A method for detecting CRP2C19 gene polymorphism based on VPCR-Cas13a
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DALIAN UNIV OF TECH
- Filing Date
- 2022-12-15
- Publication Date
- 2026-06-26
Smart Images
Figure CN116287125B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of analytical detection technology, specifically relating to a method for detecting CYP2C19 gene polymorphism based on VPCR-Cas13a. Background Technology
[0002] Stroke is the leading cause of death and disability among Chinese residents. The latest guidelines and expert consensus recommendations for the treatment of acute stroke, primary prevention, and secondary prevention all mention antiplatelet aggregation therapy. Clopidogrel, as an antiplatelet aggregation drug, is widely used in clinical treatment. Clopidogrel requires the CYP450 enzyme family to metabolize it into its active substance to exert its antiplatelet aggregation effect, with the CYP2C19 enzyme playing a major role. However, single nucleotide polymorphisms (SNPs) in the CYP2C19 gene encoding the CYP2C19 enzyme can affect the metabolism of clopidogrel. The most common genotypes are CYP2C19*2 (c.681G>A), CYP2C19*3 (c.636G>A), and CYP2C19*17 (c.806C>T), with frequencies of approximately 23.1-35%, 2-7%, and 0.5-4% respectively in the Chinese population. CYP2C19*2 and CYP2C19*3 mutations reduce the metabolic activity of the CYP2C19 enzyme in clopidogrel, requiring increased dosage or alternative medications. Conversely, CYP2C19*17 mutations increase clopidogrel metabolic activity, raising the risk of bleeding and necessitating reduced dosage. Therefore, CYP2C19 genotyping-guided individualized antiplatelet therapy is of great significance for stroke patients.
[0003] Currently, the main established genotyping methods are gene sequencing and quantitative polymerase chain reaction (qPCR). However, gene sequencing is time-consuming, complex, and costly, making it unsuitable for rapid clinical genotyping. qPCR suffers from drawbacks such as non-specific amplification and poor specificity at low template concentrations. Therefore, there is an urgent need to develop a rapid and highly specific method for detecting CYP2C19 gene polymorphisms to guide clinical medication. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides a method for detecting CYP2C19 gene polymorphism based on VPCR-Cas13a. First, this invention designs and screens crRNAs corresponding to wild-type and mutant CYP2C19*2, *3, and *17 gene polymorphic sites, respectively, to improve the specificity of Cas13a for CYP2C19 genotyping. Subsequently, V-type polymerase chain reaction (VPCR) is used to amplify the polymorphic sites (CYP2C19*2, *3, *17) of clopidogrel metabolism-related genes. RNA is generated through transcription; the target RNA can activate the CRISPR-Cas13a system, releasing a fluorescent signal, while RNA with single-base mismatches cannot activate the CRISPR-Cas13a system, and the signal is turned off, thereby achieving rapid and accurate genotyping of clopidogrel metabolism-related genes CYP2C19.
[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:
[0006] This invention provides a crRNA screening method to improve the specificity of Cas13a for detecting CYP2C19 standard ssRNA, comprising the following steps:
[0007] (1) Introduce one or more base mutations on one or both sides of the site where crRNA binds to the CYP2C19 allele.
[0008] (2) The crRNA obtained in step (1) is combined with Cas13a for the recognition and detection of CYP2C19 standard ssRNA, activating the trans-cleavage activity of Cas13a, cleaving FQ-ssRNA signal molecules, releasing fluorescence, and screening for crRNAs that can improve the specificity of Cas13a for the detection of CYP2C19 standard ssRNA.
[0009] Based on the above technical solution, the crRNA in step (1) further includes the nucleotide sequence shown in SEQ ID NO.7 to SEQ ID NO.24.
[0010] Based on the above technical solution, the standard ssRNA in step (2) further includes the nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.6.
[0011] Based on the above technical solution, further, in step (2), the FQ-ssRNA signaling molecule is FAM-UUUUU-BHQ1.
