CN112779322A - Gene mutation detection kit based on non-fluorescence labeled probe and high-resolution melting curve, detection method and application thereof - Google Patents
Gene mutation detection kit based on non-fluorescence labeled probe and high-resolution melting curve, detection method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biomedicine, in particular to a gene mutation detection kit based on a high-resolution melting curve of a non-fluorescence labeled probe, a detection method and application thereof, wherein the kit comprises a system consisting of a non-fluorescence labeled probe, two forward and reverse amplification primers, 5 '-3' exonuclease activity-deficient DNA polymerase, a fluorescent dye, a dNTP mixed solution and a PCR buffer solution; wherein: the non-fluorescent labeled probe is a non-fluorescent labeled probe with 3' end being modified and closed by a group so as to lose extension capability. The detection system of the invention replaces the expensive fluorescent probe with the common oligonucleotide for nucleic acid identification and achieves the purpose of detecting gene polymorphism, combines the non-fluorescent labeled probe with the target sequence with mutation sites, converts the sequences with different melting temperatures into the generation of signals through the analysis of a melting curve, and realizes the rapid detection of gene mutation through the non-fluorescent labeled probe.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a gene mutation detection kit based on a non-fluorescence labeled probe and a high-resolution melting curve, a detection method and application thereof.
Background
The research of gene mutation has become one of the hot spots of life science research at present, and the detection method is also developed rapidly. As described above, for the detection of gene mutation, the Southern blotting method was used before 1985 to screen out the mutation patterns such as deletion, insertion and frame-shift recombination of the gene. For mutations that cannot be detected by this method, only sophisticated and time-consuming DNA sequencing assays can be applied. Polymerase Chain Reaction (PCR) technology is the most important progress in mutation research, so that gene mutation detection technology has long-term development, almost all molecular diagnosis technologies for gene mutation detection are established on the basis of PCR at present, and new PCR-derived methods are continuously appeared, which reach more than twenty varieties at present, the automation degree is higher and higher, the analysis time is greatly shortened, and the accuracy of analysis results is also greatly improved. These include single-strand conformation polymorphism (SSCP) and Heteroduplex Analysis (HA).
At present, the PCR technology cannot be used for detecting gene mutation. In brief, PCR is the in vitro synthesis of a large amount of specific genes by using DNA polymerase, and a section of gene can be amplified to one hundred billion to one billion times of the original gene by the specific replication of the DNA polymerase, so that the PCR is easy to further analyze. The conditions that must be met for the PCR reaction are: a copied DNA template, a specific pair of primers, DNA polymerase, synthetic raw materials and a buffer system required by the reaction. PCR consists of three basic reaction steps of denaturation, annealing and extension: firstly, the denaturation of template DNA: heating the template DNA to about 94 ℃ for a certain time, and dissociating the double strands of the template DNA to form single strands so that the single strands can be combined with the primers to prepare for next reaction; annealing (renaturation) of template DNA to the primer: heating and denaturing the template DNA into single strands, cooling to about 55 ℃, and pairing and combining the primers and the complementary sequences of the template DNA single strands; extension of the primer: under the action of Taq DNA polymerase, dNTP as reaction material and target sequence as template are combined to synthesize one new semi-retained replicated chain complementary to the template DNA chain, and the three processes of repeated circular denaturation, annealing and extension are repeated to obtain more semi-retained replicated chains, which may become the template for the next circulation. The target gene to be amplified can be copied to hundreds of billions of times after 2-3 hours after 2-4 minutes of one cycle is completed.
