CN114350791A - Primer probe combination for detecting polymorphism of folate metabolism related gene and application thereof - Google Patents
Primer probe combination for detecting polymorphism of folate metabolism related gene and application thereof Download PDFInfo
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Abstract
The invention provides a primer probe combination for detecting folate metabolism related gene polymorphism and application thereof, wherein the primer probe combination for detecting folate metabolism related gene polymorphism specifically amplifies and detects polymorphism conditions of MTHFR gene C677T, MTHFR gene A1298C and MTRR gene A66G; the upstream primer of the specific primer pair for amplifying the MTHFR gene C677T comprises a nucleotide sequence shown in SEQ ID No.1, the downstream primer comprises a nucleotide sequence shown in SEQ ID No.2, and a probe for detecting the MTHFR gene C677T comprises a nucleotide sequence shown in SEQ ID No. 3. The invention also provides a kit for detecting the polymorphism of the folate metabolism related gene. The primer probe combination has the advantages of high sensitivity, good specificity, high detection efficiency, easy operation and wide application prospect.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a primer probe combination for detecting polymorphism of folate metabolism related genes and application thereof.
Background
Folic acid, also known as vitamin B9, plays an important role in cell growth and synthesis of nucleic acid, amino acid and protein. Folate deficiency can lead to hypo-and hyperhomocysteinemia, thereby increasing the risk of birth defects, cardiovascular disease, and the like in newborns. MTHFR and MTRR are key enzymes in the folate metabolism process, and the site mutation of the MTHFR gene has an effect on the enzymatic activity and the heat stability of the MTHFR, so that the folate metabolism and the methionine metabolism are influenced. The site mutation of MTRR gene can cause the activity change of MTRR enzyme, and result in folic acid deficiency or homocysteine level increase in vivo.
Therefore, the method has important guiding significance for detecting the mutation condition of the key enzyme coding gene in the folate metabolism process. CN107254520A discloses a method for detecting folate metabolism related genes, which comprises the steps of extracting DNA of oral mucosa cells of a detector, using corresponding primer pairs and using genome DNA as a template, carrying out PCR amplification on MTHFR genes C677T and A1298C and MTRR gene A66G, recovering a target band after agarose gel electrophoresis detection, carrying out sequencing reaction, and analyzing SNP conditions of 3 genotypes according to a sequencing result. However, the method needs to purify a sample, needs to sequence, is complex to operate, has high cost, needs to be matched with a professional instrument for use, has high requirements on technical personnel, and limits popularization and use of related methods.
Therefore, how to provide a product and a method for detecting folate metabolism related gene polymorphism, which can rapidly and accurately detect a sample, are easy to operate and have low cost, has become a problem to be solved.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a primer probe combination for detecting folate metabolism related gene polymorphism and application thereof, which can realize the individual detection of MTHFR gene C677T (rs1801133), A129 1298C (rs1801131) and MTRR gene A66G (rs1801394), can also realize the single-tube multiple parallel detection of the three sites, and has the advantages of short detection time, low cost, simple and convenient operation, strong specificity, high accuracy, high sensitivity and wide application prospect.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer probe combination for detecting folate metabolism related gene polymorphism, which specifically amplifies and detects polymorphism conditions of MTHFR gene C677T, MTHFR gene A1298C and MTRR gene A66G;
the upstream primer of the specific primer pair for amplifying the MTHFR gene C677T comprises a nucleotide sequence shown in SEQ ID No.1, the downstream primer comprises a nucleotide sequence shown in SEQ ID No.2, and a probe for detecting the MTHFR gene C677T comprises a nucleotide sequence shown in SEQ ID No. 3.
SEQ ID No.1：ACTTGAAGGAGAAGGTGTC；
SEQ ID No.2：CAAGTGATGCCCATGT；
SEQ ID No.3：AAGGTGTCTGCGGGAGTCGATTTCATCAT。
Preferably, the upstream primer of the specific primer pair for amplifying the MTHFR gene A1298C comprises the nucleotide sequence shown in SEQ ID No.4, the downstream primer comprises the nucleotide sequence shown in SEQ ID No.5, and the probe for detecting the MTHFR gene A1298C comprises the nucleotide sequence shown in SEQ ID No. 6.
Preferably, the upstream primer of the specific primer pair for amplifying the MTRR gene A66G comprises the nucleotide sequence shown in SEQ ID No.7, the downstream primer comprises the nucleotide sequence shown in SEQ ID No.8, and the probe for detecting the MTRR gene A66G comprises the nucleotide sequence shown in SEQ ID No. 9.