[0012] Based on the above technical solution, further, in step (2), the concentration of Cas13a is 30-50 nM, the concentration of crRNA is 10-30 nM, the concentration of target ssRNA is 50-200 pM, the ribonuclease inhibitor is 0.1-10 U / μL, and the concentration of FQ-ssRNA signaling molecule is 50-300 nM.
[0013] This invention also provides a method for detecting CYP2C19 gene polymorphism based on VPCR-Cas13a, comprising the following steps:
[0014] 1) Using the designed VPCR primers, VPCR was used to amplify the standard ssDNA corresponding to the positive strand of the CYP2C19*2, *3, and *17 gene polymorphism sites;
[0015] 2) Add the VPCR product obtained in step 1) directly to the T7 transcription-Cas13a detection system and perform genotyping based on the detected fluorescence intensity.
[0016] Based on the above technical solution, further, the standard ssDNA corresponding to the positive strand in step 1) is used as the DNA template for VPCR amplification, including the nucleotide sequences shown in SEQ ID NO.25 to SEQ ID NO.30.
[0017] Based on the above technical solution, further, the VPCR primers in step 1) include nucleotide sequences as shown in SEQ ID NO.31 to SEQ ID NO.36.
[0018] Based on the above technical solution, further, the VPCR amplification conditions in step 1) are 94℃ for 1s, 50℃ for 1s thermal cycling for 30-40 rounds, and finally 72℃ for 5min extension.
[0019] Based on the above technical solution, further, in step 1), the concentration of Taq DNA polymerase for VPCR amplification is 0.01-1 U / μL, the concentration of the upstream primer is 0.5-2 μM, the concentration of the downstream primer is 0.5-2 μM, the concentration of dNTP is 100-1000 μM, and the concentration of the standard ssDNA template is 10-30 pM.
[0020] Based on the above technical solution, further, the T7 transcription-Cas13a detection system described in step 2) mainly includes Cas13a, crRNA, T7 RNA polymerase and FQ-ssRNA signaling molecules.
[0021] Based on the above technical solution, the crRNA further includes the nucleotide sequences shown in SEQ ID NO.7, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.18, SEQ ID NO.21, and SEQ ID NO.22.
[0022] Based on the above technical solution, further, the concentration of Cas13a is 30-60 nM, the concentration of crRNA is 10-30 nM, the VPCR product is 1-10 μL, the ribonuclease inhibitor is 1-10 U / μL, the NTP is 100-1000 μM, the T7 RNA polymerase is 0.1-10 U / μL, and the concentration of FQ-ssRNA signaling molecule is 50-300 nM.
[0023] Compared with the prior art, the beneficial effects of the present invention are:
[0024] 1. This invention combines “V-type” PCR (VPCR) with Cas13a to establish a rapid, highly specific, and highly sensitive method for detecting gene polymorphism in CYP2C19. It only requires 1 second of reaction at two temperatures: denaturation and annealing. The DNA polymerase extension reaction is completed in a dynamic process with varying temperatures. Compared with traditional PCR amplification, this method can save 2 / 3 of the time and greatly improve the reaction speed.
[0025] 2. The detection method of the present invention has good specificity. The optimal crRNA sequence provided can improve the specificity of Cas13a for the target gene and can effectively distinguish the single base difference of CYP2C19*2, *3, and *17 gene polymorphic sites, resulting in more accurate genotyping detection results. This is of great significance for guiding the precise use of clopidogrel.
[0026] 3. The detection method of the present invention has the advantage of rapid response, which greatly shortens the time required by the traditional PCR-CRISPR-Cas detection method. The detection can be completed within 60 minutes. Compared with the traditional gene sequencing and Southern blotting methods, the response speed is greatly improved.
[0027] 4. The detection method of the present invention has the advantage of high-throughput detection. Utilizing a 96-well plate of an ELISA reader, it has the potential to detect multiple samples simultaneously, thereby improving the detection capability for clinical samples. Attached Figure Description
[0028] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.
