 3 The results showed that the two LCRs had similar amplification efficiencies, and when their amplification products were detected by square wave voltammetry, Figure 2 The B values ​​in the figure show similar current values ​​for MB and Fc, indicating that MB and Fc have similar electrochemical responses in R-eLCR. Figure 2 The value of C indicates that the specific amplification signal induced by perfect base pairs (AT, GC) is significantly greater than the non-specific amplification signal induced by rocking base pairs (GT, AC). These results lay the theoretical foundation for the R-eLCR constructed in this invention.

[0060] Example 3:

[0061] Clinical Sample Collection and Pretreatment: Department of Pharmacy, First Affiliated Hospital of Fujian Medical University CYP2C19*2 Whole blood samples from individuals used in the testing project (approved by the hospital's medical ethics committee,

[2019] 071) were used. An automated nucleic acid extractor and a matching total nucleic acid extraction kit (for whole blood) were used to extract whole genomic DNA, which was then stored at -20°C. DNA was retrieved from the NCBI GenBank database. CYP2C19*2 The whole genome sequence was obtained, and a gene fragment containing the mutation site was selected, totaling 1170 nt. The PCR primers are shown in Table 2, and the PCR reaction system and reaction steps are shown in Tables 3 and 4.

[0062] Example 4:

[0063] Fabrication of a ratiometric DNA electrochemical sensor for accurate determination of swing-type base pair allele typing and its application in the detection of CYP2C19*2 allele typing in human whole blood genomic DNA samples.

[0064] 1. Constructing a DNA self-assembled monolayer on a gold electrode surface, including the following steps:

[0065] (1) Pretreatment of gold electrode (AuE): The AuE was sonicated with Piranha solution (30wt% H2O2 and concentrated H2SO4 mixed at a volume ratio of 1:3) for 10 min, ultrasonically cleaned twice with deionized water for 5 min each time, polished to a mirror finish with a mixture of 0.3 μm and 0.05 μm Al2O3 and water, respectively, and then ultrasonically cleaned with anhydrous ethanol and distilled water for 2 min each. The ultrasonicated electrode was placed in 0.5 MH2SO4 and cyclically scanned in the potential range of 0~1.6 V until stable, rinsed with double distilled water, dried with N2, and ready for use.

[0066] (2) Self-assembly of CP on AuE: The AuE pretreated in step (1) was fixed on a horizontal surface. The CP (prepared according to step (10) of Example 1) was reduced at room temperature in the dark using TE+TCEP prepared according to step (5) to remove disulfide bonds. 3 µL of 350 nM capture probe (CP) was dropped onto the AuE surface and kept at room temperature in the dark for 16 h to form a thiol-DNA self-assembled monolayer (SAM). Then, it was rinsed with 10 mM PB and immersed in 100 µL of 2 mM MCH solution, sealed and kept in the dark for 2 h to replace non-specifically adsorbed DNA and passivate the remaining AuE region. After blocking with mercaptohexanol, it was rinsed with 10 mM PB, dried with N2, and ready for use.

[0067] 2. Construction of R-eLCR: The primer and probe sequences of R-eLCR are shown in Table 1. 100 µL of a reaction solution containing 1×Ampligase buffer (prepared from the 10×Ampligase buffer in step (11) of Example 1, provided by Epicentre Technologies, Inc.), 1.0 U of thermostable Ampligase ligase (provided by Epicentre Technologies, Inc.), 120 nM of four LCR primers and the target was thoroughly vortexed and then thermally cycled in an Applied Biosystems 2720 thermal cycler. The optimized LCR protocol was established as follows: ligation at 53 °C for 2 min, denaturation at 94 °C for 1 min, repeated for 30 cycles. Subsequently, an equal volume (50 µL) of LCR was... MB→A and LCR Fc→G The products were mixed, and 5 µL of 0.2 MPB was added to the mixture to hybridize with CP on AuE. The electrical signal was collected using square wave voltammetry.

