Method and application of high-resolution melting curve combined with mutation amplification system to detect EXT1 gene and / or EXT2 gene polymorphism

Abstract

The present invention provides a method and application for detecting EXT1 gene and / or EXT2 gene polymorphism by combining a high-resolution melting curve with a mutation amplification system; the method comprises using the nucleic acid of a sample to be tested as a template DNA, performing a PCR reaction and a melting curve analysis, performing real-time fluorescence collection during the PCR reaction and the melting curve process, and drawing an amplification curve and a melting curve for result interpretation. The present invention utilizes asymmetric amplification to realize a melting curve, analyzes the polymorphism of mutation sites 1018C>T and 1019G>A of the EXT1 gene, has the advantages of being simple and fast in detection method, and does not require multiple enrichment during amplification, effectively avoiding the problem of environmental aerosol contamination of the sample to be tested, and the detection result is more accurate, realizing the analysis of mutation sites of HEM pathogenic genes EXT1 and EXT2 at an early stage, and providing a reliable experimental means for the clinical detection of multiple osteochondromatosis.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method and application for detecting EXT1 gene and / or EXT2 gene polymorphism by combining a high-resolution melting curve with a mutation amplification system. Background Art

[0002] Hereditary multiple exostoses (HME) is an autosomal dominant genetic disease, a benign hamartoma. The disease is more common in males aged 0-20 years old, with a male-female ratio of about 3:1. The site of disease is often found in the long bones of the limbs, especially around the knee joint, such as the distal femur and the proximal tibia and fibula, and manifests as palpable bone masses. Studies have shown that the cause of HME is autosomal dominant inheritance, and about 62% of patients have a positive family history. Studies have shown that about 90% of HME patients can detect EXT1 gene and EXT2 gene mutations, and the correlation of EXT1 gene mutations in patients with severe HME is greater than that of ETX2 gene mutations and those without mutations. In the study of HME patients, some mutations may be related to the number and severity of the patient's tumors. HME is mostly a benign bone tumor, but the risk of deterioration in adult patients is extremely high. After deterioration, peripheral chondrosarcoma or osteosarcoma will form in the pelvis and femur in most cases, which is life-threatening. Therefore, the study of HME patients provides an important reference for clinicians to conduct more accurate intervention and treatment at an early stage. In summary, the detection of EXT1 gene and EXT2 gene mutation spectrum is of great significance in the early diagnosis and management of hereditary multiple osteochondroma, and the above research can also be expected to achieve more comprehensive and accurate genetic testing with the continuous advancement of technology, providing a strong reference value for clinical practice.

[0003] Chinese invention patent CN116287280A discloses an EXT1 or EXT2 diagnostic kit based on the mRNA level of a single-cell transcriptome and its application method. The patent uses multiple sets of primers and probes for PCR amplification, and then sequences and analyzes the products to obtain the SNP site mutation status on the EXT1 gene or EXT2 gene. The method has the characteristics of high specificity and high sensitivity, and has a high success rate in clinical detection, so it has profound application value; however, the method requires multiple enrichments during amplification, and the PCR products are inevitably exposed to the experimental environment during the enrichment process, so it is also easy to cause aerosol contamination of the experimental environment, and the final product must be sequenced, so the time required is also increased accordingly.

[0004] Therefore, it is of great significance to provide a method for detecting SNP site mutations on the EXT1 gene and / or EXT2 gene that is simple to operate, rapid and safe. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides a method and application for detecting EXT1 gene and / or EXT2 gene mutations by combining a high-resolution melting curve with a mutation amplification system. The detection method is simple and rapid, and can realize the analysis of early mutation sites of HEM pathogenic genes EXT1 gene and EXT2 gene. In particular, the analysis method does not require opening the cover, and can effectively avoid aerosol contamination during the experiment, providing a more reliable experimental means for the clinical detection of hereditary multiple osteochondromatosis.

[0006] To achieve the above purpose, the present invention provides the following technical solutions: a method for detecting EXT1 gene and / or EXT2 gene polymorphism by high-resolution melting curve combined with mutation amplification system, using the nucleic acid of the sample to be tested as template DNA, performing PCR reaction and melting curve analysis, performing real-time fluorescence collection during the PCR reaction and melting curve process, and drawing amplification curve and melting curve to interpret the results. The present invention uses asymmetric amplification to realize melting curve, analyzes the polymorphism of mutation sites 1018C>T, 1019G>A of EXT1 gene, and simultaneously analyzes EXT1 gene 1469delT and EXT2 gene 67C>T, 679G>A, 1225delG in FAM, VIC, ROX, CY5 fluorescence channels.

[0007] The interpretation of the results includes the interpretation of the mutation sites 1018C>T, 1019G>A, 1469delT of the EXT1 gene and / or the interpretation of the mutation sites 67C>T, 679G>A, 1225delG of the EXT2 gene.

[0008] Preferably, the PCR amplification reaction system includes a primer-probe combination, a PCR buffer, an enzyme solution, a positive control substance, a negative control substance, and a sample lysis solution.

