Method and application for melting curve detection without PCR amplification for SNP genotyping based on cationic polythiophene

Abstract

The present application relates to a melting curve detection method and application for SNP genotyping based on cationic polythiophene without PCR amplification. The method includes: S1: designing a probe according to the sequence of the SNP site of the target gene; S2: mixing the probe solution, the cationic polythiophene solution and the working solution of the hybridization buffer solution to obtain a first mixture; S3: performing a hybridization reaction on the first mixture to obtain a double helix complex solution; S4: mixing the genomic DNA solution of the sample to be tested with the double helix complex solution to obtain a second mixture; S5: performing a hybridization reaction on the second mixture to obtain a triple helix complex solution; gradually heating the triple helix complex solution and collecting fluorescence to obtain a melting curve; S6: analyzing the melting curve and judging the genotype of the SNP site of the target gene in the sample to be tested according to the difference in the shape of the melting peak and the Tm value of the melting peak. This method does not require PCR amplification, is simple and easy to operate, has low detection cost, short time consumption, and can provide clinical rapid detection services.

Description

Technical Field

[0001] The present application relates to the technical field of nucleic acid detection, and in particular, to a melting curve detection method and application for SNP genotyping without PCR amplification based on cationic polythiophene. Background Art

[0002] Currently, for the detection of gene SNP sites, common methods include direct sequencing method, gene chip hybridization method, MALDI-TOF MS mass spectrometry technology, PCR-Taqman MGB probe genotyping method, ARMS-PCR method, PCR high-resolution melting curve method, etc. These methods all have more or less defects in popularization, such as high reagent detection cost, complex operation, easy to produce contamination, and certain false negatives and false positives. At the same time, the above detection methods are mainly based on PCR technology, and inevitably require a PCR amplification step. The disadvantages of the PCR detection method are as follows: due to the powerful amplification efficiency of the PCR reaction, it is easy to cause cross-contamination, and extremely trace contamination can lead to false positive results; PCR amplification is very time-consuming, usually about 2-3 hours, accounting for more than half of the entire experimental process, and is not suitable for the field of rapid clinical testing; the detection cost of PCR amplification reagents is high, and the PCR reaction usually requires specific enzymes, primers or probes with special modification labels, buffer, dNTP and other reaction components. Therefore, it is necessary to establish a simple and feasible detection method for SNPs without PCR amplification, so as to improve the detection efficiency and serve rapid clinical testing.

[0003] Although some current PCR-free amplification studies have overcome the deficiencies of PCR, they still require complex steps such as enzymatic reactions, magnetic bead preparation, and complex chemical modifications. Additionally, most of the PCR-free amplification methods for nucleic acid analysis developed by current researchers mainly amplify signals. For example: hybridization capture method (WO 03 / 078966, Digene Corporation, USA), branched DNA (bDNA) method, Monica K et al. comprehensively used Luminex flow cytometry fluorescence technology and dendrimer technology to directly detect target nucleic acids (JOURNAL OF CLINICAL MICROBIOLOGY, July 2005, p. 3255-3259, Vol. 43, No. 7). In addition, Nanosphere, Inc. in the United States has developed commercial instruments and reagents that do not amplify target DNA fragments but amplify signals, enabling direct detection of target nucleic acids (www.nanosphere.us). These methods all require special supporting instruments to detect the generated signals, resulting in high costs. However, it is unrealistic for many users or in many situations to purchase expensive instruments in China. Additionally, high maintenance costs and low usage rates after purchasing the instruments also become a major problem. Chinese Patent CN 101760527 B, a nucleic acid analysis method without PCR amplification and without a detection instrument, enriches the target by a solid-phase carrier (microsphere) and is based on the characteristics of a biotin / avidin reaction carrying a detection probe for signal amplification in an immune reaction to improve the detection sensitivity, but it requires complex special labeling and cumbersome operation steps. Therefore, there is a need to develop a low-cost SNP detection method without PCR amplification.

[0004] Gout is an acute arthritis caused by the deposition of urate crystals in joints due to excessive blood uric acid levels, and allopurinol can effectively treat gout. HLA-B*5801 is a gene that can be used to predict whether allergies will occur during the use of allopurinol. HLA-B*5801 is a leukocyte differentiation antigen. In recent years, many studies at home and abroad have confirmed a strong correlation between it and the risk of severe allergies in patients using allopurinol. The 2012 American College of Rheumatology (ACR) guidelines for gout patients also pointed out that HLA-B*5801 can be used as an effective prediction method to determine whether gout patients can use allopurinol. Therefore, detecting whether gout patients carry HLA-B*5801 is of great significance for guiding drug use.

[0005] Japanese scholars' research found that the rs9263726 allele (G > A) on the PSORS1C1 gene is associated with allopurinol-induced SJS / TEN, and this SNP locus is in complete linkage with HLA-B*5801, that is, people with the GA or AA genotype of rs9263726 must be carriers of the HLA-B*5801 allele, and vice versa. This discovery means that the genotype of the SNP locus of rs9263726 can be used to replace HLA-B*5801 as a predictive indicator for allopurinol-induced severe drug eruption, for individualized medication guidance of allopurinol, and a simple and rapid method for detecting the HLA-B*5801 genotype has been found.