[0068] Table 1 Primer and probe sequences for R-eLCR

[0069] .

[0070] Notes: 1) Underlined bases indicate mutation sites or partial (12 nt) complementary sites; bolded bases are electroactive indicator modification sites; 2) CP, AP, HT1, and HT2 were used in this study. G HT2 A SP MB SP Fc All G-contained targets were provided by Sangon Biotech (Shanghai) Co., Ltd.

[0071] 3. CYP2C19*2 Clinical sample testing for allele typing includes the following steps:

[0072] (1) Collection and extraction of clinical samples: Department of Pharmacy, First Affiliated Hospital of Fujian Medical University CYP2C19*2 Whole blood samples from individuals used for testing (approved by the hospital's medical ethics committee,

[2019] 071), fully automated nucleic acid extractor and matching total nucleic acid extraction kit (human whole blood), and clinical samples of whole blood genomic DNA obtained from each individual were stored at -20 ℃.

[0073] (2) PCR amplification of human whole blood genomic DNA: Search for... CYP2C19*2The whole genome sequence was obtained, and a gene fragment containing the mutation site was selected, totaling 1170 nt. The PCR primers are shown in Table 2, and the PCR reaction system and reaction steps are shown in Tables 3 and 4.

[0074] Table 2 shows the PCR primer design.

[0075]

[0076] Note: The rs4244285-728-F and rs4244285-1202R used in this study were provided by Sangon Biotech (Shanghai) Co., Ltd.

[0077] Table 3 PCR reaction system

[0078]

[0079] Notes: Template: Human whole blood genomic DNA; dNTP: Deoxyribonucleoside triphosphate; Taq Buffer: Taq DNA polymerase buffer; Taq enzyme: Taq DNA polymerase. Both Taq DNA polymerase buffer and Taq DNA polymerase were provided by Sangon Biotech (Shanghai) Co., Ltd.

[0080] Table 4 shows the PCR reaction steps.

[0081]

[0082] (3) Electrochemical detection: MB / Fc electrical signals were collected using a three-electrode system and a CHI 760E dual potentiostat via square wave voltammetry (SWV). The AuE cells assembled in step 1 were gently rinsed along the edges with electrolyte before being placed in the electrolyte for detection. SWV: Amplitude: 25 mV, Frequency: 10~100 Hz, Scan potential: -0.5 V~ 0.6 V.

[0083] Specifically, the optimization of conditions and detection performance of the ratiometric DNA electrochemical sensor described above, which can be used for accurate determination of swing-type base pair allele typing, are explained below:

[0084] like Figure 4 The diagram shows the optimization of the capture probe CP concentration. When the CP concentration is 350 nM, the MB and Fc peaks reach their highest values. Thereafter, as the CP concentration gradually increases, the distance between DNA molecules gradually decreases, and the AP-SP... MB AP-SP Fc It is also difficult to enter.

[0085] like Figure 5 Figures A and B show the optimization of R-eLCR primer concentrations. Increasing the primer concentration can increase the enzyme digestion product (AP-SP). MBAP-SP Fc The concentration of LCR primers is increased to improve the hybridization efficiency with CP. However, if the concentration of LCR primers is too high, the non-specific amplification products will increase sharply, which will seriously interfere with the specificity and sensitivity of this method. Based on the above results, the optimized primer concentration is 120 nM.

[0086] like Figure 5 Figures C and D show the optimization of ligase dosage. In the LCR amplification system containing the A-contained target, the MB peak current increases significantly with increasing ligase dosage, up to 1.5 U, then approaches saturation. Above 1.0 U, an increasing trend in nonspecific Fc peak current is observed. However, in the LCR amplification system containing the G-contained target, a significant increasing trend in nonspecific MB peak current is observed once the ligase dosage exceeds 0.5 U. This is based on the fact that guanine mismatches (GT) are the most stable mismatched base pairs, while cytosine (AC) are the least stable. To obtain I... MB / I Fc The largest difference was found in the optimized ligase amount, which was 1.0 U.

