CN112029848A - Improved sample processing method for detecting polymorphism of C677T locus of MTHFR gene - Google Patents
Improved sample processing method for detecting polymorphism of C677T locus of MTHFR gene Download PDFInfo
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Abstract
The invention provides a sample processing method for detecting polymorphism of site C677T of MTHFR gene, which comprises the following steps: (1) adding lysis solution into a sample to be detected, and then incubating for 4-6 min at 68-75 ℃; (2) adding magnetic particles into the product obtained in the step (1), uniformly mixing, and removing the supernatant after magnetic separation; (3) washing the magnetic particles obtained in the step (2), and discarding the supernatant after magnetic separation; (4) adding an eluent into the magnetic particles obtained in the step (3), uniformly mixing, incubating at 70-75 ℃ for 4-6 min, and collecting a supernatant after magnetic separation; (5) and (5) carrying out PCR amplification on the supernatant obtained in the step (4) to obtain a treated sample. The processing method is optimized by multiple parameters such as sample cracking and PCR amplification conditions, so that the quality of a detection result can be effectively improved, the detection line is prevented from generating a background, the detection time is shortened, and the detection efficiency is improved.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an improved sample processing method for detecting polymorphism of a C677T locus of an MTHFR gene.
Background
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme for folic acid metabolism, the MTHFR gene has the full length of 20.374kb and has 12 exons, and codes a protein consisting of 656 amino acid residues, and the main function of the MTHFR gene is to convert 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, and the 5-methyltetrahydrofolate is a methyl donor for the re-methylation of Homocysteine (HCY) into methionine. Polymorphism of site C677T of MTHFR gene, i.e. 677CC type (wild type), 677CT type (heterozygous mutant type) and 677TT (homozygous mutant type). Mutation at C677T renders the enzyme heat intolerant and thus less active, with only 65% of the enzyme activity in CT individuals compared to CC individuals and only 30% in TT individuals. The inhibition of the re-methylation of homocysteine prevents its methylation to methionine, resulting in accumulation, resulting in reduced levels of folate metabolism and hyperhomocysteinemia (HHCY). The defect of MTHFR gene can cause the disturbance of a plurality of metabolic processes of the organism, including cell cycle regulation, DNA replication, DNA and protein methylation modification and the like, further cause a plurality of diseases such as neural tube defects, cancers, cardiovascular and cerebrovascular diseases and the like, and is proved to be closely related to birth defects, such as Down syndrome, cleft lip and palate, neural tube defects and other neonatal defects.
The existing kit can detect the polymorphism of the C677T locus of human peripheral blood or genomic DNA MTHFR gene extracted from human peripheral blood, provides effective, rapid and accurate clinical reference for auxiliary diagnosis and medication of patients with folic acid metabolism abnormality by the homocysteine level, and can provide more effective guidance for clinical application only by ensuring the accuracy of a detection result. Most of the conventional methods for treating the kit samples are manually operated, the detection time is long, and the final judgment result part adopts a chromatography method, but the detection line is often background, so that the judgment of the result is influenced, and the phenomenon of misjudgment of the result is caused.
Disclosure of Invention
Based on the above, the present invention aims to provide an improved sample processing method for detecting polymorphism at C677T locus of MTHFR gene, which can effectively improve the quality of detection results, avoid the occurrence of background in detection lines, shorten the detection time, and improve the detection efficiency.
The specific technical scheme is as follows:
a sample processing method for detecting polymorphism of site C677T of MTHFR gene comprises the following steps:
(1) adding lysis solution into a sample to be detected, and then incubating for 4-6 min at 68-75 ℃;
(2) adding magnetic particles capable of adsorbing DNA into the product obtained in the step (1), uniformly mixing, and removing supernatant after magnetic separation;
(3) washing the magnetic particles obtained in the step (2), and discarding the supernatant after magnetic separation;
(4) adding an eluent into the magnetic particles obtained in the step (3), uniformly mixing, incubating at 70-75 ℃ for 4-6 min, and collecting a supernatant after magnetic separation to obtain a DNA solution;
(5) carrying out PCR amplification on the DNA solution obtained in the step (4) to obtain a treated sample; the amplification primers were as follows: the amplification primers aiming at the mutant type of the C677T locus of the MTHFR gene comprise an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2; the amplification primers aiming at the wild type of the C677T locus of the MTHFR gene comprise an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4; the primers shown in SEQ ID NO. 1-SEQ ID NO.4 are modified with detectable molecular markers.