SEQ ID No.4：AGGAGCTGACCAGTGAA；
SEQ ID No.5：CATTCCGGTTTGGTTCT；
SEQ ID No.6：ACCAGTGAAGCAAGTGTCTTTGAAGTCTTCGTTC；
SEQ ID No.7：GCCATCGCAGAAGAAA；
SEQ ID No.8：GGTAAAATCCACTGTAACG；
SEQ ID No.9：CAAAGGCCATCGCAGAAGAAATGTGTGAGCAAGCT。
In the invention, the polymorphism conditions of the MTHFR gene C677T (rs1801133), A1298C (rs1801131) and the MTRR gene A66G (rs1801394) in a sample can be detected independently or in a combined mode through the mutual matching of the primer pair and the probe, the detection time is short, the operation is simple and convenient, the specificity is strong, the accuracy is high, the sensitivity is extremely high, a template with the concentration as low as 100 pg/mu L in the sample can be detected, the accurate detection can be realized only by a trace amount of sample, the probability of false negative occurrence is reduced, and the application value is high.
Preferably, the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group.
Preferably, the fluorophore comprises any one of FAM, VIC or CY 5.
Preferably, the quencher group comprises BHQ1 or BHQ 2.
Preferably, the fluorescent group of the probe for detecting the MTHFR gene C677T is FAM, and the quenching group is BHQ 1.
Preferably, the fluorescent group of the probe for detecting the MTHFR gene A1298C is VIC, and the quenching group is BHQ 1.
Preferably, the fluorescent group of the probe for detecting the MTRR gene A66G is CY5, and the quenching group is BHQ 2.
In a second aspect, the invention provides a kit for detecting polymorphism of a gene related to folate metabolism, which comprises the primer probe combination for detecting polymorphism of a gene related to folate metabolism of the first aspect.
Preferably, the final concentration of the specific primer pair is 0.006-0.3 μmol/L, such as 0.006 μmol/L, 0.01 μmol/L, 0.05 μmol/L, 0.1 μmol/L, 0.15 μmol/L, 0.2 μmol/L, 0.25 μmol/L, or 0.3 μmol/L, etc., and the final concentration of the probe is 0.01-0.06 μmol/L, such as 0.01 μmol/L, 0.02 μmol/L, 0.03 μmol/L, 0.04 μmol/L, 0.05 μmol/L, or 0.06 μmol/L, etc., and other specific points in the value range can be selected, which is not repeated herein.
Preferably, the final concentration of the upstream primer of the specific primer pair for amplifying the MTHFR gene C677T is 0.01 mu mol/L, the final concentration of the downstream primer is 0.2 mu mol/L, and the final concentration of the probe for detecting the MTHFR gene C677T is 0.05 mu mol/L.
Preferably, the final concentration of the upstream primer of the specific primer pair for amplifying the MTHFR gene A1298C is 0.06 mu mol/L, the final concentration of the downstream primer is 0.006 mu mol/L, and the final concentration of the probe for detecting the MTHFR gene A1298C is 0.01 mu mol/L.
Preferably, the final concentration of the upstream primer of the specific primer pair for amplifying the MTRR gene A66G is 0.3 mu mol/L, the final concentration of the downstream primer is 0.03 mu mol/L, and the final concentration of the probe for detecting the MTRR gene A66G is 0.06 mu mol/L.
Preferably, the kit for detecting the folate metabolism related gene polymorphism further comprises dNTPs, Taq DNA polymerase and Mg2+And PCR reaction buffer.
Preferably, the final concentration of the dNTPs is 0.1-0.5 mmol/L, for example, 0.1mmol/L, 0.2mmol/L, 0.3mmol/L, 0.4mmol/L or 0.5mmol/L, and other specific points in the numerical range can be selected, which is not described herein, and is preferably 0.2 mmol/L.
Preferably, the final concentration of the Taq DNA polymerase is 0.01-0.05U/μ L, for example, 0.01U/μ L, 0.02U/μ L, 0.03U/μ L, 0.04U/μ L, or 0.05U/μ L, and the like, and other specific values in the numerical range can be selected, which are not described in detail herein, and are preferably 0.025U/μ L.
Preferably, said Mg2+The final concentration of (b) is 1-5 mmol/L, for example, 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L or 5mmol/L, and other specific values in the numerical range can be selected, which is not described herein, and is preferably 2 mmol/L.
Preferably, the PCR reaction buffer comprises Tris-HCl and KCl.
Preferably, the final concentration of Tris-HCl is 2-7 mmol/L, for example, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L or 7mmol/L, and other specific points in the value range can be selected, which is not described herein, and is preferably 5 mmol/L.
Preferably, the final concentration of KCl is 8-12 mmol/L, for example, 8mmol/L, 9mmol/L, 10mmol/L, 11mmol/L, or 12mmol/L, and other specific points in the value range can be selected, which is not described herein again, and is preferably 10 mmol/L.