[0006] Currently, the main methods for analyzing and detecting the HLA-B*5801 allele at home and abroad are polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), sequencing method, and fluorescence quantitative PCR method. The sequencing method has high accuracy for the detection results. It relies on large-scale precision instrument equipment, fixed laboratories, and professional operators. Therefore, this detection method has high costs, cumbersome steps, and long time consumption, and it is difficult to be popularized clinically. The PCR-SSCP method is a relatively classical method. DNA is amplified by several pairs of HLA-B*5801 specific primers and analyzed through electrophoresis results. This method has a high risk of contamination and a long detection period, and it is not suitable for clinical use either. CN103805701A discloses a fluorescence quantitative PCR detection method. Although this method has the advantages of low cost, short time, and simple result analysis, since this reaction system can only amplify purified genomic DNA samples and must adopt multi-step and multi-tube operations, it is not ideal for clinical promotion either. Therefore, these conventional detection technologies are difficult to meet the requirements of the domestic population carrying the HLA-B*5801 mutant gene for rapid and accurate HLA-B*5801 gene detection.

[0007] Therefore, there is an urgent need to develop a low-cost SNP detection method without PCR amplification, which is of great significance for rapidly detecting the HLA-B*5801 genotype of gout patients. Summary of the Invention

[0008] To solve the deficiencies of the prior art, this application provides a method for SNP detection using a melting curve technology without PCR amplification. This method innovatively uses cationic polythiophene in the melting curve method to detect SNPs. The detection process does not require any chemical modification of the probe or target analyte, and does not require PCR amplification to increase the fluorescence signal change value. The detection cost is low and the operation is simple. At the same time, the accuracy of the detection results is high. Using existing conventional fluorescence quantitative PCR instruments can provide a cheap analysis system for rapid detection and identification of nucleic acids.

[0009] To this end, the first aspect of the present application provides a melting curve detection method for SNP genotyping based on cationic polythiophene without PCR amplification, and the method includes the following steps:

[0010] S1: Design a probe for the SNP site of the target gene according to the sequence of the SNP site of the target gene;

[0011] S2: After mixing the solution of the probe, the cationic polythiophene solution and the working solution of the hybridization buffer solution, obtain a first mixture; S3: Perform a hybridization reaction on the first mixture in a fluorescence quantitative PCR instrument to obtain a solution containing a double helix complex;

[0012] S4: After mixing the genomic DNA solution of the sample to be tested with the solution containing the double helix complex, obtain a second mixture; S5: Perform a hybridization reaction on the second mixture in a fluorescence quantitative PCR instrument to obtain a solution containing a triple helix complex; Gradually increase the temperature of the solution containing the triple helix complex and collect fluorescence to obtain a melting curve;

[0013] S6: Analyze the melting curve, and judge the genotype of the SNP site of the target gene in the sample to be tested according to the difference in the shape of the melting peak and the Tm value of the melting peak.

[0014] The technical principle of the method detected in the present application is as follows: Cationic polythiophene is an important fluorescent conjugated polymer with strong fluorescence. Under the induction of the substrate, the conformation or aggregation state of the polymer itself changes, and the effective conjugation length of the polymer changes due to the conformational change, which is specifically manifested as the red shift or blue shift of the absorption peak in the spectrum, the quenching and recovery of the fluorescence emission peak, etc. Therefore, DNA can be detected according to the change of the fluorescence spectrum of the polythiophene solution. Since the reason for this detectable fluorescence change of the polymer is due to its conformational change, no labeling process is required.

[0015] When cationic polythiophene forms a double helix structure with the capture probe ssDNA (probe), the emission wavelength redshifts and is accompanied by obvious fluorescence quenching. When cationic polythiophene forms a triple helix structure with the capture probe ssDNA (probe) and the single-stranded genomic DNA to be tested, the emission wavelength blueshifts and is accompanied by significant fluorescence enhancement. As the temperature increases, the probe dissociates, and the triple helix complex structure hybridized on the single-stranded genomic DNA becomes a double helix complex structure formed by the cationic polythiophene solution and the capture probe ssDNA (probe), and the fluorescence signal gradually weakens. Analyze the melting curve, and judge the genotype of the SNP site of the target gene according to the difference in the shape of the melting peak and the Tm value of the melting peak.

[0016] In this application, since the change value of the fluorescence signal intensity caused by the change in the effective conjugation length of the polymer is strong enough that it is not necessary to amplify the fluorescence signal change value through PCR, the method provided in this application can overcome the disadvantages of high detection cost, long time consumption, easy cross-contamination, false positive results, etc. in the existing conventional melting curve technology based on the PCR method.

[0017] In some embodiments, the concentration of the probe in the probe solution is 40 - 60 nM, and the length of the probe is 25 - 35 bp, and the Tm value is 50 - 80 °C.

[0018] The probe in this application does not require any labeling treatment. The probe can be a probe for the wild-type sequence of the SNP site of the target gene or a probe for the mutant sequence of the SNP site of the target gene. When the probe is a probe for the wild-type sequence of the SNP site of the target gene, the probe can specifically bind to the wild-type target gene and form a single-base mismatch with the mutant target gene. Subsequently, in the melting curve step, the mutant mismatched base sequence will dissociate preferentially compared to the wild-type completely matched base sequence, so the Tm value of the mutant sequence is lower than that of the wild-type sequence; when the probe is a probe for the mutant sequence of the SNP site of the target gene, the probe can specifically bind to the mutant target gene and form a single-base mismatch with the wild-type target gene. Subsequently, in the melting curve step, the wild-type mismatched base sequence will dissociate preferentially compared to the mutant completely matched base sequence, so the Tm value of the wild-type sequence is lower than that of the mutant sequence. Therefore, a single specific probe can be used to distinguish wild-type, homozygous mutant, and heterozygous mutant.