In some embodiments, the incubation in step (1) is performed at 69-72 ℃ for 4-6 min.
In some of these embodiments, it is preferred that the incubation in step (1) is at 70 ℃ for 5 min.
In some embodiments, the incubating of step (1) is incubating using a dry bath.
The inventor finds that the sample treated by adding the lysis solution can be better improved in sample lysis effect, the sample lysis time is shortened, and the sample detection quality is improved by incubating the sample in a dry bath at 70 ℃ for 5 min.
In some of these embodiments, step (1) simultaneously adds the lysis solution and proteinase K to the sample to be assayed.
In some of these embodiments, the sample is a whole blood sample.
In some embodiments, the washing method of step (3) is: adding 700 +/-50 ul of cleaning solution I, mixing uniformly, performing magnetic separation, then discarding the supernatant, adding 500 +/-50 ul of cleaning solution II, mixing uniformly, and performing magnetic separation, then discarding the supernatant.
In some embodiments, the step (3) washes and discards the supernatant, and then centrifuges the sample for 2-4 s, and discards the supernatant.
In some embodiments, the incubation in step (4) is performed at 70-72 ℃ for 4-6 min.
In some of these embodiments, it is preferred that the incubation in step (4) is performed at 70 ℃ for 5 min.
In some embodiments, the incubating in step (4) is incubating using a dry bath.
In some embodiments, the concentration of DNA in the PCR amplification system in step (5) is 2-3.2 ng/. mu.l.
In some embodiments, the PCR amplification conditions of step (5) are: 2min at 50 ℃; 3.5min at 95 ℃; 25-28 cycles: 94 ℃ for 5s, 60 +/-1 ℃ for 10s and 65 ℃ for 30 s; 10min at 65 ℃.
In some embodiments, the PCR amplification conditions of step (5) are: 2min at 50 ℃; 3.5min at 95 ℃; 26-27 cycles: 94 ℃ for 5s, 60 +/-1 ℃ for 10s and 65 ℃ for 30 s; 10min at 65 ℃.
In some embodiments, the detectable molecule modified on the primers shown in SEQ ID NO. 1-SEQ ID NO.4 in step (5) is digoxin.
In some of these embodiments, the magnetic separation method is to place the sample directly on the magnetizer for 5 ± 1 min.
The sample processing method is only used for the sample pretreatment step of batch, and does not relate to specific disease diagnosis.
The invention also provides a method for detecting polymorphism of C677T locus of MTHFR gene, which comprises the following steps:
s1, sample processing: processing a sample to be detected by using the sample processing method to obtain a PCR amplification product, namely the processed sample;
s2, detecting the PCR amplification product obtained in the step S1 by using a detection reagent aiming at the modified molecular marker on the PCR amplification primer, and judging the C677T site polymorphism of the MTHFR gene.
Compared with the prior art, the invention has the following beneficial effects:
the sample processing method improves the sample cracking and PCR amplification conditions, and the improved cracking method can effectively improve the sample cracking effect in a shorter time and improve the DNA yield and quality, thereby improving the subsequent PCR amplification effect and avoiding pollution; and further, the optimized PCR amplification primers and the optimized amplification program are utilized to carry out PCR amplification on the DNA extracted by the sample cracking method, so that the specificity of the PCR amplification can be effectively improved, non-specific products in PCR amplification products are removed, and the detection line of the PCR amplification products is prevented from generating a background during subsequent detection, thereby improving the detection quality and ensuring the accuracy of the detection result. In addition, the invention also optimizes the subsequent magnetic separation and washing steps aiming at the sample cracking method, thereby shortening the sample processing time and improving the detection efficiency.
Drawings
FIG. 1 is a graph showing the results of detecting polymorphism at C677T site of MTHFR gene in 10 samples in example 2 using the detection method of the present invention.
FIG. 2 is a graph showing the results of detecting polymorphism at C677T site of MTHFR gene in 10 samples in example 2 using a conventional detection method.
Detailed Description
The experimental procedures of the present invention, without specifying the specific conditions in the following examples, are generally carried out according to conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In the following examples of the present invention, the DNA extraction reagent (including lysis buffer, proteinase K, binding solution, magnetic particles, washing solution I, washing solution II, and eluent), PCR amplification reagent, and the detection card for PCR amplification product of the whole blood sample were purchased from seian magnetic nanotechnology limited.