[0029] Figure 1The diagram below illustrates the principle of the CYP2C19 gene polymorphism detection method based on VPCR-Cas13a described in Example 1. In this diagram, a is a schematic diagram of the detection method flow, b is the detection of CYP2C19 gene polymorphism, and c is the VPCR amplification cycle process.
[0030] Figure 2 Characterization of LbuCas13a as described in Example 2; where a is the SDS-PAGE characterization of LbuCas13a, and b is the fluorescence kinetic characterization of LbuCas13a in the control experiment.
[0031] Figure 3 The sequences and fluorescence intensity of the crRNAs targeting wild-type and mutant alleles screened in Example 3 are shown below; where a to c are crRNAs targeting wild-type alleles of CYP2C19*2, CYP2C19*3 and CYP2C19*17 respectively; e to f are crRNAs targeting mutant alleles of CYP2C19*2, CYP2C19*3 and CYP2C19*17 respectively.
[0032] Figure 4 The wild-type and mutant sensor sequences and fluorescence kinetics of CYP2C19*2, CYP2C19*3 and CYP2C19*17 based on Cas13a constructed in Example 4 are shown. Among them, a to c are wild-type sensor sequences targeting CYP2C19*2, CYP2C19*3 and CYP2C19*17 respectively; e to f are mutant sensors targeting CYP2C19*2, CYP2C19*3 and CYP2C19*17 respectively.
[0033] Figure 5 This is a feasibility verification of the genotyping method based on VPCR-Cas13a constructed in Example 5; where a is the feasibility of amplifying CYP2C19*2, CYP2C19*3 and CYP2C19*17 sites by the VPCR method, b is the fluorescence kinetics of detecting wild-type CYP2C19*2, CYP2C19*3 and CYP2C19*17 sites by the VPCR-Cas13 wild-type sensor, and c is the fluorescence kinetics of detecting mutant CYP2C19*2, CYP2C19*3 and CYP2C19*17 sites by the VPCR-Cas13 mutant sensor.
[0034] Figure 6 The performance characterization of the VPCR-Cas13 wild-type sensor constructed in Example 6 is shown in Figure 6; where a represents the sensitivity characterization of the wild-type sensor and b represents the specificity characterization of the wild-type sensor.
[0035] Figure 7The performance characterization of the VPCR-Cas13 mutant sensor constructed in Example 6 is shown in Figure 6; where a represents the sensitivity characterization of the mutant sensor and b represents the specificity characterization of the mutant sensor. Detailed Implementation
[0036] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.
[0037] Examples include the expression, purification, and activity verification of LbuCas13a; screening crRNA to improve the specificity of CYP2C19 gene polymorphism detection of Cas13a; kinetic characterization of CYP2C19 gene polymorphism detection based on optimal crRNA-Cas13a; construction of a CYP2C19 gene polymorphism detection method based on VPCR-Cas13a; and performance evaluation of VPCR-Cas13a wild-type and mutant sensors.
[0038] Table 1: Nucleic acid sequences used in this invention
[0039]
[0040]
[0041]
[0042]
[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0044] The Cas13a reaction buffer (1×CRB) in the following examples is: 20 mM HEPES, 50 mM potassium chloride, 5 mM magnesium chloride, 10 μg / mL bovine serum albumin, 10 μg / mL yeast tRNA, 0.01% (v / v) IGEPA CA-630, 5% glycerol, pH 7.
[0045] The PCR amplification buffer (1×PB) in the following examples is: 20mM Tris hydrochloric acid, 10mM ammonium sulfate, 1mg / mL bovine serum albumin, 100mM potassium chloride, 2mM magnesium sulfate, 1% Triton X-100, pH 8.8.
[0046] The T7 RNA polymerase transcription reaction buffer (1×TB) in the following examples is: 40mM Tris hydrochloric acid, 6mM magnesium chloride, 1mM dithiothreitol, 2mM spermidine, pH 7.9.