[0019] In this application, the length and Tm value of the probe have a greater impact on the detection result. By controlling the length and Tm value of the probe within the above ranges, it is more conducive to the degradation hybridization in the subsequent steps, thereby improving the accuracy of the detection result.

[0020] In some embodiments, the cationic polythiophene in the cationic polythiophene solution is poly(3-[(S)-5-amino-5-carboxy-3-pentyloxy]-2,5-thiophene chloride (abbreviation, POWT), and the concentration of poly(3-[(S)-5-amino-5-carboxy-3-pentyloxy]-2,5-thiophene chloride in the cationic polythiophene solution is (0.8 - 1.2)×10 -6 g / mL; preferably 1.0×10 -6 g / mL.

[0021] In this application, the concentration of POWT in the cationic polythiophene solution used in the detection process has a greater impact on the detection result. Too high or too low a concentration of POWT is not conducive to the sensitivity and accuracy of the detection result.

[0022] In some embodiments, the working solution of the hybrid buffer solution is prepared by diluting the hybrid buffer solution by 10 times. The pH value of the hybrid buffer solution is 8.0 - 9.5, which includes 180 - 220 mM of Tris-HCl, 80 - 120 mM of KCl, 80 - 120 mM of (NH 4 ) 2 SO 4 , 18 - 22 mM of MgSO 4 , 0.8 - 1.2 wt% of Triton X-100 and 0.8 - 1.2 mg / mL of BSA.

[0023] In some preferred embodiments, the pH value of the hybrid buffer solution is 8.8, which includes 200 mM of Tris-HCl, 100 mM of KCl, 100 mM of (NH 4 ) 2 SO 4 , 20 mM of MgSO 4 , 1 wt% of Triton X-100 and 1 mg / mL of BSA.

[0024] The hybrid buffer solution in this application is 10*buffer. Diluting it 10 times with purified water can obtain the working solution of the hybrid buffer solution, 1*buffer. During the hybridization reaction, the components and pH of the hybrid buffer solution both have important effects on the hybridization process. Appropriate components and pH of the hybrid buffer solution will help the conformational conversion of the system and the stability of the double-stranded structure, shortening the hybridization reaction time. The inventors of this application determined the final formulation of the hybrid buffer solution through research. The hybrid buffer solution with the above formulation is more conducive to the conformational conversion and double-stranded structure stability in the hybridization system. At the same time, it is beneficial to enhance the fluorescence emission intensity of cationic polythiophene in the system, and more helpful to reduce the interference of the fluorescence background signal, thereby being conducive to improving the accuracy and sensitivity of detection.

[0025] In the hybrid buffer solution of this application, Tris-HCl is used to adjust the pH of the hybrid buffer solution to maintain the hybridization environment at a slightly alkaline level (pH 8.0 - 9.5), which is beneficial to the stability of the double-helix complex structure in the hybridization reaction and maintains the conformation of cationic polythiophene tending to be planar.

[0026] Triton X-100 in the hybrid buffer solution of this application is a surfactant, which helps to improve the dispersion performance of cationic polythiophene, avoiding the significant quenching of fluorescence intensity due to the self-absorption effect after the aggregation of cationic polythiophene, thereby reducing the sensitivity and accuracy of the detection results.

[0027] Mg in the hybrid buffer solution of this application2+ It is provided in the form of MgSO 4 to further enhance the hybridization ability between the oligonucleotide probe and the cationic polythiophene POWT. A low M g2+ concentration can lead to a weakening of the hybridization ability between the oligonucleotide probe and POWT, dissociation of POWT, and an increase in the background signal of the melting curve reaction. An excessively high Mg 2+ concentration can result in non-specific binding between the oligonucleotide probe and single-stranded template DNA, thereby reducing the accuracy of the detection results.

[0028] In the hybridization buffer of this application, a unique K + / NH 4 + double-cation buffer is adopted. K + can promote the specificity of the binding between the probe and the template DNA, and NH 4 + has a destabilizing effect, especially on the weak hydrogen bonds between the mismatched probe and the template DNA, and can open them. The cooperation of the two enhances the specificity of the hybridization reaction, reduces the background signal of the melting curve analysis after the hybridization reaction, and enhances the change value of the fluorescence signal.

[0029] BSA in the hybridization buffer solution of this application can reduce the situation where the hybridization reactants adhere to the test tube wall and improve the hybridization reaction efficiency.

[0030] In some embodiments, the volume ratio of the probe solution, the cationic polythiophene solution, and the working solution of the hybridization buffer solution is (1-3):(1-3):1. In some preferred embodiments, the volume ratio of the probe solution, the cationic polythiophene solution, and the working solution of the hybridization buffer solution is 2:2:1.

[0031] By controlling the volume ratio of the probe solution, the cationic polythiophene solution, and the working solution of the hybridization buffer solution within the above range, this application can be more conducive to the hybridization of the probe and the cationic polythiophene to form a double helix complex.

[0032] In some embodiments, in step S3, the program of the hybridization reaction is: denaturation at 95°C for 2-8 minutes, incubation at 45-55°C for 5-15 minutes.

[0033] In some preferred embodiments, in step S3, the program of the hybridization reaction is: denaturation at 95°C for 5 minutes, incubation at 50°C for 10 minutes.