Example 1
The sample processing method for detecting polymorphism of site C677T of MTHFR gene of this embodiment includes the following steps:
(1) collecting a whole blood sample of a person to be detected, wherein the whole blood sample is 100ul, then adding 20ul of protease K and 200ul of lysis solution, uniformly mixing by vortex, and carrying out metal bath in a dry bath device at 70 ℃ for 5 min;
(2) adding 300ul of binding solution and 25ul of magnetic particles capable of adsorbing DNA into the product obtained in the step (1), uniformly mixing by vortex, directly placing on a magnetic device for separation for 5min, and discarding the supernatant;
(3) washing the magnetic particles obtained in the step (2): firstly, 700ul of cleaning fluid I is added, vortex mixing is carried out, the mixture is directly placed on a magnetizer for separation for 5min, and supernatant is discarded; then adding 500ul of cleaning solution II, uniformly mixing by vortex, directly placing on a magnetic device for separation for 5min, and removing the supernatant; centrifuging the sample for 2s in a centrifuge, and then discarding the supernatant;
(4) adding 100ul of eluent into the magnetic particles obtained in the step (3), uniformly mixing by vortex, then carrying out metal bath in a dry bath device at 70 ℃ for 5min, placing the centrifugal tube in a magnetic separator until the supernatant is clear, collecting the supernatant (namely DNA solution), and transferring the supernatant into a 2ml centrifugal tube for later use;
(5) taking the supernatant obtained in the step (4) for PCR amplification: adding the DNA solution obtained in the step (4) into the mutant type (M) amplification tube and the Wild Type (WT) amplification tube respectively, wherein the PCR amplification system in each amplification tube comprises the following components: mu.l amplification solution (containing dNTP, corresponding amplification primer, magnesium chloride and Taq buffer) 1. mu.l reaction solution (containing Taq DNase and UNG enzyme), genomic DNA solution to be detected and ddH2O (make up volume to 50. mu.l), cover the PCR tube tightly, mix by vortex, then put the PCR tube into the PCR instrument, perform PCR amplification according to the following procedure: 2min at 50 ℃; 3.5min at 95 ℃; 26 cycles: 94 ℃ for 5s, 60 ℃ for 10s and 65 ℃ for 30 s; 10min at 65 ℃; hold at 4 ℃; resulting in a treated sample (i.e., PCR amplification product). The concentration of the genome DNA to be detected in the PCR amplification system is 2.5 ng/mu l; the corresponding amplification primers were: use in mutant amplification tubesThe amplification primers aiming at the mutant type of the C677T locus of the MTHFR gene comprise an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2; amplification primers aiming at a wild type of a C677T locus of the MTHFR gene are used in a wild type amplification tube, and comprise an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4; the primers shown in SEQ ID NO. 1-SEQ ID NO.4 are modified with digoxin molecules, and the final concentration of each primer in the PCR amplification system is 0.5 uM; other components in the PCR amplification system are provided by a kit, and the dosage of the components is strictly used according to the instruction of the kit.
SEQ ID NO.1：5’-TGACCTGGTGACTGGATT-3’；
SEQ ID NO.2：5’-GGATACTAAGCCTACGACTC-3’；
SEQ ID NO.3：5’-CCATCGGTGACTCAGTGAC-3’；
SEQ ID NO.4：5’-ACGGTGATAGCCAACATTC-3’。
And after the amplification is finished, taking out the PCR product, and storing at 2-8 ℃ for subsequent detection.
Example 2
The method for detecting polymorphism of site C677T of MTHFR gene of the embodiment comprises the following steps:
s1, sample processing: processing a sample to be detected by using the sample processing method in the embodiment 1 to obtain a corresponding PCR amplification product, namely the processed sample;
s2, detecting the processed sample obtained in the step S1: and taking out the detection card from the sealed bag, respectively dripping PCR products in the mutant PCR amplification tube and the wild PCR amplification tube of the sample to be detected at the sample pad corresponding to the detection card, and judging the result after 2-5min, wherein the result is unreliable after 20 min.