At present, a sequencing method is commonly used for detecting gene mutation; gene chip hybridization; PCR-RFLP method; capillary electrophoresis analysis of PCR amplification product; PCR fluorescent quantitative probe method; the allele specific primer PCR amplification method and the like can detect the gene mutation condition in different degrees, but the methods have the defects of high instrument price, high operation difficulty, certain false negative and false positive, high detection cost, low clinical popularization degree, incapability of simultaneously detecting clinical samples in large scale and the like. Such as: the direct sequencing method is a gold standard for mutation analysis, can find known and unknown mutation sites, but each site needs PCR amplification and then sequencing, and has the advantages of higher cost, large workload, long period, high price and unsuitability for large sample analysis; the common PCR-RFLP method has simple and convenient technology and low price, is suitable for laboratory detection of a small amount of samples, but RFLP can only detect mutation with enzyme cutting sites, can not detect without the enzyme cutting sites, and wastes time and labor; compared with the traditional instrument detection method, the chip technology has the characteristics of high throughput, miniaturization, automation, pollution prevention and the like, is suitable for whole genome mutation scanning, is not suitable for detecting mutation sites of single genes, and has low precision and high price. The Taqman probe method is suitable for known mutation sites, two probes are often required, the probe sequences and reaction conditions must be optimized, and otherwise false positive occurs; the allele specific primer PCR amplification method is characterized by that according to the mutation site a specific PCR primer whose 3' end is complementary to base of mutation site or mismatched with base is designed, and the gel electrophoresis or fluorescent signal method is used to detect the existence of PCR amplification product so as to detect the mutation in genotype. The method is characterized in that two specific primers which are complementary or mismatched with the base of the 3' terminal mutation site are designed, the primer design is not good, high background cross amplification can be caused, if the SNP analysis is acceptable, the detection of the mutant of the patient gene is greatly forbidden, otherwise, higher false positive can be caused.
The above technical schemes can analyze the gene single nucleotide polymorphism, but complicated manual methods are needed, such as separation and digestion of restriction enzymes on agarose gel, and the fluorescent probe method needs to design and synthesize each SNP individually, each site detection needs at least 2 probes to complete detection, and can not complete detection in the same reaction system, and the detection of single site needs to be performed in different tubes, and meanwhile, the probes are very expensive, the detection cost is greatly increased, and the detection of multiple fluxes is not facilitated.
Disclosure of Invention
The invention provides a gene mutation detection kit based on a non-fluorescence labeling probe and a high-resolution melting curve, a detection method and application thereof, aiming at overcoming the defects that the existing means for detecting gene mutation has high instrument price, high operation difficulty, certain false negative and false positive, high detection cost, low clinical popularization degree, incapability of simultaneously detecting clinical specimens on a large scale and the like, and solving the problems of complex operation, high cost and easy occurrence of false positive in the conventional detection method.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a gene mutation detection kit based on a high-resolution melting curve of a non-fluorescence labeled probe comprises a system consisting of a non-fluorescence labeled probe, two forward and reverse amplification primers, 5 '-3' exonuclease activity-deficient DNA polymerase, a fluorescent dye, dNTP mixed liquor and PCR buffer solution;
wherein: the non-fluorescent labeled probe is a non-fluorescent labeled probe with 3' end being modified and closed by a group so as to lose extension capability.
The labeled probe in the gene mutation detection kit based on the high-resolution melting curve of the non-fluorescent labeled probe is not a fluorescent labeled probe as in the conventional method, but the non-fluorescent labeled probe is combined with the high-resolution melting curve to detect SNP, so that a single probe can simultaneously detect up to 20 SNP sites, the resolution and the sensitivity are higher, the mutation abundance lower than 1% can be detected, the kit is particularly suitable for being applied to a detection template with high heterogeneity, the enrichment detection can be carried out on mutant genes, and the detection efficiency and the cost are greatly improved.
Preferably, the non-fluorescent labeled probe may be any one of a DNA probe or an RNA probe whose 3' end is blocked by a group modification.
Preferably, the method for blocking the hydroxyl group at the 3' end of the non-fluorescently labeled probe sequence may comprise the following steps: c3spacer, amino modified C6, phosphorylation, reverse dT, 3' single base mismatch, etc.
Preferably, the non-fluorescently labeled probe may be any one of an oligonucleotide probe, a double probe, a scorpion probe, a molecular beacon probe, and a twin probe.
Preferably, the length of the non-fluorescence labeling probe is 20-40 bp.
Further preferably, the non-fluorescent labeled probe is a DNA or RNA sequence with a 3' terminal phosphorylation block, the sequence is preferably 20-30bp long, the region covered by the probe contains a mutation site existing in the target sequence, and the probe can be combined with the target sequence under a certain temperature condition and can fall off from the target sequence at a specific temperature.