[0087] like Figure 6 The figure shown is a graph illustrating the detection performance of the detection method of the present invention and the linear relationship between allele concentration and MB electrical signal. Figure 6 In the sample, A represents the three alleles (AA: CYP2C19*2*2 GG: CYP2C19*1*1 GA: CYP2C19* 1*2 )I MB / I Fc Value region division, when the clinical sample contains A-contained target (equivalent to CYP2C19*2*2 When the concentration of the A-contained target is in the range of 100 fM to 1 nM, the LCR is... MB→A The current value (I) MB Significantly higher than LCR Fc→G The current value (I) Fc ), making I MB / I Fc Greater than 2. In contrast, when clinical samples contain G-contained target (equivalent to...) CYP2C19*1*1 When the concentration of the G-contained target is in the range of 100 fM to 1 nM, the LCR is... Fc→G The current value (I) Fc Significantly higher than LCR MB→A The current value (I) MB ), making I MB / I Fc Less than 1. Furthermore, when clinical samples simultaneously contain both A-contained target and G-contained target (equivalent to...) CYP2C19*1*2 When I MB / I Fc Between 1 and 2, this is caused by a combination of various factors (similar hybridization environment, similar electrochemical responses of electrochemical indicators MB and Fc, etc.). Figure 6 B in the text stands for LCR. MB→A The current value (I) MB The concentrations of the three alleles showed a good linear relationship with the concentrations of the samples in the range of 100 fM-100 pM, and the regression equations were I... MB =279.77+20.29lgC AA (R 2 =0.9982), I MB =270.09+20.32lgC GG (R 2 =0.9997), I MB =337.22+23.46lgC GA (R 2 =0.9997), and the detection limit is 100 fM (current value close to 0 nA).

[0088] Example 5:

[0089] A ratiometric DNA electrochemical sensor for precise determination of swing-type base pair allele genotyping. CYP2C19*2 Clinical Sample Testing: To explore the applicability of the proposed R-eLCR in clinical samples, nine human whole blood clinical samples were collected from the Department of Pharmacy, First Affiliated Hospital of Fujian Medical University. SWV detection was performed on the PCR products. The control group consisted of PCR products containing no target strand. Figure 7 As shown in A and B, based on I from 9 clinical samples MB / I Fc value, CYP2C19*2 The three genotypes can be clearly distinguished, and the detection results are consistent with the gold standard for genotype detection (sequencing method), indicating that the ratio-type DNA electrochemical sensor for accurate determination of swing-type base pair allele genotyping in this invention is stable and reliable.