In this example, the polymorphism at the site C677T of MTHFR gene in the sample to be detected, which is the same as the detection method in this example, is also detected by using the existing detection method, which includes the following steps:
s1, sample processing:
(1) collecting 100ul of a whole blood sample of a person to be detected, adding 20ul of protease K and 200ul of lysis solution, uniformly mixing by vortex, and carrying out water bath at 56 ℃ for 20 min;
(2) adding 300ul of binding solution and 25ul of magnetic particles capable of adsorbing DNA into the product obtained in the step (1), uniformly mixing by vortex, standing at room temperature for 5min, separating on a magnetic device for 5min, and removing supernatant;
(3) washing the magnetic particles obtained in the step (2): adding 400ul of cleaning solution I, uniformly mixing by vortex, performing magnetic separation until the supernatant is clear, and discarding the supernatant; then adding 400ul of cleaning solution I, uniformly mixing by vortex, carrying out magnetic separation until the supernatant is clear, and discarding the supernatant; adding 500ul of cleaning solution II, uniformly mixing by vortex, performing magnetic separation until the supernatant is clear, and discarding the supernatant; opening the centrifugal tube cover, and airing the centrifugal tube cover for 5min at room temperature;
(4) adding 100ul of eluent into the magnetic particles obtained in the step (3), uniformly mixing by vortex, then carrying out water bath at 56 ℃ for 5min, placing the centrifugal tube in a magnetic separator to clarify the supernatant, collecting the supernatant (namely DNA solution), and transferring the supernatant into a 2ml centrifugal tube for later use;
(5) and (3) carrying out PCR amplification on the DNA solution obtained in the step (4): adding the DNA solution obtained in the step (4) into the mutant type (M) amplification tube and the Wild Type (WT) amplification tube respectively, wherein the PCR amplification system in each amplification tube comprises the following components: mu.l amplification solution (containing dNTP, corresponding amplification primer, magnesium chloride and Taq buffer) 1. mu.l reaction solution (containing Taq DNase and UNG enzyme), genomic DNA solution to be detected and ddH2O (make up volume to 50. mu.l), cover the PCR tube tightly, mix by vortex, then put the PCR tube into the PCR instrument, perform PCR amplification according to the following procedure: 2min at 50 ℃; 3.5min at 95 ℃; 31 cycles: 94 ℃ for 5s, 60 ℃ for 10s and 65 ℃ for 30 s; 10min at 65 ℃; hold at 4 ℃. The concentration of the genomic DNA to be detected in the PCR amplification system is 5ng/ul, the corresponding amplification primers (including amplification primers aiming at wild type and mutant type) and other components in the PCR amplification system are provided by a kit, and the dosage of the components is strictly used according to the kit instruction (the dosage is the same as that in the embodiment 1). And taking out a PCR product after amplification is finished, namely the processed sample.
S2, detecting the processed sample obtained in the step S1: and taking out the detection card from the sealed bag, respectively dripping PCR products in the mutant PCR amplification tube and the wild PCR amplification tube of the sample to be detected at the sample pad corresponding to the detection card, and judging the result after 2-5min, wherein the result is unreliable after 20 min.
The two detection methods are respectively used for detecting 200 samples of which the polymorphism condition of the C677T locus of the MTHFR gene is confirmed, and the results show that the detection results of the 200 samples of the detection method are completely consistent with the known results, and the accuracy is up to 100 percent, while the detection results of 10 samples of the existing detection method are not consistent with the known results, and misjudgment is carried out on the polymorphism of the C677T locus of the MTHFR gene due to the background of the detection line, which is specifically shown in Table 1.
TABLE 1 results of polymorphism detection of C677T site of MTHFR gene in 10 samples described above
| Sample numbering | Knowing the results of the assay | The method of the invention detects the result | The detection result of the existing |
|
| 1 | | TT | CT | |
| 2 | | TT | CT | |
| 3 | | TT | CT | |
| 4 | | Invalidation | CC | |
| 5 | | CC | CT | |
| 6 | | TT | CT | |
| 7 | | CC | CT | |
| 8 | | TT | CT | |
| 9 | | CC | CT | |
| 10 | TT | TT | CT |
FIG. 1 shows a test card for 10 samples according to the present invention, and FIG. 2 shows a test card for 10 samples according to the conventional test method. As can be seen from FIGS. 1 and 2, the detection method of the present invention has clean and clear detection line, no background generation (FIG. 1), and can intuitively and accurately judge the detection result; however, the detection result graph detection line of the existing detection method has a background (fig. 2), which causes the misjudgment of the detection result of the polymorphism at the C677T site of the MTHFR gene. In addition, compared with the existing detection method, the detection method of the invention has the advantages of shorter detection time and higher detection efficiency.
Example 3
The sample processing method simultaneously optimizes the sample cracking and PCR amplification conditions, thereby effectively improving the quality of the detection result, avoiding the background of the detection line, shortening the detection time and improving the detection efficiency.