In a third aspect, the present invention provides a method for using the kit for detecting a polymorphism of a gene associated with folate metabolism according to the second aspect, wherein the method for using the kit comprises:
and amplifying and detecting a sample to be detected by using the kit for detecting the polymorphism of the folate metabolism-related gene, and then analyzing.
Preferably, the procedure of amplification and detection comprises:
pre-denaturation:
94-97 deg.C, 2-5 min, such as 94 deg.C, 94.5 deg.C, 95 deg.C, 95.5 deg.C, 96 deg.C, 96.5 deg.C or 97 deg.C, and such as 2min, 2.5min, 3min, 3.5min, 4min, 4.5min or 5min, and other specific values in the value range can be selected, and are not described in detail herein;
and (3) cyclic extension:
94-97 ℃ for 5-15 s, the temperature can be 94 ℃, 94.5 ℃, 95 ℃, 95.5 ℃, 96 ℃, 96.5 ℃ or 97 ℃ for example, the time can be 5s, 6s, 7s, 8s, 9s, 10s, 11s, 12s, 13s, 14s or 15s for example, and other specific point values in the numerical range can be selected, which is not described again;
56-60 ℃ and 25-35 seconds, wherein the temperature can be 56 ℃, 56.5 ℃, 57 ℃, 57.5 ℃, 58 ℃, 58.5 ℃, 59 ℃, 59.5 ℃ or 60 ℃ and the like, the time can be 25s, 26s, 27s, 28s, 29s, 30s, 31s, 32s, 33s, 34s or 35s and the like, and other specific point values in the numerical range can be selected, and are not described in detail herein;
circulating for 35-40 times, for example, 35 times, 36 times, 37 times, 38 times, 39 times or 40 times;
annealing and extending:
94-97 deg.C for 1-3 min, wherein the temperature can be 94 deg.C, 94.5 deg.C, 95 deg.C, 95.5 deg.C, 96 deg.C, 96.5 deg.C or 97 deg.C, and the time can be 1min, 1.5min, 2min, 2.5min or 3min, and other specific values in the value range can be selected, which is not described in detail herein;
38-42 ℃ for 0.5-2 min, the temperature can be 38 ℃, 38.5 ℃, 39 ℃, 39.5 ℃, 40 ℃, 40.5 ℃, 41 ℃, 41.5 ℃ or 42 ℃ and the like, the time can be 0.5min, 1min, 1.5min or 2min and the like, and other specific values in the numerical range can be selected, which is not described in detail herein;
collecting signals:
40-85 ℃, for example, the temperature can be 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃ or 85 ℃, and other specific point values in the numerical range can be selected, and are not described in detail herein;
the temperature rise rate is 0.01-0.06 ℃/s, for example, 0.01 ℃/s, 0.02 ℃/s, 0.03 ℃/s, 0.04 ℃/s, 0.05 ℃/s or 0.06 ℃/s, and other specific values in the numerical range can be selected, which is not described in detail herein, and is preferably 0.06 ℃/s.
Preferably, the basis for the analysis comprises:
the Tm value of the C677T locus of the MTHFR gene is 72 +/-1.5 ℃, and the MTHFR gene is judged as T; the Tm value is 66 +/-1.5 ℃, and the sample is judged to be C;
the Tm value of the site A1298C of the MTHFR gene is 70 +/-1.5 ℃, and the MTHFR gene is judged as C; the Tm value is 64 +/-1.5 ℃, and the judgment is A;
the Tm value of the site A66G of the MTRR gene is 72 +/-1.5 ℃, and the result is judged as G; the Tm value was 66. + -. 1.5 ℃ and the result was judged as A.
In the present invention, the melting curve is a curve indicating the degree of degradation of a DNA double strand with an increase in temperature. The temperature at which the DNA double strand is degraded in half is the melting temperature (Tm). The Tm values of different DNA sequences are different, and the gene polymorphism can be detected based on the principle, so that the method has the advantages of high accuracy, good specificity and the like.
As a preferred technical scheme, the use method of the kit for detecting the folate metabolism related gene polymorphism comprises the following steps:
(1) the kit for detecting the polymorphism of the folate metabolism related gene is used for amplifying and detecting a sample to be detected, and the amplification and detection program comprises the following steps:
pre-denaturation: 94-97 ℃ for 2-5 min;
and (3) cyclic extension: 94-97 ℃ for 5-15 s; at the temperature of 58-62 ℃ for 25-35 s; circulating for 35-40 times;
annealing and extending: 94-97 ℃ for 1-3 min; 38-42 ℃ for 0.5-2 min;
collecting signals: the temperature is 40-85 ℃, wherein the heating rate is 0.01-0.06 ℃/s;
(2) and (3) analysis:
the Tm value of the C677T locus of the MTHFR gene is 72 +/-1.5 ℃, and the MTHFR gene is judged as T; the Tm value is 66 +/-1.5 ℃, and the sample is judged to be C;
the Tm value of the site A1298C of the MTHFR gene is 70 +/-1.5 ℃, and the MTHFR gene is judged as C; the Tm value is 64 +/-1.5 ℃, and the judgment is A;
the Tm value of the site A66G of the MTRR gene is 72 +/-1.5 ℃, and the result is judged as G; the Tm value was 66. + -. 1.5 ℃ and the result was judged as A.