Preferably, the polymerase is any one of klenow enzyme, pfu DNA polymerase, and Vent DNA polymerase.
Further preferably, the commercial polymerases comprise: roche ActiTaq exo DNA Polymerase, NEB Hemo Klen Taq, and the like.
Still more preferably, the DNA polymerase having no exonuclease activity is Roche Acti-taq DNA polymerase.
Preferably, the fluorescent dye is a nucleic acid-specific saturated dye with an excitation wavelength of 440-470nm and an emission wavelength of 470-520 nm.
Further preferably, the fluorescent dye is one of LC Green, Syto9 or Eva Green.
Still further preferably, the nucleic acid saturating dye is LC Green, which does not inhibit the PCR reaction when incorporated into a DNA strand.
Preferably, the concentration ratio of the two forward and reverse amplification primers is asymmetric, and the concentration ratio of the reverse primer to the forward primer is (10-20): 1.
as the improvement of the gene mutation detection of the invention, the two primers of the invention have the function of conventional sequence amplification, and because a melting curve method is adopted, a probe is required to be combined with an amplified product, the amplification of the invention adopts asymmetric amplification, namely the concentration ratio of the two primers is asymmetric, so that the concentration ratio of the two primers is preferably 10: 1 or 20: 1.
preferably, the dNTP mixture has a concentration of 25mM and contains equal amounts of dATP, dCTP, dGTP and dTTP.
A gene mutation detection method based on a high-resolution melting curve of a non-fluorescence labeled probe comprises the following steps: (S.1) extracting and purifying sample nucleic acid: extracting DNA from tissue, excrement and peripheral blood, measuring its concentration, and storing at-20 deg.C;
(S.2) preparing the gene mutation detection kit;
take 10. mu.L of reaction system (concentration and content of each component in the reaction system) as an example:
PCR reaction buffer: 8.03 uL
Forward primer (10 μ M): 0.01 μ L
Reverse primer (10 μ M): 0.1. mu.L
Non-fluorescently labeled probe (10 μ M): 0.1. mu.L
dNTP mix (25 mM): 0.01 μ L
Polymerase: 0.25 μ L
Nucleic acid dye: 0.5. mu.L
A sample to be tested: 1 mu L of the solution;
(S.3) adding the sample into the prepared gene mutation detection kit;
(S.4) fluorescent PCR amplification: placing reaction tubes of multiple mutation sites to be detected on a conventional LC-480 fluorescent quantitative PCR instrument, setting the reaction conditions to be pre-denatured at 95 ℃ for 5min, carrying out 55 cycles in an amplification stage, wherein the cycles comprise denaturation at 95 ℃ for 10s, annealing at 54 ℃ for 15s, and extension at 68 ℃ for 25s, directly carrying out melting curve detection after the cycle is finished, firstly carrying out denaturation at 95 ℃ for 40s, then rapidly cooling to 40 ℃ and keeping for 3min, and then carrying out melting at the heating rate of 0.11 ℃/s, wherein the detection temperature range is 40-95 ℃;
(S.5) analyzing through a high-resolution melting curve, and directly judging the type of the mutation corresponding to the temperature data when a melting curve peak with a specific temperature appears.
The application of the gene mutation detection kit based on the high-resolution melting curve of the non-fluorescence labeled probe in-vitro diagnosis.
In the invention, the concentration ratio of the primers and the design of the probes are critical, so that dimers among the primers and the probes are avoided, in addition, the blocking efficiency of the probes is high, otherwise, nonspecific hybrid peaks can occur. DNA polymerase has a certain selectivity for primer sequence, so it is important to optimize the match between primer and polymerase, and more efficient primer combinations should be selected.
The invention has the following technical advantages:
(1) the specificity is strong, because of adopting the probe method to detect the mutation site, the specificity of the probe determines the specificity of detection, and the traditional dye method has no selectivity to nucleic acid, so the specificity can not be ensured.
(2) The sensitivity is high, the probe is specificity-controlled, the detection adopts a dye method, the characteristic of more prominent signal is achieved, and the detection sensitivity is higher than that of the traditional fluorescent PCR method.
(3) The detection process is a closed tube reaction, so that the cross contamination is obviously reduced, and the truth and reliability of the result are ensured.