[0009] Preferably, in the primer-probe composition, an artificial mutation site is introduced at the 5' adjacent base of the mutation site to be detected and analyzed by the probe to amplify the mutation signal and improve the sensitivity of the detection; at the same time, the base of the artificial mutation site is modified with a locked nucleic acid. Specifically, the artificial mutation site includes: taking the probes with sequence numbers such as SEQ ID NO.: 15 and SEQ ID NO.: 20 as examples, the wild sequences of the mutation sites of the above two sequences are CGTGGT+ C+GC , after mutation, it should be CGTGGT+ C+AC , CGTGGT+ T+GC, CGTGGT+ T+AC Any of the three, but in actual detection, the detection and analysis of only a single base mutation is more difficult, so the present invention replaces the base T before the mutation site with A, that is, the wild sequence CGTGG T +C+GC was changed to the artificial mutant sequence CGTGG A +C+GC); and adjacent to the possible mutation site, when the original gene mutates, the bases at the adjacent position also mismatch due to artificial mutation, thereby amplifying the mutation signal and improving the recognition strength of the primer or probe and the sensitivity of detection.

[0010] Preferably, the primer-probe combination is separately purified to HPLC grade.

[0011] Preferably, the sample lysate is a mixture comprising EDTA, Triton X-100, NaCl, PMSF, Leupeptin, and SDS.

[0012] Preferably, the PCR amplification reaction system includes a first PCR amplification reaction system and a second PCR amplification reaction system.

[0013] Preferably, the first PCR amplification reaction system includes A reaction solution, PCR buffer, enzyme solution, positive control substance, and negative control substance.

[0014] Preferably, the second PCR amplification reaction system includes B reaction solution, PCR buffer, enzyme solution, positive control substance, and negative control substance.

[0015] Preferably, the primer-probe combination comprises a non-mutated primer-probe combination and a mutated primer-probe combination.

[0016] Preferably, the non-mutated primer-probe combination comprises primers containing non-mutated EXT1 gene or EXT2 gene.

[0017] Preferably, the mutation primer-probe combination includes primers for the mutated EXT1 gene or EXT2 gene.

[0018] Preferably, the probe is a common probe.

[0019] Preferably, the A reaction solution contains the non-mutated primer-probe combination.

[0020] Preferably, the B reaction solution contains the mutation primer-probe combination.

[0021] Preferably, the mutation sites of the EXT1 gene or EXT2 gene primers containing mutation detection include 1018C>T, 1019G>A, 1469delT of the EXT1 gene and 67C>T, 679G>A, 1225delG of the EXT2 gene;

[0022] Preferably, in the non-mutated primer-probe combination, the sequences of the primers are shown in SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, SEQ ID NO.: 8, and SEQ ID NO.: 9;

[0023] Preferably, the primer sequences in the mutation primer-probe combination are shown in SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 7, SEQ ID NO.: 10 to SEQ ID NO.: 14;

[0024] Preferably, the sequence of the probe is shown in SEQ ID NO.: 15 to SEQ ID NO.: 20.

[0025] Preferably, the method for detecting EXT1 gene and / or EXT2 gene polymorphism by combining high-resolution melting curve with mutation amplification system comprises the following steps:

[0026] S01, nucleic acid extraction: take sample lysis solution, add 1 mL of plasma sample to be tested, heat at 95-100℃ for 20 min, let stand and take the supernatant for use;

[0027] S02, reagent preparation: take reaction solution A, reaction solution B, PCR buffer, and enzyme solution, and prepare reaction mixture A and reaction mixture B respectively;

[0028] S03, amplification detection: the nucleic acid sample and negative control extracted in step S01 are added to the reaction mixture A and reaction mixture B obtained in step S02, and the wild-type positive control and mutant positive control are added to prepare the first PCR amplification reaction system and the second PCR amplification reaction system, and the detection is performed on a fluorescent quantitative PCR instrument;

[0029] Step S04, interpreting and analyzing the test results.

[0030] Preferably, in S2, the PCR buffer contains KCl, dNTPs, MgSO 4 , Tween-20, Tris-HCl mixture;

[0031] Preferably, the enzyme solution is a TE buffer solution containing Taq DNA polymerase; the concentration of the enzyme is 8U

[0032] Preferably, in S03, the wild-type positive control is a TE buffer solution of a wild-type plasmid containing EXT1 gene and EXT2 gene; the mutant positive control is a TE buffer solution of a mutant plasmid containing EXT1 gene and EXT2 gene, and the mutation refers to 1018C>T, 1019G>A, 1469delT mutations in the EXT1 gene and 67C>T, 679G>A, 1225delG mutations in the EXT2 gene.

[0033] Preferably, the amplified sequence of the wild-type plasmid in the positive control is as shown in SEQ ID NO.21.

[0034] Preferably, the amplified sequence of the mutant plasmid is shown as SEQ ID NO.22.

[0035] Preferably, the negative control is sterile water for injection;

[0036] Preferably, the wild-type plasmid concentration in the wild-type positive control and the mutant plasmid concentration in the mutant positive control are both 10 6 ±5000 copies / mL.

[0037] Preferably, the PCR reaction program is: 95°C×1min, 1cycle; 95°C×5s, 60°C×30s, 45cycles; 95°C×5s, 1cycle; 30-45°C heating for melting curve, with a heating rate of 0.03°C / s; wherein, in the 60°C×30s and 30-45°C heating processes, FAM, VIC, ROX, and CY5 fluorescence channels are used to collect fluorescence.