[0034] In this application, denaturing at 95°C for 2 to 8 minutes can fully denature the probes in the hybridization reaction system, which is more conducive to subsequent binding to cationic polythiophene. Incubating at 45 to 55°C for 5 to 15 minutes can enable the fully denatured probes in the hybridization reaction system to form a stable double helix complex PT1 with cationic polythiophene through electrostatic attraction, which makes the POWT conformation tend to be planar, and the self-absorption effect leads to a decrease in fluorescence intensity.

[0035] In some embodiments, in step S4, the concentration of DNA in the genomic DNA solution of the test sample is 20 to 70 ng / μL, and the volume ratio of the genomic DNA solution of the test sample to the solution containing the double helix complex is (1 to 2):1, preferably 1.5:1.

[0036] In this application, the concentration and purity of the genomic DNA of the test sample affect the accuracy of the detection result. Only after the concentration and purity of the genomic DNA of the test sample meet the requirements can it be effectively detected. In this application, the OD 260 / OD 280 value of the genomic DNA solution of the test sample should be between 1.6 and 2.0, and the concentration should be between 20 and 70 ng / μL, preferably 50 ng / μL.

[0037] In this application, the method for extracting genomic DNA from the test sample is a conventional method for those skilled in the art. For example, a commercial extraction kit can be used to extract the genomic DNA from the test sample.

[0038] In addition, by controlling the volume ratio of the genomic DNA solution of the test sample and the solution containing the double helix complex within the above range in this application, it is more conducive to the hybridization of genomic DNA with the probe and cationic polythiophene, and then a triple helix complex is formed.

[0039] In some embodiments, in step S5, the procedure of the hybridization reaction during the melting curve acquisition is: denaturing at 95°C for 2 to 8 minutes, incubating at 45 to 55°C for 1 to 5 minutes, heating from 45 to 55°C to 85 to 95°C, and collecting fluorescence signals at a heating rate of 0.04°C / s to 0.06°C / s.

[0040] In some preferred embodiments, in step S5, the procedure of the hybridization reaction during the melting curve acquisition is: denaturing at 95°C for 5 minutes, incubating at 50°C for 2 minutes, heating from 50°C to 90°C, and collecting fluorescence signals at a heating rate of 0.06°C / s.

[0041] In this application, denaturing at 95 °C for 2 - 8 minutes can denature and unwind the double-stranded genomic DNA in the hybridization system into single-stranded DNA, and separate the probe in the double-helix complex PT1 from the cationic polythiophene. Subsequently, incubating at 45 - 55 °C for 1 - 5 minutes enables the separated probe to preferentially bind to the unwound genomic single-stranded DNA to form a double-stranded helix (dsDNA). Then, the dsDNA and the cationic polythiophene POWT form a triple-helix complex PT2, with the conformation tending to be non-planar and the fluorescence intensity of the system increasing. In this application, fluorescence signals are collected during the heating process. As the temperature gradually rises, the triple-helix complex structure PT2 hybridized on the single-stranded genomic DNA changes into the double-helix complex structure PT1 formed by the cationic polythiophene and the capture probe ssDNA (probe). The conformation of the cationic polythiophene POWT changes from tending to be non-planar to tending to be planar, and the fluorescence signal intensity gradually decreases. The melting curve can be obtained based on the fluorescence signals collected during the heating program. Therefore, the hybridization reaction program in step S5 is also called the melting curve reaction program.

[0042] The method described in this application can directly detect nucleic acid targets without PCR amplification during the detection process, overcoming the drawback of the high detection cost of PCR amplification reagents in the PCR amplification step, reducing reagent costs and detection costs, lowering the detection fees for patients, having low technical requirements for operators, wide adaptability, simple operation and clear interpretation in clinical detection, and being suitable for promotion to clinical units or basic medical units for relevant rapid POCT detection. At the same time, the detection time of this method is short and the speed is fast. The sample can be detected within half an hour after being put into the instrument. Without using expensive detection instruments, it can provide a low-cost analysis system for the rapid detection and identification of nucleic acids, and is widely applicable to promotion and application in various medical, scientific research and other fields.

[0043] It should be noted that: the method described in this application is for non-disease treatment and diagnosis purposes.

[0044] The second aspect of this application provides an application of the melting curve detection method for SNP genotyping based on cationic polythiophene without PCR amplification as described in the first aspect of this application in the detection of HLA-B*5801 genotype based on the POCT mode.

[0045] The method described in this application can quickly and accurately determine the genotype of the SNP locus of the target gene, and the genotype of the SNP locus of rs9263726 is completely linked to HLA-B*5801. People with the GA or AA genotype of rs9263726 must be carriers of the HLA-B*5801 allele. Therefore, the genotype of the SNP locus of rs9263726 in gout patients can be detected by using the method described in this application, and indirectly used for the detection of the HLA-B*5801 genotype in gout patients. At the same time, since the method described in this application is suitable for clinical units or basic medical units to perform relevant rapid POCT tests, the method described in this application can be well applied to the detection of the HLA-B*5801 genotype based on the POCT mode.

[0046] In some embodiments, the sequence of the probe used in the detection is as shown in SEQ ID NO.1.

[0047] In this application, by using the probe shown in SEQ ID NO.1 for detection, the accuracy of the detection result can be further improved.

[0048] Similarly, applying the method described in this application to the detection of the HLA-B*5801 genotype is only to obtain the HLA-B*5801 genotype of gout patients, not for the purpose of disease diagnosis.