In the invention, by optimizing the amplification system and the reaction program, the specificity and the sensitivity of the reaction are further improved, the detection limit is lower, and the use scene is wider.
In a fourth aspect, the present invention provides an application of any one or a combination of at least two of the primer probe combination for detecting a polymorphism of a folate metabolism-related gene according to the first aspect, the kit for detecting a polymorphism of a folate metabolism-related gene according to the second aspect, and the method for using the kit for detecting a polymorphism of a folate metabolism-related gene according to the third aspect in detection of a polymorphism of a folate metabolism-related gene.
Compared with the prior art, the invention has the following beneficial effects:
(1) the operation is simple and convenient: various components are mixed to prepare a detection reaction solution, and a single or a plurality of genotypes can be detected on a machine only by adding a sample, so that the operation is simple and convenient, and the manual operation error is reduced;
(2) the detection time is shorter: the existing method is a three-step method, the detection time is 120min or longer, the detection method adopted by the invention is a two-step method, the detection can be completed only by 80min, the detection time is greatly saved, and the detection flux is improved;
(3) the sensitivity is high: the sensitivity of the conventional detection method is about 1 ng/mu L, and the technical scheme of the application obviously improves the detection sensitivity, is as low as 100 pg/mu L, can detect the gene polymorphism condition of trace DNA in a sample, and provides favorable conditions for popularization and use of related products.
Drawings
FIG. 1 is a graph of a melting curve of a sample L1 detected by a two-step method in test example 1 of the present invention;
FIG. 2 is a graph showing a melting curve of a sample L1 at an annealing temperature of 60 ℃ in test example 1 of the present invention;
FIG. 3 is a graph of a melting curve of sample L1 with an annealing time of 30s according to test example 1 of the present invention;
FIG. 4 is a graph of a melting curve of a sample L1 taken through 35 cycles in test example 1;
FIG. 5 is a photograph showing a melting curve at MTRR A66G site of sample X1 in test example 3 of the present invention;
FIG. 6 is a photograph showing a melting curve of a kit of lot 1 of sample M2 in test example 3 of the present invention.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
The material and the method are as follows:
the 9 genotype samples containing 3 loci were from clinical whole blood samples.
Taq DNA polymerase was purchased from Novozam.
Example 1
The present example provides a primer probe set for detecting folate metabolism related gene polymorphism, which specifically amplifies and detects polymorphism of MTHFR gene C677T, MTHFR gene A1298C and MTRR gene A66G;
amplifying an upstream primer of a specific primer pair of the MTHFR gene C677T to be a nucleotide sequence shown in SEQ ID No.1, a downstream primer to be a nucleotide sequence shown in SEQ ID No.2, and detecting a probe of the MTHFR gene C677T to be a nucleotide sequence shown in SEQ ID No. 3;
amplifying an upstream primer of a specific primer pair of the MTHFR gene A1298C to be a nucleotide sequence shown in SEQ ID No.4, a downstream primer of the specific primer pair of the MTHFR gene A1298C to be a nucleotide sequence shown in SEQ ID No.5, and detecting a probe of the MTHFR gene A1298C to be a nucleotide sequence shown in SEQ ID No. 6;
amplifying an upstream primer of a specific primer pair of the MTRR gene A66G to be a nucleotide sequence shown in SEQ ID No.7, amplifying a downstream primer of the specific primer pair of the MTRR gene A66G to be a nucleotide sequence shown in SEQ ID No.8, and detecting a probe of the MTRR gene A66G to be a nucleotide sequence shown in SEQ ID No. 9;
SEQ ID No.1：ACTTGAAGGAGAAGGTGTC；
SEQ ID No.2：CAAGTGATGCCCATGT；
SEQ ID No.3：AAGGTGTCTGCGGGAGTCGATTTCATCAT；
SEQ ID No.4：AGGAGCTGACCAGTGAA；
SEQ ID No.5：CATTCCGGTTTGGTTCT；
SEQ ID No.6：ACCAGTGAAGCAAGTGTCTTTGAAGTCTTCGTTC；
SEQ ID No.7：GCCATCGCAGAAGAAA；
SEQ ID No.8：GGTAAAATCCACTGTAACG；
SEQ ID No.9：CAAAGGCCATCGCAGAAGAAATGTGTGAGCAAGCT。
the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group, wherein:
detecting a fluorescent group of the probe of the MTHFR gene C677T as FAM and a quenching group as BHQ 1;
detecting a fluorescent group of a probe of the MTHFR gene A1298C as VIC, and a quenching group as BHQ 1;
the fluorescent group of the probe for detecting the MTRR gene A66G is CY5, and the quenching group is BHQ 2.