[0047] Example 1: Technical route for constructing a CYP2C19 gene polymorphism detection method based on the PCR-T7 transcription-Cas13a system
[0048] The experimental principle of this invention is as follows: Figure 1 As shown, based on the above principles, the following technical approach is designed:
[0049] (1) Express and purify LbuCas13a, and test the bioactivity of Cas13a;
[0050] (2) Screening for specific crRNAs to improve the specificity of CRISPR-Cas13a in detecting wild-type / mutant alleles corresponding to CYP2C19*2, *3, *17 polymorphic sites, so that the signal response can reach the best signal-to-noise ratio.
[0051] (3) Design PCR primers to characterize the PCR amplification products of CYP2C19 gene polymorphic sites;
[0052] (4) Construct a PCR-T7 transcription-Cas13a detection system to detect wild-type / mutant alleles corresponding to the CYP2C19*2, *3, and *17 gene polymorphic sites, verify the feasibility, and evaluate the detection performance such as sensitivity and specificity.
[0053] Example 2: Expression, purification, and activity verification of LbuCas13a
[0054] (1) Screening for crRNAs targeting wild-type alleles
[0055] The p2CT-His-MBP-Lbu_C2c2_WT (Addgene#83482) expression vector was transformed into *E. coli* BL21(DE3) cells and grown in 2×YT broth at 37°C until the OD600 reached 0.6–0.8. Cells were induced with 0.5 mM IPTG and then transferred to 16°C for overnight expression. Cells were collected by centrifugation, and the cell particles were resuspended in lysis buffer (50 mM Tris-Cl pH 7.0, 500 mM NaCl, 5% glycerol, 1 mM TCEP, 0.5 mM PMSF, and EDTA-free protease inhibitor), sonicated on ice for 20 min, and centrifuged at 13,000 rpm for 30 min. The supernatant of the lysate was mixed with Ni-NTA affinity resin (GE Healthcare, USA) and incubated on ice for approximately 30 min. Soluble His6-MBP-TEV-Cas13a was separated by metal ion affinity chromatography. To remove the His6-MBP tag, the protein-containing eluent was incubated overnight with TEV protease at 4°C while dialyzing into ion exchange buffer (50 mM Tris-Cl pH 7.0, 250 mM KCl, 5% glycerol, 1 mM TCEP). The cleaved protein was loaded onto a HiTrap SP column (GE Healthcare) and eluted on a linear KCl gradient (0.25–1.5 M). Cas13a was pooled, concentrated, and further purified by size exclusion chromatography on a HiLoad Superdex 200 (GE Healthcare) in gel filtration buffer (20 mM Tris-Cl pH 7.0, 200 mM KCl, 5% glycerol, 1 mM TCEP), and then stored at -80°C.
[0056] Subsequently, the expressed and purified product was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as follows: Figure 2 As shown in figure a, the purified protein has a molecular weight of approximately 135 kDa, which is consistent with the molecular weight of LbuCas13a.
[0057] (2) Cas13a activity verification
[0058] First, 4 pmol of Cas13a and 2 pmol of crRNA (R2M1) were added to a 100 μL 1×CRB reaction system containing 1 U / μL of a ribonuclease inhibitor (RRI), and incubated at 37°C for 10 minutes to prepare the Cas13a-crRNA complex. Then, 10 fmol of CYP2C19 wild-type standard ssRNA (WT-ssRNA2) and 15 pmol of FQ-ssRNA reporter group were added. Fluorescence intensity was measured in real-time at 37°C for 120 minutes using a Spark multi-mode microplate reader, with fluorescence measurements performed every 5 minutes (λ). ex : 485nm; λ em (520nm).
[0059] Control experiment: Except for not adding Cas13a / crRNA / WT-ssRNA2, the other steps are the same as above.
[0060] Experimental results are as follows Figure 2 As shown in b, the trans-cleavage activity of Cas13a can only be activated when crRNA is present with the target RNA molecule, which is consistent with the literature reports, indicating that the LbuCas13a protein was successfully synthesized and has good biological activity.
[0061] Example 3: Screening crRNA to improve the specificity of CYP2C19 gene polymorphism detection of Cas13a.