[0038] Preferably, the method for interpreting the first PCR amplification reaction system includes:

[0039] When the melting curve temperature of the FAM fluorescence channel peaks at 40±0.5°C, it indicates that there is no mutation at sites 1018 and 1019 of the EXT1 gene;

[0040] When the Ct value of the amplification curve of the FAM fluorescence channel is <38, it indicates that the 1469 site of the EXT1 gene has not mutated;

[0041] When the Ct value of the amplification curve of the VIC fluorescence channel is less than 38, it indicates that the 67th site of the EXT2 gene has not mutated;

[0042] When the Ct value of the amplification curve of the ROX fluorescence channel is <38, it indicates that the 679 site of the EXT2 gene has not mutated;

[0043] When the Ct value of the amplification curve of the CY5 fluorescence channel is less than 38, it indicates that the 1225 site of the EXT2 gene has not mutated.

[0044] Alternatively, the method for interpreting the second PCR amplification reaction system includes:

[0045] When the melting curve temperature of the FAM fluorescence channel peaks at 34-36°C, it indicates that mutations have occurred at sites 1018 and 1019 of the EXT1 gene;

[0046] When the Ct value of the amplification curve of the FAM fluorescence channel is less than 38, it indicates that the 1469 site of the EXT1 gene has mutated;

[0047] When the Ct value of the VIC fluorescence channel amplification curve is less than 38, it indicates that the 67th site of the EXT2 gene has mutated;

[0048] When the Ct value of the amplification curve of the ROX fluorescence channel is <38, it indicates that the 679 site of the EXT2 gene has not mutated;

[0049] When the Ct value of the amplification curve of the CY5 fluorescence channel is less than 38, it indicates that the 1225 site of the EXT2 gene has mutated;

[0050] Alternatively, if the detection results of the first PCR system and the second PCR system are both judged to be positive at the same site, it indicates that a heterozygous mutation has occurred at the site.

[0051] As one of the purposes of the invention, the present invention also provides a diagnostic kit, including an inner lining and an outer packaging box; the inner lining includes multiple container holes, which are respectively used to hold A reaction solution, B reaction solution, PCR buffer, enzyme solution, wild-type positive control, mutant positive control and negative control, etc.

[0052] As one of the purposes, the present invention also provides a method for detecting EXT1 gene and / or EXT2 gene polymorphism by combining the above-mentioned high-resolution melting curve with a mutation amplification system for application in detecting EXT1 gene and / or EXT2 gene for non-disease diagnosis purposes.

[0053] The technical solution provided by the present invention has the following beneficial effects compared with the prior art:

[0054] 1. The present invention adopts asymmetric amplification combined with a mutation amplification system, applies melting curve detection to analyze the polymorphism of 1018C>T, 1019G>A of EXT1 gene, and utilizes the principle of mutation amplification system to analyze the 1469delT mutation of EXT1 gene and the 67C>T, 679G>A, 1225delG polymorphism of EXT2 gene in FAM, VIC, ROX, and CY5 fluorescence channels. Not only is the detection method simple, but it only needs to add the nucleic acid sample to be tested and then run according to the preset reaction program on the fluorescent PCR instrument. There is no need to perform multiple enrichment during amplification, and there is no need to open the cover during the detection process, which effectively avoids the problem of environmental aerosol contamination of the sample to be tested, and the detection result is more accurate.

[0055] 2. By adopting the technical solution of the present invention, an artificial mutation site is introduced at the 5' adjacent base of the mutation site detected and analyzed by the probe, and the base of the mutation site is modified with a locked nucleic acid to enhance the recognition strength of the primer or probe. At this time, if the site mutates, the introduced artificial mutation site will promote the recognition effect of the entire primer or probe on the mutation, thereby achieving the accuracy of the detection result and significantly improving the accuracy of the detection.

[0056] 3. The method provided by the present invention for detecting EXT1 gene and / or EXT2 gene polymorphism using a high-resolution melting curve combined with a mutation amplification system can achieve early diagnosis of EXT1 gene 1018C>T, 1019G>A, 1469delT and EXT2 gene 67C>T, 679G>A, 1225delG, and has low detection cost, requiring only the cost of two sets of reaction reagents to achieve result judgment, and the detection can be performed on a conventional fluorescent quantitative PCR instrument, with high versatility and simple result interpretation. Most importantly, there is no need to open the cover during the detection process, and there is no risk of contamination. It can be applied to all medical institutions with clinical PCR laboratories, providing a reliable experimental means for the diagnosis of clinical hereditary multiple osteochondromatosis, and is suitable for large-scale promotion and application.

[0057] 4. For early prevention and treatment and early screening of HME patients, especially for infants and young children, early detection and early intervention can be carried out to prevent and reduce the incidence of HME. The method for genetic testing of multiple osteochondromatosis provided by the present invention can accurately analyze and detect EXT1 gene and EXT2 gene mutations in HME patients, which is of great preventive and clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the result that no mutation occurred at sites 1018 and 1019 of the EXT1 gene in Example 1 of the present invention, and a peak value appeared at 40±0.5°C in the figure.

[0059] Figure 2This is the result that no mutation occurred at position 1469 of the EXT1 gene in Example 1 of the present invention, and Ct in the figure is less than 38.

[0060] Figure 3 This is the result that no mutation occurred at site 67 of the EXT2 gene in Example 1 of the present invention, and Ct in the figure is less than 38.

[0061] Figure 4 This is the result that no mutation occurred at position 679 of the EXT2 gene in Example 1 of the present invention, and Ct in the figure is less than 38.