[0049] The beneficial technical effects of this application are as follows:

[0050] (1) The method described in this application innovatively uses cationic polythiophene in the melting curve method to detect SNPs. During the detection process, the reason for the change in the detectable fluorescence signal of the cationic polythiophene polymer is based on the conformational change of the conjugated main chain of the cationic polythiophene when mixed with single-stranded or double-stranded (hybridized) oligonucleotides. Therefore, no chemical modification is required for the probe or the target analyte. It solves the problem that the traditional melting curve analysis methods of TaqMan probes or molecular beacons require special fluorescence group and quenching group labeling at both ends of the probe, and these traditional methods all have certain limitations, such as the small signal difference between the hybridized state and the free state of TaqMan probes.

[0051] (2) The method provided in this application can directly detect nucleic acid targets without PCR amplification, has a short detection time, high speed, and can achieve instant detection. All 96 samples can be detected within half an hour after being put on the machine. Therefore, it can provide doctors with medication reference in the first time and reduce the medication risk of patients.

[0052] (3) The method provided in this application can overcome the disadvantages of high detection cost, complex labeling, long time consumption, easy cross-contamination, and false positive results in the conventional melting curve technology based on the PCR method.

[0053] (4) The method provided by this application has low technical requirements for operators, wide adaptability, simple operation and clear interpretation in clinical testing, and is suitable for promotion to clinical units or basic medical units for relevant rapid POCT testing.

[0054] (5) The detection method provided by this application can improve the accuracy of reagent detection results, accurately distinguish wild-type homozygotes, mutant homozygotes and heterozygotes of SNP gene loci, and has the advantages of convenient operation, small interference, high sensitivity and strong specificity.

[0055] (6) The specific probe provided by this application has high sensitivity, strong specificity and strong anti-interference ability, can effectively distinguish background signals, has clear and simple interpretation, and realizes accurate identification of target targets through closed-tube detection throughout the process.

[0056] (7) This application is widely applicable to molecular fields with rapid detection requirements such as personalized medicine, respiratory pathogens, HPV genotyping detection, and deafness gene detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The flow chart and schematic diagram of detecting a sample to be detected by using the method described in this application in Example 1.

[0058] Figure 2 The melting curve graph of the GG wild type at the rs9263726 locus in Sample 2.

[0059] Figure 3 The melting curve graph of the AA mutant at the rs9263726 locus in Sample 3.

[0060] Figure 4 The melting curve graph of the GA heterozygous type at the rs9263726 locus in Sample 4.

[0061] Figure 5 The first-generation sequencing result graph of the GG wild type at the rs9263726 locus in Sample 2.

[0062] Figure 6 The first-generation sequencing result graph of the AA mutant at the rs9263726 locus in Sample 3.

[0063] Figure 7 The first-generation sequencing result graph of the GA heterozygous type at the rs9263726 locus in Sample 4.

[0064] Figure 8 The comparison graph of the melting curve results of Sample 5 when the hybridization buffer solution contains 100 mM NaCl and 100 mM KCl respectively.

[0065] Figure 9When the hybridization buffer solution contains 100 mM (NH 4 ) 2 SO 4 and 50 mM (NH 4 ) 2 SO 4 respectively, it is a comparison graph of the melting curve results of sample 21.

[0066] Figure 10 When the hybridization buffer solution contains 10 mM MgSO 4 , 20 mM MgSO 4 and 30 mM MgSO 4 respectively, it is a comparison graph of the melting curve results of sample 6.

[0067] Figure 11 When the concentration of the probe in the probe solution is 50 nM and 10 nM respectively, it is a comparison graph of the melting curve results of sample 7.

[0068] Figure 12 When the volumes of the added genomic DNA solution are 15 μL and 30 μL respectively, it is a comparison graph of the melting curve results of sample 14. Detailed implementation mode

[0069] To make the present application easier to understand, the following will further elaborate on the present application in combination with embodiments. These embodiments are only illustrative and are not limited to the application scope of the present application. The raw materials or components used in the present application can be obtained through commercial channels or conventional methods without special instructions.

[0070] Embodiment 1: Rapid detection of HLA-B*5801 genotype based on the POCT mode

[0071] The process and schematic diagram for detecting the sample to be tested are as Figure 1 shown.

[0072] Step 1: According to the reference sequence of the rs9263726 locus disclosed in the nucleic acid sequence database GeneBank of the National Center for Biotechnology Information (NCBI) in the United States, the MEGA4 software was used for homologous sequence alignment, and the PrimerExpress 3.0 software of ABI Company was used to design specific probes for detecting the HLA-B*5801 allele respectively. The relevant specific sequences are as follows:

[0073] Wild-type probe: 5’-ACTCCGACTTCCGCAAGGCCTTCCGGCACT-3’ (SEQ ID NO:1);

[0074] Step 2: DNA extraction. Collect clinical blood samples from 30 patients with hyperuricemia (the samples are from a central hospital), and use a commercial blood extraction kit to extract genomic DNA from the 30 blood samples respectively. After the genomic DNA is extracted, use an ultraviolet spectrophotometer to measure the concentration and purity of the extracted DNA. The extracted DNA needs to be measured for concentration and purity with an ultraviolet spectrophotometer. The purity of the DNA, the value of OD 260 / OD 280 should be between 1.6 and 2.0, and the concentration should be between 20 and 70 ng / μL. Samples with unqualified DNA quality cannot be used for detection. The DNA with qualified quality inspection is stored for future use at -20°C. The extraction results of genomic DNA from 30 clinical blood samples in this example are shown in Table 1. Mark each sample during the extraction process to avoid sample confusion.