Example 2
The present embodiment provides a kit for detecting a polymorphism of a gene related to folate metabolism, the kit for detecting a polymorphism of a gene related to folate metabolism includes the primer probe combination for detecting a polymorphism of a gene related to folate metabolism described in embodiment 1, wherein:
the final concentration of the upstream primer of the specific primer pair for amplifying the MTHFR gene C677T is 0.01 mu mol/L, the final concentration of the downstream primer is 0.2 mu mol/L, and the final concentration of the probe for detecting the MTHFR gene C677T is 0.05 mu mol/L;
the final concentration of the upstream primer of the specific primer pair for amplifying the MTHFR gene A1298C is 0.06 mu mol/L, the final concentration of the downstream primer is 0.006 mu mol/L, and the final concentration of the probe for detecting the MTHFR gene A1298C is 0.01 mu mol/L;
the final concentration of the upstream primer of the specific primer pair for amplifying the MTRR gene A66G is 0.3 mu mol/L, the final concentration of the downstream primer is 0.03 mu mol/L, and the final concentration of the probe for detecting the MTRR gene A66G is 0.06 mu mol/L.
The kit for detecting the folic acid metabolism related gene polymorphism also comprises dNTPs, Taq DNA polymerase and Mg2+And buffering of PCR reactionA solution, wherein:
the final concentration of the dNTPs is 0.2 mmol/L;
the final concentration of the Taq DNA polymerase is 0.025U/mu L;
the Mg2+The final concentration of (a) is 2 mmol/L;
the PCR reaction buffer solution comprises Tris-HCl and KCl;
the final concentration of Tris-HCl is 5 mmol/L;
the final concentration of KCl is 10 mmol/L.
Test example 1
In this test example, the kit for detecting polymorphisms of genes associated with folate metabolism prepared in example 2 was used to verify the effect of different amplification procedures on the detection results.
Influence of two-step amplification and three-step amplification
(1) And (3) amplifying by taking 9 genotype samples which are subjected to sequencing verification and contain 3 loci as templates, wherein an amplification system comprises the following steps:
the procedure for the two-step amplification is as follows:
pre-denaturation: at 95 ℃ for 3 min;
and (3) cyclic extension: 95 ℃ for 10 s; 56 ℃ for 50 s; circulating for 45 times;
annealing and extending: at 95 ℃ for 2 min; at 40 ℃ for 1 min;
collecting signals: 40-85 ℃, wherein the heating rate is 0.03 ℃/s.
The procedure for the three-step amplification was as follows:
pre-denaturation: at 95 ℃ for 3 min;
and (3) cyclic extension: 95 ℃ for 10 s; 56 ℃ for 20 s; 72 ℃ for 30 s; circulating for 45 times;
annealing and extending: at 95 ℃ for 2 min; at 40 ℃ for 1 min;
collecting signals: 40-85 ℃, wherein the heating rate is 0.03 ℃/s.
The detection results are shown in table 1, wherein the melting curve of the sample L1 detected by the two-step method is shown in fig. 1, and the detection results of the other samples and conditions are similar thereto, and are not repeated here.
TABLE 1
As can be seen from Table 1, 2 reaction procedures can realize detection, but compared with a three-step method, the two-step method has simpler procedure, shorter detection time, improved detection efficiency and higher application value.
Influence of annealing temperature
And (3) amplifying by taking 9 genotype samples which are subjected to sequencing verification and contain 3 sites as templates, and verifying the influence of the annealing temperature on the detection result.
The amplification system is the same as that in the verification of the influence of the two-step amplification and the three-step amplification,
the amplification procedure was as follows:
pre-denaturation: at 95 ℃ for 3 min;
and (3) cyclic extension: 95 ℃ for 10 s; gradient temperature, 50 s; circulating for 45 times;
annealing and extending: at 95 ℃ for 2 min; at 40 ℃ for 1 min;
collecting signals: 40-85 ℃, wherein the heating rate is 0.06 ℃/s.
Wherein the gradient temperature is 54 ℃, 56 ℃, 58 ℃, 60 ℃ and 62 ℃ in sequence.
The detection results are shown in table 2, wherein the melting curve of the sample L1 at the annealing temperature of 60 ℃ is shown in fig. 2, and the detection results of the other samples and conditions are similar thereto, and are not repeated here.