[0062] Figure 5 This is the result that no mutation occurred at site 1225 of the EXT2 gene in Example 1 of the present invention, and Ct in the figure is less than 38.

[0063] Figure 6 This is the result of 1018C>T occurring in the EXT1 gene in Example 1 of the present invention, and a peak value appears at 34.5±0.5°C in the figure.

[0064] Figure 7 This is the result of 1019G>A occurring in the EXT1 gene in Example 1 of the present invention, and a peak value appears at 35.5±0.5°C in the figure.

[0065] Figure 8 This is the result of 1469delT in the EXT1 gene in Example 1 of the present invention, where Ct is less than 38.

[0066] Fig. 9 This is the result of 67C>T occurring in the EXT2 gene in Example 1 of the present invention, where Ct<38.

[0067] Fig.10 This is the result of 679G>A occurring in the EXT2 gene in Example 1 of the present invention, where Ct<38.

[0068] Fig.11 This is the result of 1225delG occurring in the EXT2 gene in Example 1 of the present invention, where Ct is less than 38.

[0069] Fig.12 Schematic diagram of the structure of the kit of Example 1 of the present invention. DETAILED DESCRIPTION

[0070] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0071] The first object of the present invention is to provide a method for detecting EXT1 gene and / or EXT2 gene polymorphism using a high-resolution melting curve combined with a mutation amplification system. The second object of the present invention is to address the above-mentioned problems existing in the prior art and provide a kit for detecting EXT1 gene and / or EXT2 gene polymorphism using a high-resolution melting curve combined with a mutation amplification system.

[0072] The first object of the present invention can be achieved by the following technical solutions: A method for detecting EXT1 gene and / or EXT2 gene polymorphism using a high-resolution melting curve combined with a mutation amplification system, comprising a first PCR amplification reaction system and a second PCR amplification reaction system;

[0073] The first PCR amplification reaction system includes A reaction solution, PCR buffer, enzyme solution, wild-type positive control substance, and negative control substance.

[0074] The second PCR amplification reaction system includes B reaction solution, PCR buffer, enzyme solution, mutant positive control substance, and negative control substance.

[0075] Preferably, the first PCR amplification reaction system and the second PCR amplification reaction system are used together with the same sample lysis solution.

[0076] Preferably, the sample lysate is a mixture of 1.2 mM EDTA, 1% Triton X-100, 120 mM NaCl, 2 mM PMSF, 3.5 μg / mL Leupeptin, and 0.15% SDS.

[0077] Preferably, the A reaction solution contains primers and probes for detecting unmutated EXT1 gene or EXT2 gene.

[0078] Preferably, the B reaction solution contains primers and probes for detecting mutations in the EXT1 gene or EXT2 gene.

[0079] Preferably, the sequences of the primers in the reaction solution A and the reaction solution B are shown in SEQ ID NO.: 1 to SEQ ID NO.: 14.

[0080] The sequences of the probes in the A reaction solution and the B reaction solution are shown in SEQ ID NO.: 15 to SEQ ID NO.: 20.

[0081] The PCR buffer contains KCl, dNTPs, MgSO 4 , Tween-20, Tris-HCl mixture;

[0082] Preferably, the enzyme solution is a solution containing Taq DNA polymerase with a concentration of 8U.

[0083] Preferably, it also includes an inner liner and an outer packaging box; the inner liner is provided with a plurality of container holes, and the plurality of container holes are used to respectively place a reaction solution tube A, a reaction solution tube B, a PCR buffer tube, an enzyme solution tube, a wild-type positive control tube, a mutant positive control tube, a negative control tube, and a sample lysis solution tube.

[0084] Preferably, the wild-type positive control is a TE buffer solution of a wild-type plasmid containing the EXT1 gene and / or the EXT2 gene; the mutant positive control is a TE buffer solution of a mutant plasmid containing the EXT1 gene and / or the EXT2 gene, and the mutation refers to 1018C>T, 1019G>A, 1469delT mutations in the EXT1 gene and 67C>T, 679G>A, 1225delG mutations in the EXT2 gene.

[0085] Preferably, the amplified sequence of the wild-type plasmid in the positive control is as shown in SEQ ID NO.: 21.

[0086] Preferably, the amplified sequence of the mutant plasmid is shown in SEQ ID NO.: 22.

[0087] Preferably, the negative control is sterile water for injection.

[0088] Preferably, the wild-type plasmid concentration in the wild-type positive control and the mutant plasmid concentration in the mutant positive control are both 10 6 ±5000 copies / mL.

[0089] In some specific embodiments, the present invention further provides a method for detecting EXT1 gene and / or EXT2 gene polymorphism using a high-resolution melting curve combined with a mutation amplification system, comprising the following steps:

[0090] Step S01, nucleic acid extraction: take sample lysis solution, add 1 mL of plasma sample to be tested, heat at 95-100°C for 20 min, let stand and take the supernatant for use;

[0091] Step S02, reagent preparation: taking reaction solution A, reaction solution B, PCR buffer, and enzyme solution to prepare reaction mixture A and reaction mixture B;

[0092] Step S03, amplification detection: the nucleic acid sample and negative control extracted in step S01 are added to the A reaction mixture and the B reaction mixture obtained in step S02, and the wild-type positive control and the mutant positive control are added to prepare the A-PCR system and the B-PCR system, and the detection is performed on a 4-color fluorescence quantitative PCR instrument;

[0093] Step S04, analyzing the test results:

[0094] Preferably, in step S04, the A-PCR system result analysis is as follows:

[0095] When the melting curve temperature of the FAM fluorescence channel peaks at 40±0.5°C, it indicates that there is no mutation at sites 1018 and 1019 of the EXT1 gene;

[0096] When the Ct value of the amplification curve of the FAM fluorescence channel is <38, it indicates that the 1469 site of the EXT1 gene has not mutated;

[0097] When the Ct value of the amplification curve of the VIC fluorescence channel is less than 38, it indicates that the 67th site of the EXT2 gene has not mutated;

[0098] When the Ct value of the amplification curve of the ROX fluorescence channel is <38, it indicates that the 679 site of the EXT2 gene has not mutated;

[0099] When the Ct value of the amplification curve of the CY5 fluorescence channel is <38, it indicates that the 1225 site of the EXT2 gene has not mutated;

[0100] Preferably, in step S04, the B-PCR system result analysis is as follows:

[0101] When the melting curve temperature of the FAM fluorescence channel peaks at 34-36°C, it indicates that mutations have occurred at sites 1018 and 1019 of the EXT1 gene;

[0102] When the Ct value of the amplification curve of the FAM fluorescence channel is less than 38, it indicates that the 1469 site of the EXT1 gene has mutated;

[0103] When the Ct value of the VIC fluorescence channel amplification curve is less than 38, it indicates that the 67th site of the EXT2 gene has mutated;

[0104] When the Ct value of the amplification curve of the ROX fluorescence channel is <38, it indicates that the 679 site of the EXT2 gene has not mutated;

[0105] When the Ct value of the amplification curve of the CY5 fluorescence channel is less than 38, it indicates that a mutation has occurred at the 1225 site of the EXT2 gene.

[0106] Preferably, in step S04, if the detection results of the A-PCR system and the B-PCR system are both judged to be positive, it indicates that no heterozygous mutation has occurred.

[0107] Preferably, in step S02, 10 μL of A reaction solution, 5 μL of enzyme solution, and 5 μL of PCR buffer are taken to prepare 20 μL of A reaction mixed liquid; 10 μL of LB reaction solution, 5 μL of enzyme solution, and 5 μL of PCR buffer are taken to prepare 20 μL of LB reaction mixed liquid.

[0108] In step S03, 5 μL of the extraction product, positive control substance, and negative control substance obtained in step S01 are taken and added to 20 μL of the PCR qualitative reaction liquid obtained in step S02 to prepare a total of 25 μL of the PCR system.

[0109] Compared with the prior art, the present invention uses a high-resolution melting curve combined with a mutation amplification system to develop a kit and method for detecting the polymorphism of the EXT1 gene and / or EXT2 gene, and only two tubes of reagents are used to achieve early diagnosis of the polymorphism analysis of the EXT1 gene and / or EXT2 gene, providing a reliable experimental means for the clinical detection of hereditary multiple osteochondromatosis.

[0110] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0111] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. The PCR reagents in the present invention are all from Shanghai Jianyi Biotechnology Co., Ltd.

[0112] The technical solution of the present invention is further described in detail below through specific embodiments.

[0113] Example 1

[0114] This embodiment provides a method for detecting EXT1 gene and / or EXT2 gene polymorphism by combining high-resolution melting curve with mutation amplification system.

[0115] 1. Primer and probe design

[0116] The design of the probe of the present invention specifically includes introducing an artificial mutation site at the 5' adjacent base of the mutation site, and at the same time performing locked nucleic acid modification on the mutation site base to enhance the recognition strength of the primer and / or probe; at this time, if the site mutates, the artificially introduced mutation will promote the entire primer or probe to recognize the mutation site, thereby achieving the accuracy of the detection result. See Table 1 for the base sequences of the primers and probes used in this embodiment, where "+" indicates locked nucleic acid modification.

[0117] The preferred primer and probe sequences of this example are shown in Table 1. All primers and probes were commissioned to be synthesized by Shanghai Bioengineering.

[0118] Specifically, the artificial mutation sites include: Taking the probes shown in sequence numbers such as SEQ ID NO.: 15 and SEQ ID NO.: 20 as examples, the wild sequences of the mutation sites of the above two sequences are CGTGGT+ C+GC , after mutation, it should be CGTGGT+ C+ AC , CGTGGT+ T+GC , CGTGGT+ T+AC Any of the three, but in actual detection, the detection and analysis of only a single base mutation is more difficult, so the present invention replaces the base T before the mutation site with A, that is, the wild sequence CGTGG T +C+GC was changed to the artificial mutant sequence CGTGG A +C+GC); and adjacent to the possible mutation site, when the original gene mutates, the bases at the adjacent position also mismatch due to artificial mutation, thereby amplifying the mutation signal and improving the recognition strength of the primer or probe.

[0119] The synthesized primers and probes were purified to HPLC level and then used.

[0120] Table 1 Names and sequences of primers and probes

[0121]

[0122]

[0123] 2. Reagents

[0124] The detection system includes reaction solution A, reaction solution B, PCR buffer, enzyme solution, wild-type positive control, mutant positive control, negative control, and sample lysate. Specifically, reaction solution A contains primers and probes for detecting unmutated EXT1 gene and / or EXT2 gene, and reaction solution B contains primers and probes for detecting mutated EXT1 gene and / or EXT2 gene. The mutation sites include 1018C>T, 1019G>A, 1469delT of EXT1 gene and 67C>T, 679G>A, 1225delG of EXT2 gene.