(4) The method is simple and quick to operate, can finish the detection of a plurality of sites within 2 hours, and is simple and visual in result interpretation and convenient to analyze.
(5) The probe design is simple, and the resolution can be up to within 1 degree, so that the difficulty of the probe design is greatly reduced. It is easier to clinically cope with a wide variety of mutation types.
(6) Compared with a sequencing method, the method has the advantages of lower detection cost, short time consumption, high sensitivity (the sequencing sensitivity is low, and the gene abundance is generally required to be more than 10% for detection), and simple interpretation.
Drawings
FIG. 1 is a diagram showing the results of genotyping SNP site rs1801133 of folate key enzyme gene using whole blood sample as template.
FIG. 2 is a graph showing the results of genotyping the SNP site rs671 of the gene ALDH2, which is a key drug metabolism gene of a nitroglycerin tablet, using a whole blood sample as a template.
Detailed Description
The invention is further described with reference to the drawings and the specific embodiments. Those skilled in the art will be able to implement the invention based on these teachings. Moreover, the embodiments of the present invention described in the following description are generally only some embodiments of the present invention, and not all embodiments. Therefore, all other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any creative effort shall fall within the protection scope of the present invention.
Example 1:
composition of human MTHFR gene polymorphism detection kit (non-labeled probe method)
(1) The SNP locus rs1801133 on the MTHFR coding gene is typed by a high-resolution melting curve gene mutation detection method based on a non-fluorescent labeled probe. According to the distribution of the mutation sites on the corresponding gene sequences, non-labeled probes covering the mutation sites are designed, and then primer pairs for specifically amplifying target sequences are respectively designed according to the distribution of the probes, wherein the sites to be detected are contained, and the length of an amplification fragment between primers corresponding to the MTHFR gene sequences is 105 bp.
The primer sequence is as follows:
primer 1: TGAGGCTGACCTGAAGCAC
Primer 2: GCCTTCACAAAGCGGAAGA
Non-labeled probe sequence:
1, probe 1: TCTGCGGGAGCCGATTTCATC
(2) The preparation process of the kit is as follows:
preparing a PCR reaction solution:
reaction solution for each individual site:
| buffer solution for PCR reaction | 8.03μL |
| Forward primer (10. mu.M) (primer 1) | 0.01μL |
| Reverse primer (10. mu.M) (primer 2) | 0.1μL |
| Non-labeled Probe (10. mu.M) (Probe 1) | 0.1μL |
| dNTP mix (25mM) | 0.01μL |
| Polymerase enzyme | 0.25μL |
| Nucleic acid dyes | 0.5μL |
| Sample to be tested | 1μL |
A method for using the kit.
The specific detection steps of the detection kit for detecting the polymorphism of the human MTHFR gene of the embodiment are as follows: DNA extraction
A blood sample DNA extraction kit is adopted to extract a sample (the sample is EDTA anticoagulated whole blood), and the specific operation refers to the DNA kit extraction product instruction.
Sample DNA quality detection
After the sample DNA is obtained, the quality of the sample is controlled by measuring the ratio of the concentration to the OD260/OD280, the sample is finally added into a reaction system, the optimal reaction result can be obtained when the ratio of the OD260/OD280 is 1.7-2.0, and the concentration is more appropriate to be 5-20 ng/mu L.
PCR reaction
Preparing a total pipe according to the volume and the sequence of each part of reaction liquid in the above, preparing according to the number of detected people, inverting and uniformly mixing after the preparation is finished, subpackaging into each PCR reaction tube, respectively adding a template to be detected, adding an LC-480 fluorescent quantitative PCR instrument after the reaction tubes are covered, and carrying out PCR reaction by adopting the following procedures:
and analyzing the melting curve by using a PCR instrument corresponding software, wherein a fluorescence channel selected during analysis is SYBR Green I.
4. Interpretation of results
And (3) comparing the Tm value of the sample to be tested to judge the genotype of the sample according to the melting point (Tm) of a double-stranded hybrid formed by hybridizing the probe with the target sequences of the corresponding known mutant type and the wild type, namely judging that the sample belongs to the genotype when the Tm value of the sample to be tested is consistent with the Tm value of a standard product of the known genotype. And (4) judging a result standard: MTHFR (C/T)57.63 deg.C/50.79 deg.C.