TABLE 2
As can be seen from Table 2, at 54 ℃, there was no melting peak at MTRR A66G site, and no Tm value; under the condition of 62 ℃, the AG genotype only has 1 Tm value, and cannot be accurately typed; under the condition of 56-60 ℃, the typing effect of 3 sites is good. Wherein the detection time under the condition of 60 ℃ is the shortest, and the detection time is the most preferable.
Influence of annealing time
And (3) amplifying by taking 9 genotype samples which are subjected to sequencing verification and contain 3 sites as templates, and verifying the influence of annealing time on the detection result.
The amplification system is the same as that in the verification of the influence of the two-step amplification and the three-step amplification,
the amplification procedure was as follows:
pre-denaturation: at 95 ℃ for 3 min;
and (3) cyclic extension: 95 ℃ for 10 s; gradient time at 60 ℃; circulating for 45 times;
annealing and extending: at 95 ℃ for 2 min; at 40 ℃ for 1 min;
collecting signals: 40-85 ℃, wherein the heating rate is 0.03 ℃/s.
Wherein the gradient time is 25s, 30s, 35s, 40s and 50s in sequence.
The detection results are shown in table 3, wherein the melting curve of sample L1 with 30s annealing time is shown in fig. 3, and the melting curves of the rest samples and different conditions are similar to them, and are not repeated here.
TABLE 3
As can be seen from Table 3, all samples can be accurately typed under the condition of 25-50 s, but the longer the extension time is, the longer the time required for detection is, and the detection efficiency is reduced. And from the viewpoint of comprehensive detection effect and detection time, 30s is the optimal annealing time.
Influence of the number of cycles
And (3) amplifying by taking 9 genotype samples which are subjected to sequencing verification and contain 3 sites as templates, and verifying the influence of the cycle number on the detection result.
The amplification system is the same as that in the verification of the influence of the two-step amplification and the three-step amplification,
the amplification procedure was as follows:
pre-denaturation: at 95 ℃ for 3 min;
and (3) cyclic extension: 95 ℃ for 10 s; 30s at 60 ℃; the number of gradient cycles;
annealing and extending: at 95 ℃ for 2 min; at 40 ℃ for 1 min;
collecting signals: 40-85 ℃, wherein the heating rate is 0.03 ℃/s.
Wherein the number of gradient cycles is 30, 35, 40 and 45 in this order.
The detection results are shown in table 4, wherein the melting curve of sample L1 obtained by 35 cycles is shown in fig. 4, and the melting curves of the rest samples and different conditions are similar to the melting curves, which are not repeated herein.
TABLE 4
As can be seen from Table 4, detection was achieved using 4 amplification stage cycle numbers, with 35 cycle numbers being the best overall detection and shorter detection times being the most preferred.
Influence of the rate of temperature rise
And (3) amplifying by taking 9 genotype samples which are subjected to sequencing verification and contain 3 sites as templates, and verifying the influence of the temperature rise rate on the detection result.
The amplification system is the same as that in the verification of the influence of the two-step amplification and the three-step amplification,
the amplification procedure was as follows:
pre-denaturation: at 95 ℃ for 3 min;
and (3) cyclic extension: 95 ℃ for 10 s; 30s at 60 ℃; circulating for 35 times;
annealing and extending: at 95 ℃ for 2 min; at 40 ℃ for 1 min;
collecting signals: and (3) gradient heating rate of 40-85 ℃.
Wherein the gradient heating rate is 0.01 ℃/s, 0.03 ℃/s, 0.05 ℃/s and 0.06 ℃/s in sequence.
The results are shown in Table 5.
TABLE 5
As is clear from Table 5, the detection was achieved under all of the 4 temperature-rising rate conditions described above, and the detection time was the shortest when the temperature-rising rate was 0.06 ℃/s, and the most preferable.