[0125] Further, the sequences of the primers in the reaction solution A and the reaction solution B are shown in SEQ ID NO.: 1 to SEQ ID NO.: 14; wherein SEQ ID NO.: 1 to SEQ ID NO.: 10, SEQ ID NO.: 15 to SEQ ID NO.: 19 are primers and probe sequences for detecting unmutated EXT1 gene and / or EXT2 gene contained in the reaction solution A, and SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 11 to SEQ ID NO.: 14, SEQ ID NO.: 16 to SEQ ID NO.: 20 are primer sequences for detecting mutated EXT1 gene and / or EXT2 gene contained in the reaction solution B. SEQ ID NO.: 15 and SEQ ID NO.: 20 are modified with locked nucleic acid.

[0126] PCR buffer contains MgSO 4 , dNTPs and other ionic environments required for PCR. In this example, 10× Taq DNA Ploymewase, catalog number AP101.

[0127] The enzyme solution contains Taq DNA polymerase; its concentration is 8U.

[0128] See Table 2 for the preparation method of reaction solution A and reaction solution B.

[0129] Table 2 Ratio of reaction solution A and reaction solution B

[0130]

[0131]

[0132] The positive control is a plasmid containing the wild-type (W) and mutant (M) amplified sequences of the EXT1 gene and / or EXT2 gene; the negative control is sterile water for injection.

[0133] The amplified sequences of the two targets (wild-type plasmid W and mutant M plasmid) in the positive control are shown in SEQ ID NO.: 21 to SEQ ID NO.: 22, as shown in Table 3.

[0134] Table 3 Sequence table of wild-type plasmid W and mutant M plasmid

[0135]

[0136]

[0137] Table 4 Proportions of positive controls

[0138]

[0139] 3. Detection methods

[0140] (1) Nucleic acid extraction: Take the sample lysis solution, add 1 mL of the plasma sample to be tested, heat at 95-100°C for 20 min, let it stand, and take the supernatant for later use.

[0141] (2) Reagent preparation: Take 10 μL of A reaction solution and B reaction solution, add 5 μL of PCR buffer and 5 μL of enzyme solution respectively, and prepare 20 μL of A reaction mixture liquid and 20 μL of B reaction mixture respectively.

[0142] (3) Amplification detection: Take 10 μL of the extraction product obtained in step S01, 10 μL of the positive control substance, and 10 μL of the negative control substance, and add them to 20 μL of the A reaction mixture liquid and 20 μL of the B reaction mixture liquid obtained in the above step S02, respectively, to prepare 30 μL A-PCR system and 30 μL B-PCR system, and detect on a fluorescent quantitative PCR instrument with more than 4 colors (MacroSLAN-96P fluorescent quantitative PCR instrument). The specific reaction procedure is as shown in Table 5.

[0143] Table 5 Reaction procedure

[0144]

[0145] Result analysis and interpretation:

[0146] The results of the A-PCR system are analyzed as follows:

[0147] When the melting curve temperature of the FAM fluorescence channel peaks at 40±0.5°C, it indicates that there is no mutation at sites 1018 and 1019 of the EXT1 gene;

[0148] When the Ct value of the amplification curve of the FAM fluorescence channel is <38, it indicates that the 1469 site of the EXT1 gene has not mutated;

[0149] When the Ct value of the amplification curve of the VIC fluorescence channel is less than 38, it indicates that the 67th site of the EXT2 gene has not mutated;

[0150] When the Ct value of the amplification curve of the ROX fluorescence channel is <38, it indicates that the 679 site of the EXT2 gene has not mutated;

[0151] When the Ct value of the amplification curve of the CY5 fluorescence channel is less than 38, it indicates that the 1225 site of the EXT2 gene has not mutated.

[0152] The results of the B-PCR system are analyzed as follows:

[0153] When the melting curve temperature of the FAM fluorescence channel peaks at 34-36°C, it indicates that mutations have occurred at sites 1018 and 1019 of the EXT1 gene;

[0154] When the Ct value of the amplification curve of the FAM fluorescence channel is less than 38, it indicates that the 1469 site of the EXT1 gene has mutated;

[0155] When the Ct value of the VIC fluorescence channel amplification curve is less than 38, it indicates that the 67th site of the EXT2 gene has mutated;

[0156] When the Ct value of the amplification curve of the ROX fluorescence channel is <38, it indicates that the 679 site of the EXT2 gene has not mutated;

[0157] When the Ct value of the amplification curve of the CY5 fluorescence channel is less than 38, it indicates that the 1225 site of the EXT2 gene has mutated;

[0158] Furthermore, in step S04, if the detection results of the A-PCR system and the B-PCR system are both judged to be positive, it means that the mutation has not become a heterozygous mutation;

[0159] See also Figure 1-Figure 11 , are respectively diagrams showing the amplification curve results of the nucleic acid sample to be tested in the FAM channel, VIC channel, and ROX channel in this embodiment.