[0062] Since Cas13a lacks the ability to recognize single bases, mismatches are often introduced into the crRNA to improve specificity. This invention first designs a mutation involving one or two bases flanking the allele binding site of the crRNA. Subsequently, through experimental verification, crRNAs that can improve the signal-to-noise ratio of Cas13a's response to single-base mismatches are screened. The specific experimental steps are as follows:
[0063] (1) Screening for crRNAs targeting wild-type alleles
[0064] First, 4 pmol of Cas13a and 2 pmol of crRNA (R2M1 / R2M2 / R2M3 / R3M1 / R3M2 / R3M3 / R17M1 / R17M2 / R17M3) were added to a 100 μL 1×CRB reaction system containing 1 U / μL of ribonuclease inhibitor (RRI), and incubated at 37°C for 10 minutes to prepare the Cas13a-crRNA complex. Then, 10 fmol of CYP2C19 wild-type standard ssRNA (WT-ssRNA2 / WT-ssRNA3 / WT-ssRNA17) and 15 pmol of FQ-ssRNA reporter group were added, and the reaction was carried out at 37°C for 2 hours. The fluorescence intensity (λ) was then measured using a Spark multi-mode microplate reader.ex : 485nm; λ em (520nm).
[0065] Control experiment: Except for the addition of 10 fmol of CYP2C19 mutant standard ssRNA (Mut-ssRNA2 / Mut-ssRNA3 / Mut-ssRNA17), the other steps are the same as above.
[0066] The results are as follows Figure 3 As shown in a to 3c, when detecting wild-type targets of CYP2C19*2, *3, and *17, using the designed R2M1, R3M3, and R17M2 as crRNAs can achieve the best signal-to-noise ratio for Cas13a detection. The fluorescence intensity of wild-type CYP2C19*2, *3, and *17 is ~17, ~8, and ~57 times higher than that of mutants, respectively.
[0067] (2) Screening for crRNAs targeting mutant alleles
[0068] First, 4 pmol of Cas13a and 2 pmol of crRNA (Mut_R2M1 / Mut_R2M2 / Mut_R2M3 / Mut_R3M1 / Mut_R3M2 / Mut_R3M3 / Mut_R17M1 / Mut_R17M2 / Mut_R17M3) were added to a 100 μL 1×CRB reaction system containing 1 U / μL of ribonuclease inhibitor (RRI), and incubated at 37°C for 10 minutes to prepare the Cas13a-crRNA complex. Then, 10 fmol of CYP2C19 mutant standard ssRNA (Mut-ssRNA2 / Mut-ssRNA3 / Mut-ssRNA17) and 15 pmol of FQ-ssRNA reporter group were added, and the reaction was carried out at 37°C for 2 hours. The fluorescence intensity (λ) was then measured using a Spark multi-mode microplate reader. ex : 485nm; λ em (520nm).
[0069] Control experiment: Except for the addition of 10 fmol of CYP2C19 wild-type standard ssRNA (WT-ssRNA2 / WT-ssRNA3 / WT-ssRNA17), the other steps are the same as above.
[0070] The results are as follows Figure 3 As shown in c to 3e, when detecting mutant targets of CYP2C19*2, *3, and *17, using the designed Mut_R2M3, Mut_R3M3, and Mut_R17M1 as crRNA can achieve the best signal-to-noise ratio for Cas13a detection. The fluorescence intensity of the CYP2C19*2, *3, and *17 mutants is ~7, ~6, and ~11 times higher than that of the wild type, respectively.
[0071] Example 4: Kinetic Characterization of CYP2C19 Gene Polymorphism Detection Based on Optimal crRNA-Cas13a
[0072] (1) Kinetic characterization of CYP2C19 wild-type allele detection based on the optimal crRNA-Cas13a system
[0073] First, 4 pmol of Cas13a and 2 pmol of crRNA (R2M1 / R3M3 / R17M2) were added to a 100 μL 1×CRB reaction system containing 1 U / μL of ribonuclease inhibitor (RRI), and incubated at 37°C for 10 minutes to prepare the Cas13a-crRNA complex. Then, 10 fmol of CYP2C19 wild-type standard ssRNA (WT-ssRNA2 / WT-ssRNA3 / WT-ssRNA17) and 15 pmol of FQ-ssRNA reporter group were added. Immediately, fluorescence intensity was measured in real-time at 37°C for 120 minutes using a Spark multi-mode microplate reader, with fluorescence measurements every 5 minutes (λ). ex : 485nm; λ em (520nm).