Comparative example 1
The comparative example provides a primer probe combination for detecting the polymorphism of the folate metabolism related gene, and the primer probe combination for detecting the polymorphism of the folate metabolism related gene specifically amplifies and detects the polymorphism conditions of the MTHFR gene C677T, the MTHFR gene A1298C and the MTRR gene A66G;
amplifying an upstream primer of a specific primer pair of the MTHFR gene C677T to be a nucleotide sequence shown in SEQ ID No.10, a downstream primer of the specific primer pair to be a nucleotide sequence shown in SEQ ID No.11, and detecting a probe of the MTHFR gene C677T to be a nucleotide sequence shown in SEQ ID No. 12;
amplifying an upstream primer of a specific primer pair of the MTHFR gene A1298C to be a nucleotide sequence shown in SEQ ID No.13, a downstream primer of the specific primer pair of the MTHFR gene A1298C to be a nucleotide sequence shown in SEQ ID No.14, and detecting a probe of the MTHFR gene A1298C to be a nucleotide sequence shown in SEQ ID No. 15;
amplifying an upstream primer of a specific primer pair of the MTRR gene A66G to be a nucleotide sequence shown in SEQ ID No.16, amplifying a downstream primer of the specific primer pair of the MTRR gene A66G to be a nucleotide sequence shown in SEQ ID No.17, and detecting a probe of the MTRR gene A66G to be a nucleotide sequence shown in SEQ ID No. 18;
SEQ ID No.10：AAGGAGAAGGTGTCTGCGGGA；
SEQ ID No.11：TGCCCATGTCGGTGCATGCCTT；
SEQ ID No.12：CGTGATGATGAAATCGACTCCCGC；
SEQ ID No.13：GGAGCTGACCAGTGAA；
SEQ ID No.14：ACTCCAGCATCACTCACTTT；
SEQ ID No.15：AAGACACTTGCTTCACTGGTCA；
SEQ ID No.16：CACTGTTACATGCCTTGAA；
SEQ ID No.17：AATCCACTGTAACGGCTCTA；
SEQ ID No.18：GCTCACACATTTCTTCTGCGATG。
the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group, wherein:
detecting a fluorescent group of the probe of the MTHFR gene C677T as FAM and a quenching group as BHQ 1;
detecting a fluorescent group of a probe of the MTHFR gene A1298C as VIC, and a quenching group as BHQ 1;
the fluorescent group of the probe for detecting the MTRR gene A66G is CY5, and the quenching group is BHQ 2.
Comparative example 2
The present comparative example provides a kit for detecting a polymorphism of a gene related to folate metabolism, the kit for detecting a polymorphism of a gene related to folate metabolism comprising the primer probe combination for detecting a polymorphism of a gene related to folate metabolism described in comparative example 1, wherein:
the final concentration of the upstream primer of the specific primer pair for amplifying the MTHFR gene C677T is 0.06 mu mol/L, the final concentration of the downstream primer is 0.6 mu mol/L, and the final concentration of the probe for detecting the MTHFR gene C677T is 0.08 mu mol/L;
the final concentration of the upstream primer of the specific primer pair for amplifying the MTHFR gene A1298C is 0.8 mu mol/L, the final concentration of the downstream primer is 0.08 mu mol/L, and the final concentration of the probe for detecting the MTHFR gene A1298C is 0.1 mu mol/L;
the final concentration of the upstream primer of the specific primer pair for amplifying the MTRR gene A66G is 0.6 mu mol/L, the final concentration of the downstream primer is 0.06 mu mol/L, and the final concentration of the probe for detecting the MTRR gene A66G is 0.12 mu mol/L.
The kit for detecting the folic acid metabolism related gene polymorphism also comprises dNTPs, Taq DNA polymerase and Mg2+And a PCR reaction buffer, the components and concentrations of which were the same as in example 2.
Test example 2
In this test example, the effect of the sequences of different primer pairs and probes on the detection result at different template concentrations was compared using the kits for detecting polymorphisms of folate metabolism-related genes prepared in example 2 and comparative example 2.
(1) Amplifying by using 9 genotype samples which are subjected to sequencing verification and contain 3 loci as templates,
the amplification system was as follows:
wherein the gradient concentration of the template is 20 ng/. mu.L, 10 ng/. mu.L, 5 ng/. mu.L, 2 ng/. mu.L and 1 ng/. mu.L in sequence.
The procedure for amplification was as follows:
pre-denaturation: at 95 ℃ for 3 min;
and (3) cyclic extension: 95 ℃ for 10 s; 30s at 60 ℃; circulating for 35 times;
annealing and extending: at 95 ℃ for 2 min; at 40 ℃ for 1 min;
collecting signals: 40-85 ℃, wherein the heating rate is 0.06 ℃/s.
(2) And (3) analysis:
the basis for the analysis includes:
the Tm value of the C677T locus of the MTHFR gene is 72 +/-1.5 ℃, and the MTHFR gene is judged as T; the Tm value is 66 +/-1.5 ℃, and the sample is judged to be C;
the Tm value of the site A1298C of the MTHFR gene is 70 +/-1.5 ℃, and the MTHFR gene is judged as C; the Tm value is 64 +/-1.5 ℃, and the judgment is A;
the Tm value of the site A66G of the MTRR gene is 72 +/-1.5 ℃, and the result is judged as G; the Tm value was 66. + -. 1.5 ℃ and the result was judged as A.
The results are shown in Table 6.