[0160] Specifically, Figure 1 This is the result of no mutation at sites 1018 and 1019 of the EXT1 gene, with a peak at 50 ± 0.5 °C in the figure; Figure 2 This is the result that there is no mutation at position 1469 of the EXT1 gene, and Ct is less than 38 in the figure; Figure 3 This is the result that there is no mutation at site 67 of the EXT2 gene, and Ct is <38 in the figure; Figure 4 This is the result that there is no mutation at site 679 of the EXT2 gene, and Ct is <38 in the figure; Figure 5 This is the result that there is no mutation at site 1225 of the EXT2 gene, and Ct is <38 in the figure; Figure 6 This is the result of 1018C>T in the EXT1 gene, with a peak at 50±0.5℃ in the figure; Figure 7 This is the result of 1019G>A in the EXT1 gene, with a peak at 50±0.5℃ in the figure; Figure 8 This is the result of 1469delT in the EXT1 gene, with Ct < 38 in the figure; Fig. 9 This is the result of 67C>T in EXT2 gene, Ct<38 in the figure; Fig.10 This is the result of 679G>A in EXT2 gene, Ct<38 in the figure; Fig.11This is the result of 1225delG occurring in the EXT2 gene, with Ct < 38 in the figure.

[0161] Performance evaluation:

[0162] Sensitivity:

[0163] Specifically, the wild-type positive control and mutant positive control of the present invention were graded diluted and tested. The test results showed that when the concentration was above 2000 copies / mL, the detection rate could reach more than 95%, and the wild-type or mutant sites could be accurately distinguished. Therefore, the lower limit of the nucleic acid concentration detected by the present invention was 2000 copies / mL.

[0164] Specificity:

[0165] Specifically, the primers and probes designed by the present invention were compared and analyzed in the NCBI database, and the analysis results showed that the matching rate with EXT1 and EXT2 genes was 100%, and no genes other than EXT1 and EXT2 genes were detected.

[0166] Example 2

[0167] This embodiment provides a kit for diagnosing HEM pathogenic genes EXT1 and EXT2.

[0168] Specifically, the kit includes an inner liner and an outer packaging box.

[0169] See also Fig.12 , is a schematic diagram of the structure of the unfolded inner liner of the test kit. After folding the diagram, the box body structure of the test kit with a three-layer structure is obtained. The inner liner is provided with 8 container holes for placing A reaction solution, B reaction solution, PCR buffer, enzyme solution, wild-type positive control, mutant positive control, negative control, and sample lysate respectively. The 8 container holes can be respectively recorded as: A reaction solution tube placement hole, B reaction solution tube placement hole, PCR buffer tube placement hole, enzyme solution tube placement hole, wild-type positive control placement hole, mutant positive control placement hole, negative control tube placement hole, sample lysate placement hole.

[0170] This kit is stored at -20℃, and repeated freezing and thawing should be minimized.

[0171] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or by being able to achieve the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. A method for detecting EXT1 gene and / or EXT2 gene polymorphism by high resolution melting curve combined with mutation amplification system, characterized in that: The nucleic acid of the sample to be tested is used as the template DNA, and a PCR reaction is performed through a PCR amplification reaction system. During the PCR reaction and melting curve process, real-time fluorescence collection is performed, and an amplification curve and a melting curve are drawn, and the results are interpreted; The interpretation of the results includes the interpretation of the mutation sites 1018C>T, 1019G>A, 1469delT of the EXT1 gene, and / or the interpretation of the mutation sites 67C>T, 679G>A, 1225delG of the EXT2 gene.

2. The method for detecting EXT1 gene and / or EXT2 gene polymorphism by high resolution melting curve combined with mutation amplification system according to claim 1, characterized in that: The PCR amplification reaction system includes a primer-probe combination, a PCR buffer, an enzyme solution, a positive control substance, a negative control substance, and a sample lysis solution; In the primer-probe composition, at least one artificial mutation site is introduced into the adjacent base at the 5' end of the mutation site to be detected and analyzed by the probe, and the base of the artificial mutation site is simultaneously modified with a locked nucleic acid; The primer-probe combinations were purified to HPLC grade respectively. The sample lysis solution is a mixture containing EDTA, Triton X-100, NaCl, PMSF, Leupeptin, and SDS.

3. The method for detecting EXT1 gene and / or EXT2 gene polymorphism by high resolution melting curve combined with mutation amplification system according to claim 2, characterized in that: The PCR amplification reaction system includes a first PCR amplification reaction system and a second PCR amplification reaction system; The first PCR amplification reaction system includes A reaction solution, PCR buffer, enzyme solution, wild-type positive control substance, and negative control substance; The second PCR amplification reaction system includes B reaction solution, PCR buffer, enzyme solution, mutant positive control substance, and negative control substance; The primer-probe combination includes a primer-probe combination for detecting unmutated sites and a primer-probe combination for detecting mutated sites; The primer-probe combination for detecting non-mutation includes primers and probes for detecting non-mutation of EXT1 gene or EXT2 gene; The mutation detection primer-probe combination includes primers and probes for detecting mutations in the EXT1 gene or the EXT2 gene; A reaction solution contains the primer-probe combination for detecting non-mutation; The B reaction solution contains the mutation detection primer probe combination.

4. The method for detecting EXT1 gene and / or EXT2 gene polymorphism by high resolution melting curve combined with mutation amplification system according to claim 3, characterized in that: The mutation sites of the EXT1 gene or EXT2 gene primers for detecting mutations include 1018C>T, 1019G>A, 1469delT of the EXT1 gene and 67C>T, 679G>A, 1225delG of the EXT2 gene; In the unmutated primer-probe combination, the sequences of the primers are shown in SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, and SEQ ID NO.: 10; the sequences of the probes are shown in SEQ ID NO.: 15, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19; The primer sequences in the mutation primer-probe combination are shown in SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 6, SEQ ID NO.: 13, SEQ ID NO.: 8, SEQ ID NO.: 9, and SEQ ID NO.: 14; the probe sequences are shown in SEQ ID NO.: 20, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.:

19.