The detection is carried out by the scheme, a result chart is shown in figure 1, and the result shows that for the MTHFR rs1801133 locus, a melting peak exists in the sample to be detected at 57.63 ℃, and the sample is the MTHFR rs1801133 wild type; the sample to be detected has melting peaks at 50.79 ℃ and 57.63 ℃, and the sample is MTHFR rs1801133 heterozygote; and if the sample to be detected has a melting peak at 50.79 ℃, the sample is the MTHFR rs1801133 mutant type.
5. Conclusion
The detection method can quickly and accurately detect the folate metabolism key enzyme gene MTHFR, and has important guiding significance for clinical folic acid intake of patients. MTHFR mutation can cause folic acid metabolism abnormality, reduce active folic acid level and increase homocysteine (Hcy) level, and the increase of plasma Hcy level is closely related to the occurrence of various birth defects such as neural tube malformation, congenital heart disease and Down's syndrome, and pregnancy diseases such as pregnancy hypertension and recurrent abortion. Compared with the traditional high-resolution melting curve method, the non-labeled probe adopts a specific probe and a dye with strong signal, has higher sensitivity and can more accurately type the MTHFR gene. The results show that the kit can be used for clinical detection by using the human MTHFR gene polymorphism detection kit, and has a high clinical application prospect in prevention of various diseases possibly caused by folic acid metabolism abnormality.
Example 2:
composition of human ALDH2 gene polymorphism detection kit (non-labeled probe method)
(1) The SNP locus rs671 on the ALDH2 coding gene is typed by a high-resolution melting curve gene mutation detection method based on a non-fluorescent labeled probe. According to the distribution of the mutation sites on the corresponding gene sequences, non-labeled probes covering the mutation sites are designed, then primer pairs for specifically amplifying target sequences are respectively designed according to the distribution of the probes, wherein the sites to be detected are contained, and the length of an amplification fragment between primers corresponding to the ALDH2 gene sequences is 149 bp.
The primer sequence is as follows:
primer 3: GAGCCCAGTCACCCTTTG
Primer 4: ACCAGCAGACCCTCAAGC
Non-labeled probe sequence:
and (3) probe 2: CAGGCATACACTGAAGTGAA
(2) The preparation process of the kit is as follows:
preparing a PCR reaction solution:
reaction solution for each individual site:
a method for using the kit.
The specific detection steps of the detection kit for detecting the human ALDH2 gene polymorphism of the embodiment are as follows: DNA extraction
A blood sample DNA extraction kit is adopted to extract a sample (the sample is EDTA anticoagulated whole blood), and the specific operation refers to the DNA kit extraction product instruction.
Sample DNA quality detection
After the sample DNA is obtained, the quality of the sample is controlled by measuring the ratio of the concentration to the OD260/OD280, the sample is finally added into a reaction system, the optimal reaction result can be obtained when the ratio of the OD260/OD280 is 1.7-2.0, and the concentration is more appropriate to be 5-20 ng/mu L.
PCR reaction
Preparing a total pipe according to the volume and the sequence of each part of reaction liquid in the above, preparing according to the number of detected people, inverting and uniformly mixing after the preparation is finished, subpackaging into each PCR reaction tube, respectively adding a template to be detected, adding an LC-480 fluorescent quantitative PCR instrument after the reaction tubes are covered, and carrying out PCR reaction by adopting the following procedures:
and analyzing the melting curve by using a PCR instrument corresponding software, wherein a fluorescence channel selected during analysis is SYBR Green I.
4. Interpretation of results
And (3) judging the genotype of the sample by comparing the Tm values of the samples to be detected according to the melting points (Tm) of double-stranded hybrids formed by the hybridization of the two probes and the corresponding target sequences of the known mutant type and the wild type, namely judging that the sample belongs to the genotype when the Tm value of the samples to be detected is consistent with the Tm value of a standard product of the known genotype. And (4) judging a result standard: ALDH2(A/G)59.42 ℃/52.43 ℃.