[0075] Table 1: Extraction results of genomic DNA from 30 clinical blood samples

[0076]

[0077]

[0078] Step 3: Centrifuge the synthesized 2 OD dry powder wild-type probe at 12,000 rpm for 3 min, add Vt1 (Vt1 = the nanomole number N shown on the label × 10, unit: μL) volume of purified water, vortex for 20 s, and centrifuge briefly to prepare a 100 μM probe mother solution. Dilute the probe mother solution to 50 nM with 1*TE to obtain the probe solution for use.

[0079] Step 4: Prepare an aqueous solution of cationic polythiophene POWT at 1.0×10 -6 g / mL with pure water as the solvent; at the same time, prepare a 10* hybridization buffer solution for the hybridization of cationic polythiophene POWT and the wild-type probe. The formula of the 10* hybridization buffer solution is as follows:

[0080]

[0081] Dilute the 10* hybridization buffer solution with purified water to the working solution of the hybridization buffer solution, that is, 1* hybridization buffer solution, and store it for future use.

[0082] Step 5: According to the hybridization reaction system described in Table 1, calculate the number of reactions required according to the number of test samples. For example, if the number of samples is n, then n + 2 reactions need to be done (including a positive control reaction and a no-template control reaction), calculate the volume of the hybridization reaction solution required, and prepare the reaction system. After the reaction system is mixed evenly, centrifuge it at 3000 rpm for 30 seconds, and aliquot it into PCR tubes, 25 μl per tube. The specific hybridization reaction system is shown in Table 2 below.

[0083] Table 2: Hybridization reaction system

[0084]

[0085]

[0086] Step 6: The hybridization reaction solution described in step 5 is subjected to hybridization reaction in a fluorescent quantitative PCR instrument to obtain a stable solution containing a double helix complex. The specific hybridization reaction procedure is shown in Table 3 below.

[0087] Table 3: Hybridization reaction procedure

[0088] Step Procedure Temperature (°C) Time (min:sec) Number of cycles 1 Pre-denaturation 95 5:00 1 2 Incubation 50 10:00 1

[0089] Step 7: After the reaction is completed, 15 μL of qualified genomic DNA solution and 10 μL of the solution containing the double helix complex formed in step 6 are respectively taken and mixed thoroughly (the volume ratio of the genomic DNA solution to the solution containing the double helix complex is 1.5:1), and then placed in a PCR instrument for hybridization reaction and melting curve analysis. The specific melting curve reaction procedure is shown in Table 4 below.

[0090] Table 4: Melting curve reaction program

[0091]

[0092] Step 8: According to the shape of the target gene melting peak and the Tm value range of the melting peak, the genotype of the rs9263726 site in the genomic DNA of the sample to be tested is interpreted, thereby indirectly obtaining the HLA-B*5801 genotype. The interpretation method is shown in Table 5 below.

[0093] Table 5: HLA-B*5801 gene melting peak Tm value range and genotype comparison table

[0094]

[0095] The first-generation sequencing method was used to test 30 clinical samples, and then the accuracy of the results of the present application method was compared and verified.

[0096] Among them, the detection process of first-generation sequencing is as follows:

[0097] In a clean bench, prepare the PCR reaction mixture according to Table 6 below. After oscillation and instant centrifugation, divide the mixture into the corresponding number of PCR 8-tube strips according to the number of samples, 23 μL per well, and mark them.

[0098] Table 6: First-generation sequencing reaction system

[0099] Serial number Name Working concentration Final concentration 1 person dose 1 CXF1 25 μM 0.2 μM 0.2 μL 2 CXR1 25 μM 0.2 μM 0.2 μL 3 2*Rapid Taq Master Mix 1 mL - 12.5 μL 4 ddH2O - - 10.1 μL 5 Template - - 2 μL

[0100] After the reaction mixture is prepared, it is transferred to the sample preparation room through the transfer window. In the biological safety cabinet, the extracted DNA sample is added to an eight-well tube, 2 μL per well. After tightly covering the tube cap, it is centrifuged instantaneously. During the operation, bubbles should be avoided to ensure that there is no liquid adhering to the tube wall. Then it is transferred to the PCR amplification area and amplified according to the PCR amplification procedure in Table 7 below. After the amplification is completed, the reaction solution is labeled and entrusted to a third-party biological company for sequencing.

[0101] Table 7: PCR Sequencing Amplification Procedure

[0102]

[0103] The melting curve detection results of the HLA-B*5801 allele in the clinical samples of 30 patients with hyperuricemia using the method of this application were compared and statistically analyzed with the detection results of first-generation sequencing. The results are shown in Tables 8 and 9 below.

[0104] Table 8: Comparison Results of the Detection Results of the Method of this Application and First-generation Sequencing for 30 Clinical Samples

[0105]

[0106]

[0107] Table 9: Statistical Results of the Detection Results of the Method of this Application and First-generation Sequencing for 30 Clinical Samples

[0108]

[0109] As shown in Tables 8 and 9, after comparison, the positive coincidence rate and negative coincidence rate of the results of detecting the gene HLA-B*5801 by the method described in this application and the detection results of first-generation sequencing are both 100%. It shows that the method of this application has high detection accuracy and strong operability. Figures 5 - 7 Figure of the first-generation sequencing results for clinical samples 2 - 4, compared with the method of this application Figures 2 - 4 It can be seen that the melting curve detection results of the HLA-B*5801 of the samples by the method of this application are completely consistent with the first-generation sequencing results, and the accuracy meets the requirements of clinical applications. In addition, the method of this application performs melting curve detection on 30 samples simultaneously, with a total time consumption of less than half an hour. The detection time is greatly shortened compared with the sequencing method, and up to 96 samples can be detected simultaneously at most, and the detection efficiency is increased several times.