5. The method for detecting EXT1 gene and / or EXT2 gene polymorphism by high resolution melting curve combined with mutation amplification system according to any one of claims 1 to 4, characterized in that: The steps include: S01, nucleic acid extraction: take sample lysis solution, add 1 mL of plasma sample to be tested, heat at 95-100°C for 20 min, let stand and take the supernatant for use; S02, reagent preparation: take reaction solution A, reaction solution B, PCR buffer, and enzyme solution, and prepare reaction mixture A and reaction mixture B respectively; S03, amplification detection: the nucleic acid sample and negative control extracted in step S01 are added to the reaction mixture A and the reaction mixture B obtained in step S02, and the wild-type positive control and the mutant positive control are added to prepare the first PCR amplification reaction system and the second PCR amplification reaction system, and the detection is performed on a 4-color fluorescence quantitative PCR instrument; S04, interpreting and analyzing the test results.

6. The method for detecting EXT1 gene and / or EXT2 gene polymorphism using a high-resolution melting curve combined with a mutation amplification system according to claim 5, characterized in that: In S2, the PCR buffer is a mixture containing KCl, dNTPs, MgSO4, Tween-20, and Tris-HCl; The enzyme solution is a mixture containing Taq DNA polymerase and glycerol; the concentration of the enzyme is 8U; In S03, the wild-type positive control is a TE buffer solution of a wild-type plasmid containing EXT1 gene and EXT2 gene; the mutant positive control is a TE buffer solution of a mutant plasmid containing EXT1 gene and EXT2 gene, wherein the mutation refers to 1018C>T, 1019G>A, 1469delT mutations in EXT1 gene and 67C>T, 679G>A, 1225delG mutations in EXT2 gene; The amplified sequence of the wild-type plasmid is shown in SEQ ID NO.21; The amplified sequence of the mutant plasmid is shown in SEQ ID NO.22; The negative control was sterile water for injection; The wild-type plasmid concentration in the wild-type positive control and the mutant plasmid concentration in the mutant positive control were both 10 6 ±5000 copies / mL.

7. The method for detecting EXT1 gene and / or EXT2 gene polymorphism using a high-resolution melting curve combined with a mutation amplification system according to any one of claim 5, characterized in that: The PCR reaction program is: 95°C×1min, 1 cycle; 95°C×5s, 60°C×30s, 45 cycles; 95°C×5s, 1 cycle; 30-45°C heating to make a melting curve, with a heating rate of 0.03°C / s; wherein, in the 60°C×30s and 30-45°C heating processes, FAM, VIC, ROX, and CY5 fluorescence channels are used to collect fluorescence.

8. The method for detecting EXT1 gene and / or EXT2 gene polymorphism by high resolution melting curve combined with mutation amplification system according to claim 5, characterized in that: The method for interpreting the first PCR amplification reaction system includes: When the melting curve temperature of the FAM fluorescence channel peaks at 40 ± 0.5 °C, it indicates that there is no mutation at sites 1018 and 1019 of the EXT1 gene; When the Ct value of the amplification curve of the FAM fluorescence channel is less than 38, it indicates that the 1469 site of the EXT1 gene has not mutated; When the Ct value of the amplification curve of the VIC fluorescence channel is less than 38, it indicates that the 67th site of the EXT2 gene has not mutated; When the Ct value of the amplification curve of the ROX fluorescence channel is <38, it indicates that the 679 site of the EXT2 gene has not mutated; When the Ct value of the amplification curve of the CY5 fluorescence channel is <38, it indicates that the 1225 site of the EXT2 gene has not mutated; The method for interpreting the second PCR amplification reaction system includes: When the melting curve temperature of the FAM fluorescence channel peaks at 34-36°C, it indicates that mutations have occurred at positions 1018 and / or 1019 of the EXT1 gene; When the Ct value of the amplification curve of the FAM fluorescence channel is less than 38, it indicates that the 1469 site of the EXT1 gene has mutated; When the Ct value of the VIC fluorescence channel amplification curve is less than 38, it indicates that the 67th site of the EXT2 gene has mutated; When the Ct value of the amplification curve of the ROX fluorescence channel is <38, it indicates that the 679 site of the EXT2 gene has not mutated; When the Ct value of the amplification curve of the CY5 fluorescence channel is less than 38, it indicates that the 1225 site of the EXT2 gene has mutated; Alternatively, if the detection results of the first PCR system and the second PCR system are both judged to be positive at the same site, it indicates that a heterozygous mutation has occurred at the site.

9. A diagnostic kit, characterized in that It comprises an inner liner and an outer packaging box; the inner liner comprises a plurality of container holes, which are respectively used to contain reaction solution A, reaction solution B, PCR buffer, enzyme solution, wild-type positive control substance, mutant-type positive control substance, negative control substance, and sample lysate.

10. A method for detecting EXT1 gene and / or EXT2 gene polymorphism using the high-resolution melting curve combined with mutation amplification system as described in any one of claims 1 to 8, or use of the kit as described in claim 9 in detecting EXT1 gene and / or EXT2 gene for purposes other than disease diagnosis.

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