In this example, the influence of incubation conditions during sample lysis, DNA usage amount during PCR amplification, and PCR amplification cycle number on the detection effect were mainly studied, and 30 whole blood samples of known polymorphism at C677T site of MTHFR gene were processed by the sample processing method of the present invention and the sample processing methods of the control groups 1 to 6, respectively. The incubation conditions for sample lysis, the amount of PCR-amplified DNA used, and the number of PCR amplification cycles in each sample treatment set are shown in Table 2, and the other steps are the same as those in the sample treatment method described in example 1.
TABLE 2 methods of sample processing for each group
After 30 whole blood samples were treated by the above-mentioned methods for treating each sample to obtain PCR amplification products, the polymorphism at C677T site of MTHFR gene was detected in the PCR amplification products in the same manner as in step S2 of example 2.
The detection results show that when the PCR amplification products obtained by processing the samples by using the method are detected, the detection line backgrounds of 30 samples are clear and clean, the detection results are accurate and correct, and the detection results completely conform to the known results; when PCR amplification products obtained by processing samples by the method of the control groups 1-6 are detected, the detection lines of 2-4 samples of each control group generate backgrounds, so that the detection results are misjudged. The sample processing method is obtained by the inventor after various optimization and improvement, and the detection effect is reduced by changing a certain step.
Example 4
The mutant type and wild type amplification primers aiming at the C677T locus of the MTHFR gene are obtained by the inventor through elaborate design and a large number of experiments, and the amplification primers can effectively avoid non-specific amplification, improve the detection quality of PCR amplification products and ensure the accuracy of detection results.
This example studies the detection results of the amplification primers of the present invention and the amplification primers designed according to the conventional primer design principle on the polymorphism at the C677T site of MTHFR gene. Experiment groups 1 to 6 were set, the amplification primers used in each experiment group are shown in table 3, and the sample treatment method in each experiment group was the same as the sample treatment method described in example 1 except that the amplification primers used in each experiment group were shown in table 3:
TABLE 3 amplification primers used in Experimental groups 1-6
After 15 samples of whole blood with known MTHFR gene C677T site polymorphism were processed by the above-mentioned methods for sample treatment in each experimental group to obtain PCR amplification products, the PCR amplification products were subjected to MTHFR gene C677T site polymorphism detection in the same manner as in step S2 of example 2.
The results are shown in table 4:
TABLE 4 test results
As can be seen from the detection results in Table 4, the detection result of the polymorphism at the C677T locus of the MTHFR gene in the experimental group 1 (using the amplification primer of the invention) is completely consistent with the known result, and the accuracy is as high as 100%; the detection results of some samples are misjudged due to the background of the detection lines in the experiment groups 2-6, so that the detection accuracy is reduced. The results show that the optimized amplification primer has better specificity and higher accuracy for detecting the polymorphism of the C677T locus of the MTHFR gene, and can avoid the influence of non-specific amplification on the detection result.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Jilin gold area medical laboratory Co., Ltd; guangzhou city gold domain transformation medical research institute Co., Ltd
<120> an improved sample processing method for detecting polymorphism of C677T site of MTHFR gene
<130> 2020-09-07
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<400> 16
gccacgcatg tgccatattt gt 22
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 17
ggtgactcag tgacctggtg ac 22
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 18
gccacgcatg tgccatattt gt 22
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 19
atcggtgact cagtgacctg gt 22
<210> 20
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 20
gccacgcatg tgccatattt gt 22
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 21
ccaaccctca agcgaagact ga 22
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 22
agcctacgac tcccagaaag gt 22
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 23
gacctggtga ctggattctc gg 22
<210> 24
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 24
ggcacacggt gatagccaac at 22
Claims (12)
1. A sample processing method for detecting polymorphism of site C677T of MTHFR gene is characterized by comprising the following steps:
(1) adding lysis solution into a sample to be detected, and then incubating for 4-6 min at 68-75 ℃;
(2) adding magnetic particles capable of adsorbing DNA into the product obtained in the step (1), uniformly mixing, and removing supernatant after magnetic separation;
(3) washing the magnetic particles obtained in the step (2), and discarding the supernatant after magnetic separation;
(4) adding an eluent into the magnetic particles obtained in the step (3), uniformly mixing, incubating at 70-75 ℃ for 4-6 min, and collecting a supernatant after magnetic separation to obtain a DNA solution;
(5) carrying out PCR amplification on the DNA solution obtained in the step (4) to obtain a treated sample; the amplification primers were as follows: the amplification primers aiming at the mutant type of the C677T locus of the MTHFR gene comprise an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2; the amplification primers aiming at the wild type of the C677T locus of the MTHFR gene comprise an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4; the primers shown in SEQ ID NO. 1-SEQ ID NO.4 are modified with detectable molecular markers.