[0110] Example 2: Rapid Detection of HLA-B*5801 Genotype Based on the POCT Mode

[0111] The formula of the hybridization buffer solution 10*buffer used in the detection process is as follows:

[0112]

[0113] Test sample: Sample 5 in Example 1;

[0114] Detection steps: Basically the same as in Example 1, except that the formulation of the hybridization buffer 10*buffer in Example 1 is replaced with the formulation of the above-mentioned hybridization buffer 10*buffer in this example.

[0115] Detection result: The melting curve of Sample 5 obtained after the detection is as Figure 8 shown. As can be seen from Figure 8 , for the same test sample 5, after replacing 100 mM NaCl in the hybridization buffer with 100 mM KCl, there are significant differences in the melting curve detection results, that is, after replacing with 100 mM KCl, the melting peak height of the melting curve increases by about 100. It shows that compared with NaCl, adding KCl to the hybridization buffer is more conducive to enhancing the fluorescence intensity of the melting curve analysis after the hybridization reaction and reducing the background signal of the melting curve analysis after the hybridization reaction, thereby improving the sensitivity and accuracy of the detection results.

[0116] Example 3: Rapid detection of HLA-B*5801 genotype based on the POCT mode

[0117] The formulation of the hybridization buffer 10*buffer used in the detection process is as follows:

[0118]

[0119] Test sample: Sample 21 in Example 1;

[0120] Detection steps: Basically the same as in Example 1, except that the formulation of the hybridization buffer 10*buffer in Example 1 is replaced with the formulation of the above-mentioned hybridization buffer 10*buffer in this example.

[0121] Detection result: The melting curve of Sample 21 obtained after the detection is as Figure 9 shown. As can be seen from Figure 9 , for the same test sample 21, the melting peak height of the melting curve of the sample treated with the hybridization buffer containing 100 mM (NH 4 ) 2 SO 4 is about 30 higher than that of the melting curve of the sample treated with the hybridization buffer containing 50 mM (NH 4 ) 2 SO 4 . It shows that when (NH 4 ) 2 SO4 When the concentration is 100 mM, it is more conducive to enhancing the fluorescence signal intensity of the melting curve analysis after the hybridization reaction, thereby improving the sensitivity and accuracy of the detection results.

[0122] Example 4: Rapid detection of HLA-B*5801 genotype based on the POCT mode

[0123] The formulation of the hybridization buffer solution 10*buffer used in the detection process is as follows:

[0124] Group A: Hybridization buffer containing 10 mM MgSO 4

[0125]

[0126] Group B: Hybridization buffer containing 30 mM MgSO 4

[0127]

[0128] Detection sample: Sample 6 in Example 1;

[0129] Detection steps: Basically the same as in Example 1, except that the formulation of the hybridization buffer solution 10*buffer in Example 1 is replaced with the formulations of the above two hybridization buffer solutions 10*buffer in this example.

[0130] Detection result: The melting curve of sample 6 obtained after the detection is as Figure 10 shown. From Figure 10 it can be seen that for the same sample 6, after being treated with the hybridization buffer containing 10 mM MgSO 4 , 20 mM MgSO 4 and 30 mM MgSO 4 , there are obvious differences in the results of the melting curves. That is, the melting peak height of the melting curve of the sample treated with the hybridization buffer containing 20 mM MgSO 4 is increased by about 37 compared with the melting peak height of the melting curve of the sample treated with the hybridization buffer containing 10 mM MgSO 4 . And the melting curve of the sample treated with the hybridization buffer containing 30 mM MgSO 4 has a non-specific melting peak with a melting peak height of 23.76 at 65.6 °C, and the Tm value of the melting peak is significantly shifted to the left by 0.5 °C compared with the target signal melting peak, and the melting peak height is reduced by about 23 compared with the melting curve of the sample treated with the hybridization buffer containing 20 mM MgSO 4 . It shows that when MgSO 4When the concentration is 20 mM, it is more conducive to improving the sensitivity and accuracy of the detection results.

[0131] Example 5: Rapid Detection of HLA-B*5801 Genotype Based on POCT Mode

[0132] Detection sample: Sample 7 in Example 1;

[0133] Detection steps: Basically the same as in Example 1, except that in step 3, the probe mother liquor is diluted to 10 nM with 1*TE to obtain the probe solution for use. In step 5, 10 μL of the 10 nM probe solution, 10 μL of the cationic polythiophene POWT solution, and 5 μL of the hybrid buffer working solution 1*buffer are fully mixed and homogenized.

[0134] Detection result: The melting curve of sample 7 obtained after the detection is as Figure 11 shown. From Figure 11 it can be seen that for the same sample 7, the melting peak height of the melting curve of the 50 nM probe solution is increased by about 144 compared with that of the 10 nM probe solution, and the fluorescence signal intensity difference is significant. It shows that when the concentration of the probe in the probe solution is 50 nM, it is more conducive to enhancing the fluorescence signal intensity of the melting curve analysis after the hybridization reaction, thereby improving the sensitivity and accuracy of the detection results.