[0074] Control experiment: Except for the addition of 10 fmol of CYP2C19 mutant standard ssRNA (Mut-ssRNA2 / Mut-ssRNA3 / Mut-ssRNA17), the other steps are the same as above.
[0075] The results are as follows Figure 4 As shown in a to 4c, when detecting wild-type targets of CYP2C19*2, *3, and *17, R2M1, R3M3, and R17M2, which have the best signal-to-noise ratio, were selected as crRNAs. The addition of wild-type standard ssRNA activated Cas13a to trans-cleave the FQ-ssRNA signaling molecule, and the fluorescence intensity continuously increased over time, reaching saturation after 120 minutes. However, the mutant standard ssRNA could not activate Cas13a and did not release a fluorescent signal.
[0076] (2) Kinetic characterization of CYP2C19 mutant allele detection based on the optimal crRNA-Cas13a system
[0077] First, 4 pmol of Cas13a and 2 pmol of crRNA (Mut_R2M3 / Mut_R3M3 / Mut_R17M1) were added to a 100 μL 1×CRB reaction system containing 1 U / μL of ribonuclease inhibitor (RRI), and incubated at 37°C for 10 minutes to prepare the Cas13a-crRNA complex. Then, 10 fmol of CYP2C19 mutant standard ssRNA (Mut-ssRNA2 / Mut-ssRNA3 / Mut-ssRNA17) and 15 pmol of FQ-ssRNA reporter group were added. Immediately, fluorescence intensity was measured in real-time at 37°C for 120 minutes using a Spark multi-mode microplate reader, with fluorescence measurements performed every 5 minutes (λ). ex : 485nm; λ em (520nm).
[0078] Control experiment: Except for the addition of 10 fmol of CYP2C19 wild-type standard ssRNA (WT-ssRNA2 / WT-ssRNA3 / WT-ssRNA17), the other steps are the same as above.
[0079] The results are as follows Figure 4 As shown in e-4g, when detecting mutant targets of CYP2C19*2, *3, and *17, Mut_R2M3, Mut_R3M3, and Mut_R17M1, which have the best signal-to-noise ratio, were selected as crRNAs. The addition of mutant standard ssRNA activated Cas13a to trans-cleave the FQ-ssRNA signaling molecule, and the fluorescence intensity continuously increased over time, reaching saturation after 120 minutes. In contrast, wild-type standard ssRNA could not activate Cas13a and did not release a fluorescent signal.
[0080] Example 5: Construction of a CYP2C19 gene polymorphism detection method based on VPCR-Cas13a
[0081] (1) VPCR amplification of standard ssDNA corresponding to the polymorphic sites of CYP2C19*2, *3, and *17 genes.
[0082] Each 50 μL volume contained 5 U Taq polymerase, 1 μM FP (FP2 / FP3 / FP17), 1 μM RP (RP2 / RP3 / RP17), 500 μM dNTPs, 20 pM of different standard ssDNAs (WT-ssDNA2, WT-ssDNA3, WT-ssDNA17, Mut-ssDNA2, Mut-ssDNA3, Mut-ssDNA17), and 1×PB. Subsequently, 30 thermal cycles of 94℃ for 1 s and 50℃ for 1 s were performed on a Bio-Rad T100 PCR instrument, followed by a final extension at 72℃ for 5 min (time ~30 min). PCR products were separated and characterized by 2% agarose gel electrophoresis, and the results are shown below. Figure 5 As shown in Figure a, the wild-type and mutant standard ssDNA corresponding to CYP2C19*2, *3, and *17 can all be amplified by PCR, producing clear bands, and the negative control wells do not contain PCR products.