2. The method for processing the sample for detecting the polymorphism at the C677T site of MTHFR gene according to claim 1, wherein the step (1) is performed by incubating at 69-72 ℃ for 4-6 min.
3. The method for processing a sample for detecting polymorphism at site C677T of MTHFR gene according to claim 2, wherein the incubation in step (1) is performed by using a dry bath.
4. The method for processing a sample to detect polymorphism at site C677T of MTHFR gene according to claim 1, wherein the washing method in step (3) comprises: adding 700 +/-50 ul of cleaning solution I, mixing uniformly, performing magnetic separation, then discarding the supernatant, adding 500 +/-50 ul of cleaning solution II, mixing uniformly, and performing magnetic separation, then discarding the supernatant.
5. The method for processing a sample for detecting polymorphism at site C677T of MTHFR gene according to claim 4, wherein the step (3) comprises washing and discarding the supernatant, centrifuging the sample for 2-4 s, and discarding the supernatant.
6. The method for processing the sample for detecting the polymorphism at the C677T site of MTHFR gene according to claim 1, wherein the step (4) is performed by incubating at 70-72 ℃ for 4-6 min.
7. The method for processing the sample for detecting the polymorphism at the C677T site of MTHFR gene according to claim 6, wherein the incubation in step (4) is performed by using a dry bath.
8. The method for processing a sample for detecting polymorphism at C677T site of MTHFR gene according to claim 1, wherein the concentration of DNA in the PCR amplification system in step (5) is 2-3.2 ng/μ l.
9. The method for processing a sample for detecting polymorphism at C677T site of MTHFR gene according to claim 1 or 8, wherein the PCR amplification conditions in step (5) are as follows: 2min at 50 ℃; 3.5min at 95 ℃; 25-28 cycles: 94 ℃ for 5s, 60 +/-1 ℃ for 10s and 65 ℃ for 30 s; 10min at 65 ℃.
10. The method for processing a sample for detecting polymorphism at site C677T of MTHFR gene according to claim 9, wherein the PCR amplification conditions in step (5) are as follows: 2min at 50 ℃; 3.5min at 95 ℃; 26-27 cycles: 94 ℃ for 5s, 60 +/-1 ℃ for 10s and 65 ℃ for 30 s; 10min at 65 ℃.
11. The method for processing a sample for detecting polymorphism at C677T site of MTHFR gene according to claim 1, wherein the detectable molecule modified on the primers shown in SEQ ID NO. 1-SEQ ID NO.4 in step (5) is digoxin.
12. The method for processing the sample for detecting the polymorphism at the C677T site of MTHFR gene according to any one of claims 1 to 11, wherein the magnetic separation method comprises separating the sample by directly placing the sample on a magnetic device for 5 ± 1 min.
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| CN105349532A (en) * | 2015-12-15 | 2016-02-24 | 杭州千基生物科技有限公司 | Method and kit for extracting free nucleic acid by using paramagnetic particle method |
| CN106498074A (en) * | 2016-11-25 | 2017-03-15 | 金磁（苏州）纳米科技有限公司 | Hands-free cut-off connects the quick ELISA test strip method of people's mthfr gene SNP partings of amplification |
| CN110964842A (en) * | 2019-12-31 | 2020-04-07 | 广州金域医学检验集团股份有限公司 | Sample processing reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis |
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| CN105349532A (en) * | 2015-12-15 | 2016-02-24 | 杭州千基生物科技有限公司 | Method and kit for extracting free nucleic acid by using paramagnetic particle method |
| CN106498074A (en) * | 2016-11-25 | 2017-03-15 | 金磁（苏州）纳米科技有限公司 | Hands-free cut-off connects the quick ELISA test strip method of people's mthfr gene SNP partings of amplification |
| CN110964842A (en) * | 2019-12-31 | 2020-04-07 | 广州金域医学检验集团股份有限公司 | Sample processing reagent and kit for detecting mycobacterium tuberculosis nucleic acid and nucleic acid amplification method of mycobacterium tuberculosis |
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