[0135] Example 6: Rapid Detection of HLA-B*5801 Genotype Based on POCT Mode

[0136] Detection sample: Sample 14 in Example 1;

[0137] Detection steps: Basically the same as in Example 1, except that in step 6, 30 μL of the genomic DNA solution with a DNA concentration of 50 ng / μL is fully mixed and homogenized with 10 μL of the solution containing the double helix complex (the volume ratio of the genomic DNA solution to the solution containing the double helix complex is 3:1).

[0138] Detection result: The melting curve of sample 14 obtained after the detection is as Figure 12 shown. From Figure 12 it can be seen that for the same sample 15, the melting peak height of the melting curve of the 15 μL genomic DNA solution is increased by about 25 compared with that of the 30 μL genomic DNA solution, and the fluorescence signal intensity difference is significant. When the volume ratio of the genomic DNA solution to the solution containing the double helix complex is controlled at 1.5:1, it is more conducive to enhancing the fluorescence signal intensity of the melting curve analysis after the hybridization reaction, thereby improving the sensitivity and accuracy of the detection results.

[0139] It should be noted that the above-described embodiments are only used to explain the present application and do not constitute any limitation to the present application. The present application has been described by referring to exemplary embodiments, but it should be understood that the words used therein are descriptive and explanatory words rather than limiting words. Modifications can be made to the present application within the scope of the claims of the present application as provided, and the present invention can be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. A method for detecting melting curves without PCR amplification based on cationic polythiophene for SNP genotyping for non-disease diagnosis purposes, Characterized in that, The method comprises the following steps: S1: According to the sequence of the SNP site of the target gene, design a probe for the SNP site of the target gene; S2: After mixing the solution of the probe, the solution of cationic polythiophene and the working solution of the hybridization buffer solution, obtain a first mixture; S3: Perform a hybridization reaction on the first mixture in a fluorescence quantitative PCR instrument to obtain a solution containing a double helix complex; S4: After mixing the genomic DNA solution of the test sample with the solution containing the double helix complex, obtain a second mixture; S5: Perform a hybridization reaction on the second mixture in a fluorescence quantitative PCR instrument to obtain a solution containing a triple helix complex; gradually heat the solution containing the triple helix complex and collect fluorescence to obtain a melting curve; S6: Analyze the melting curve and judge the genotype of the SNP site of the target gene in the test sample according to the difference in the shape of the melting peak and the Tm value of the melting peak; The working solution of the hybrid buffer solution is prepared by diluting the hybrid buffer solution by 10 times. The pH value of the hybrid buffer solution is 8.0-9.5, and it includes 180-220 mM of Tris-HCl, 80-120 mM of KCl, 80-120 mM of (NH 4 ) 2 SO 4 , 18-22 mM of MgSO 4 , 0.8-1.2 wt% of Triton X-100 and 0.8-1.2 mg / mL of BSA; The cationic polythiophene in the cationic polythiophene solution is poly(3-[(S)-5-amino-5-carboxy-3-pentyloxy]-2,5-thiophene chloride).

2. The method according to claim 1, Characterized in that, The concentration of the probe in the solution of the probe is 40-60 nM, and the length of the probe is 25-35 bp, and the Tm value is 50-80 °C.

3. The method according to claim 1 or 2, Characterized in that, The concentration of poly(3-[(S)-5-amino-5-carboxy-3-pentyloxy]-2,5-thiophene chloride) in the cationic polythiophene solution is (0.8~1.2)×10 -6 g / mL.

4. The method according to claim 3, Characterized in that, The concentration of poly(3-[(S)-5-amino-5-carboxy-3-pentyloxy]-2,5-thiophene chloride) in the cationic polythiophene solution is 1.0×10 -6 g / mL.

5. The method according to claim 1 or 2, Characterized in that, The pH value of the hybrid buffer solution is 8.8, which includes 200 mM Tris-HCl, 100 mM KCl, 100 mM (NH 4 ) 2 SO 4 , 20 mM MgSO 4 , 1 wt% Triton X-100 and 1 mg / mL BSA.

6. The method according to claim 1 or 2, Characterized in that, The volume ratio of the solution of the probe, the solution of cationic polythiophene and the working solution of the hybridization buffer solution is (1-3):(1-3):

1.

7. The method according to claim 6, Characterized in that, The volume ratio of the solution of the probe, the solution of cationic polythiophene and the working solution of the hybridization buffer solution is 2:2:

1.

8. The method according to claim 1 or 2, Characterized in that, In step S3, the hybridization reaction program is: denaturation at 95 °C for 2-8 minutes, incubation at 45-55 °C for 5-15 minutes.

9. The method according to claim 1 or 2, Characterized in that, In step S4, the concentration of DNA in the genomic DNA solution of the test sample is 20-70 ng / μL, and the volume ratio of the genomic DNA solution of the test sample to the solution containing the double helix complex is (1-2):

1.

10. The method according to claim 9, Characterized in that, The volume ratio of the genomic DNA solution of the test sample to the solution containing the double helix complex is 1.5:

1.

11. The method according to claim 1 or 2, Characterized in that, In step S5, the procedures of the hybridization reaction and temperature increase during the melting curve acquisition are as follows: denaturation at 95°C for 2 to 8 minutes, incubation at 45 to 55°C for 1 to 5 minutes, temperature increase from 45 to 55°C to 85 to 95°C, and fluorescence signals are collected at a temperature increase rate of 0.04°C / s to 0.06°C / s.

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