The detection is carried out by the scheme, the result chart is shown in fig. 2, and the result shows that for the ALDH2 rs671 locus, a melting peak exists in the sample to be detected at 59.42 ℃, and the sample is ALDH2 rs671 wild type; the sample to be detected has melting peaks at 52.43 ℃ and 59.42 ℃, and the sample is ALDH2 rs671 heterozygote; and if the sample to be detected has a melting peak at 52.43 ℃, the sample is ALDH2 rs671 mutant.
5. Conclusion
The detection method can quickly and accurately detect the mutation of the key enzyme gene ALDH2 for the drug metabolism of the nitroglycerin tablets, and provides reference for clinical doctors to the dosage of the nitroglycerin tablets of patients. Nitroglycerin plays an important role in biotransformation of pulmonary and circulatory vascular beds, and if a patient carries ALDH2 gene mutation, transformation of nitroglycerin in vivo can be blocked, and the effect of a medicament is difficult to play. The non-labeled probe has the characteristics of high sensitivity and strong specificity, and compared with the common fluorescent probe method, the non-labeled probe does not need a fluorescent labeled probe, thereby reducing the synthesis cost of the probe and the synthesis time. Meanwhile, the method uses a fluorescent real-time PCR instrument, does not need special instruments and is convenient to popularize. The results show that the human ALDH2 gene polymorphism detection kit can be used for clinical detection, and has a high clinical application prospect for individualized treatment of patients and diagnosis and treatment of angina pectoris, myocardial infarction and other diseases.
Sequence listing
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Claims (10)
1. A gene mutation detection kit based on a high-resolution melting curve of a non-fluorescence labeled probe is characterized by comprising a system consisting of a non-fluorescence labeled probe, two forward and reverse amplification primers, 5 '-3' exonuclease activity-deficient DNA polymerase, a fluorescent dye, dNTP mixed liquor and PCR buffer solution;
wherein: the non-fluorescent labeled probe is a non-fluorescent labeled probe with 3' end being modified and closed by a group so as to lose extension capability.
2. The kit for detecting gene mutation based on high-resolution melting curve of non-fluorescently labeled probe according to claim 1, wherein the non-fluorescently labeled probe can be any one of DNA probe or RNA probe with 3' end blocked by group modification.
3. The kit for detecting gene mutation based on high-resolution melting curve of non-fluorescently labeled probe according to claim 2, wherein the non-fluorescently labeled probe can be any one of oligonucleotide probe, double probe, scorpion probe, molecular beacon probe, twin probe.
4. The kit for detecting gene mutation based on the high-resolution melting curve of the non-fluorescence labeled probe according to claim 1, 2 or 3, wherein the length of the non-fluorescence labeled probe is 20-40 bp.
5. The kit for detecting gene mutation based on high-resolution melting curve of non-fluorescently labeled probe according to claim 1, wherein the polymerase is any one of klenow enzyme, pfu DNA polymerase, and Vent DNA polymerase.
6. The kit for detecting gene mutation based on the high-resolution melting curve of the non-fluorescently labeled probe as claimed in claim 1, wherein the fluorescent dye is a nucleic acid-specific saturation dye with an excitation wavelength of 440-470nm and an emission wavelength of 470-520 nm.
7. The kit for detecting gene mutation based on high-resolution melting curve of non-fluorescent labeled probe as claimed in claim 6, wherein said fluorescent dye is one of LC Green, Syto9 or Eva Green.
8. The kit for detecting gene mutation based on the high-resolution melting curve of the non-fluorescence labeled probe according to claim 1, wherein the concentration ratio of the two forward and reverse amplification primers is asymmetric, and the concentration ratio of the reverse primer to the forward primer is (10-20): 1.
9. a gene mutation detection method based on a high-resolution melting curve of a non-fluorescence labeled probe is characterized by comprising the following steps:
(S.1) extracting and purifying sample nucleic acid;
(S.2) preparing a gene mutation detection kit according to any one of claims 1 to 8;
(S.3) adding the sample into the prepared gene mutation detection kit;
(S.4) performing fluorescent PCR amplification;
(S.5) analysis is carried out by means of a high-resolution melting curve.
10. Use of the gene mutation detection kit based on the non-fluorescence labeled probe with the high resolution melting curve according to any one of claims 1 to 8 in vitro diagnosis.
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