[0083] (2) T7 transcription-Cas13a detection
[0084] First, 4 pmol Cas13a and 2 pmol crRNA (R2M1 / R3M3 / R17M2 / Mut_R2M3 / Mut_R3M3 / Mut_R17M1) were added to a 100 μL 1×CRB reaction system containing 1 U / μL of ribonuclease inhibitor (RRI), and incubated at 37°C for 10 minutes to prepare the Cas13a-crRNA complex. Then, 5 μL of the above VPCR product, 2 μL of 25 mM NTP, 2 μL of 50 U / μL T7 RNA polymerase, and 15 pmol of FQ-ssRNA reporter were added. Immediately, fluorescence intensity was measured in real-time at 37°C for 20 minutes using a Spark multi-mode microplate reader, with fluorescence measurements performed every 2.5 minutes (λ). ex : 485nm; λ em (520nm).
[0085] The results are as follows Figure 5 As shown in b and 5c, the VPCR products of wild-type and mutant standard ssDNA corresponding to CYP2C19*2, *3, and *17 can be directly used for T7 transcription-Cas13a detection. The reaction generally reaches the optimal signal-to-noise ratio within 15 minutes, achieving target detection. The entire process from VPCR amplification to T7 transcription-Cas13a detection takes less than 60 minutes, demonstrating rapid response capabilities. Furthermore, by using a 96-well plate with a microplate reader, multiple samples can be tested simultaneously, showcasing its potential for clinical application.
[0086] Example 6: Performance Characterization of CYP2C19 Gene Polymorphism Detection Based on VPCR-Cas13a
[0087] (1) Performance characterization of CYP2C19 wild-type gene sensor based on VPCR-Cas13a
[0088] First, 4 pmol of Cas13a and 2 pmol of crRNA (R2M1 / R3M3 / R17M2) were added to a 100 μL 1×CRB reaction system containing 1 U / μL of ribonuclease inhibitor (RRI), and incubated at 37°C for 10 minutes to prepare the Cas13a-crRNA complex. Subsequently, different concentrations of WT-ssRNA2, WT-ssRNA3, WT-ssRNA17, Mut-ssRNA2, Mut-ssRNA3, Mut-ssRNA17, and 15 pmol of FQ-ssRNA reporter group were added, and the reaction was carried out at 37°C for 2 hours. The fluorescence intensity (λ) was then measured using a Spark multi-mode microplate reader. ex : 485nm; λ em (520nm).
[0089] The results are as follows Figure 6 As shown, the detection method for CYP2C19 wild-type constructed based on VPCR-Cas13a has a sensitivity of 100 copies / μL and responds only to WT-ssDNA2 / WT-ssDNA3 / WT-ssDNA17, while showing no response to other standard ssDNAs, thus exhibiting good selectivity.
[0090] (2) Performance characterization of the CYP2C19 mutant gene sensor based on VPCR-Cas13a
[0091] Except for adding 2 pmol crRNA (Mut_R2M3 / Mut_R3M3 / Mut_R17M1) respectively, the other steps are the same as in Example 6(1).
[0092] The results are as follows Figure 7 As shown, the CYP2C19 mutant detection method constructed based on VPCR-Cas13a has a sensitivity of 100 copies / μL and responds only to Mut-ssDNA2 / Mut-ssDNA3 / Mut-ssDNA17, while showing no response to other standard ssDNAs, thus exhibiting good selectivity.
[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. VPCR primers, crRNA, and ssDNA were used in the preparation of reagents for detecting CYP2C19.
2.
3. The application of 17-gene polymorphism in products is characterized by, The nucleotide sequences of ssDNA are shown in SEQ ID NO.25~SEQ ID NO.30; the nucleotide sequences of VPCR primers are shown in SEQ ID NO.31~SEQ ID NO.36; and the nucleotide sequences of crRNA are shown in SEQ ID NO.7, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.18, SEQ ID NO.21 and SEQ ID NO.22.