[0090] The DNA sequences in the sequence listing provided by this invention are known, and the letters and numbers before the first "-" and after the last "-" are omitted. The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A detection method for accurate genotyping of the CYP2C19*2 allele, characterized in that... The steps include: using a ratiometric DNA electrochemical sensor for detection; (1) constructing a ratiometric DNA electrochemical sensor R-eLCR using ligase chain reaction (LCR); (2) selecting ferrocene (Fc) and methylene blue (MB) as electrochemical response signals for LCR, and experimentally verifying that Fc and MB have similar electrochemical signal responses in R-eLCR; (3) designing two LCRs, namely LCR, with CYP2C19*2 as the detection target. Fc→G and LCR MB→A Based on its current ratio (I) MB / I Fc The region is divided into three current ratio intervals, namely I MB / I Fc <1, 1<I MB / I Fc <2,I MB / I Fc >2, in human whole blood genomic DNA testing, it can specifically distinguish the three genotypes of the CYP2C19*2 allele, namely CYP2C19*1*1, CYP2C19*2*2, and CYP2C19*1*2; in designing two LCRs (LCRs) Fc→G and LCR MB→A When probing: For probe LCR MB→A Of the four primer probes designed, probe HT1 and probe HT2 A Each of these represents half of the CYP2C19*2 allele-specific gene fragment A-contained target, while the auxiliary probe AP and the MB-modified signal probe SP are also present. MB With probes HT1 and HT2 respectively A Complementary, the specific sequences and modifying groups of the four primers and probes are shown in Table 1; for probe LCR Fc→G Of the four primer probes designed, probes HT1 and HT2... G Each of these represents half of the CYP2C19*1 allele-specific gene fragment G-contained target, while the auxiliary probe AP and the Fc-modified signal probe SP are also present. Fc With probes HT1 and HT2 respectively G The specific sequences and modification groups of the four primer probes are shown in Table 1. Among them, probe HT1 and probe AP are common probes of the two LCRs. The 5' end of the auxiliary probe AP has a 12 nt sequence that is complementary to the 3' end of CP. The 5' end of CP is modified with a -SH group, so that after the signal probes SP and AP of the two LCRs are connected, the electroactive indicator MB / Fc approaches the electrode surface in an inverted form, undergoes electron transfer, and generates the corresponding electrochemical signal. Table 1 Primer and probe sequences for R-eLCR 。 2. A method for preparing a ratiometric DNA electrochemical sensor for precise genotyping of the CYP2C19*2 allele, characterized in that... The following steps are included: constructing a ratiometric DNA electrochemical sensor R-eLCR; (1) 100 µL of reaction solution containing 1×Ampligase buffer, 1.0 U Ampligase thermostable ligase, 120 nM of four LCR primers and targets is thoroughly vortexed, and then LCR thermal cycling reaction is performed to obtain mixed LCR amplification products; the R-eLCR process is as follows: hybridization ligation at 53 ℃ for 2 min, denaturation at 94 ℃ for 1 min, repeated for 30 cycles; thereafter, an equal volume of LCR MB→A and LCR Fc→G The products were mixed, and 5 µL of 0.2 M phosphate buffer was added to the mixture; (2) Self-assembly of the capture probe CP on the gold electrode (AuE): 3 µL of 350 nM CP was dropped onto the AuE surface and kept in the dark at room temperature for 16 h to form a thiol-DNA self-assembled monolayer; then, it was rinsed with 10 mM phosphate buffer and immersed in 100 µL of 2 mM mercaptohexanol (MCH) solution, sealed and kept in the dark for 2 h to replace the non-specifically adsorbed DNA and passivate the remaining AuE region to obtain the modified AuE; (3) 3 µL of the mixed LCR amplification product obtained in step (1) was dropped onto the modified AuE surface obtained in step (2) at room temperature for 1 h, and finally electrochemical determination was performed by square wave voltammetry; In the design of two LCRs (LCRs) Fc→G and LCR MB→A When probing: For probe LCR MB→A Of the four primers and probes designed, probes HT1 and HT2A each contain half of the CYP2C19*2 allele-specific gene fragment A-contained target, while the auxiliary probes AP and MB-modified signal probe SP... MB With probes HT1 and HT2 respectively A Complementary, the specific sequences and modifying groups of the four primers and probes are shown in Table 1; for probe LCR Fc→G Of the four primer probes designed, probes HT1 and HT2... G Each of these represents half of the CYP2C19*1 allele-specific gene fragment G-contained target, while the auxiliary probe AP and the Fc-modified signal probe SP are also present. Fc With probes HT1 and HT2 respectively G The specific sequences and modification groups of the four primer probes are shown in Table 1. Among them, probe HT1 and probe AP are common probes of the two LCRs. The 5' end of the auxiliary probe AP has a 12 nt sequence that is complementary to the 3' end of CP. The 5' end of CP is modified with a -SH group, so that after the signal probes SP and AP of the two LCRs are connected, the electroactive indicator MB / Fc approaches the electrode surface in an inverted form, undergoes electron transfer, and generates the corresponding electrochemical signal. Table 1 Primer and probe sequences for R-eLCR 。 3. The preparation method according to claim 2 yields a ratiometric DNA electrochemical sensor that can be used for precise typing of the CYP2C19*2 allele.

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