TABLE 6
As can be seen from Table 6, under the condition that the sample concentration is 20 ng/. mu.L, the 2 kits can accurately type the 9 genotypes of the 3 loci, and the specificity is good; when the sample concentration is less than or equal to 10 ng/. mu.L, nonspecific melting peaks (2 Tm values exist) appear in CC and TT genotypes of MTHFR C677T site in the comparative example 2, and meanwhile, a melting peak map (only 1 Tm value or no Tm value) cannot be accurately obtained from AG genotypes of MTRR A66G site, so that accurate typing cannot be realized; in contrast, in example 2, all genotypes can be accurately typed under the condition that the sample concentration is as low as 1 ng/. mu.L, the melting peak diagram is specific, the lower limit of detection is obviously lower than that in comparative example 2, and the accuracy, the specificity and the sensitivity of the primer probe combination in example 2 are better than those in comparative example 2.
Test example 3
This test example examined the detection limit and reproducibility of the kit for detecting a polymorphism of a gene related to folic acid metabolism in example 2.
Detection limit detection
Samples containing the 3 genes and 9 genotypes were diluted to concentrations of 10 ng/. mu.L, 1 ng/. mu.L, 100 pg/. mu.L and 10 pg/. mu.L in this order, and the assay was repeated 3 times for each concentration.
The amplification system and procedure were the same as in test example 2.
The results are shown in Table 7, wherein the melting curve of MTRR A66G site of sample X1 is shown in FIG. 5, and the detection results of the rest samples and sites are similar, and are not repeated here.
TABLE 7
As can be seen from Table 7, the samples can be accurately typed under the condition that the concentration is more than or equal to 100 pg/mu L, which indicates that the lower limit of detection of the kit is not more than 100 pg/mu L.
Repeatability detection
And 3 batches of kits are prepared, and 3 batches of kits are respectively adopted for detecting the repetitive reference substance containing the 9 genotypes of the 3 genes by 2 operators and 2 operating times, and each sample is repeatedly detected for 3 times.
The amplification system and procedure were the same as in the detection limit test.
The results are shown in Table 8, wherein the melting curve of the kit of batch 1 of sample M2 is shown in FIG. 6, and the detection results of the other samples are similar, and are not repeated here.
TABLE 8
As can be seen from Table 8, the detection results are consistent with the sequencing results, and CV of Tm values in batches and CV of Tm values between batches are both less than 1%, indicating that the kit for detecting the polymorphism of the gene related to folic acid metabolism has good repeatability.
Test example 4
In this test example, the kit for detecting polymorphism of a gene related to folate metabolism in example 2 was used to detect an unknown sample.
Nucleic acid samples of 20 clinical blood samples were selected and tested, and the amplification system and procedure were the same as in test example 2.
The results are shown in Table 9.
TABLE 9
The samples are sequenced and compared with the data in the table 9, and the result shows that the detection result is consistent with the sequencing result, so that the kit is proved to have good accuracy and can be applied to actual clinical detection.
In conclusion, the primer probe combination for detecting the folate metabolism related gene polymorphism has good sensitivity and specificity, can be used for carrying out single or combined detection on the gene polymorphism conditions of corresponding sites, and has the advantages of short time, easy operation and low detection cost; by optimizing a detection reaction system and an amplification program, the specificity and the sensitivity of the reaction are further improved, samples with the concentration as low as 100 pg/mu L can be accurately detected, the accuracy is good, and the method has practical application value.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Sequence listing
<110> Shanghai Bao Teng biomedical science and technology Co., Ltd
SHANGHAI BIOTECAN MEDICAL DIAGNOSTICS Co.,Ltd.
Shanghai Zhangjiang Medical Innovation Research Institute
<120> primer probe combination for detecting folate metabolism related gene polymorphism and application thereof
<130> 2021
<160> 18
<170> PatentIn version 3.3
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Claims (10)
1. A primer probe combination for detecting the polymorphism of the folate metabolism related gene is characterized in that the primer probe combination for detecting the polymorphism of the folate metabolism related gene specifically amplifies and detects the polymorphism conditions of MTHFR gene C677T, MTHFR gene A1298C and MTRR gene A66G;
the upstream primer of the specific primer pair for amplifying the MTHFR gene C677T comprises a nucleotide sequence shown in SEQ ID No.1, the downstream primer comprises a nucleotide sequence shown in SEQ ID No.2, and a probe for detecting the MTHFR gene C677T comprises a nucleotide sequence shown in SEQ ID No. 3.
2. The primer probe combination for detecting the folate metabolism related gene polymorphism according to claim 1, wherein the upstream primer of the specific primer pair for amplifying the MTHFR gene A1298C comprises the nucleotide sequence shown in SEQ ID No.4, the downstream primer comprises the nucleotide sequence shown in SEQ ID No.5, and the probe for detecting the MTHFR gene A1298C comprises the nucleotide sequence shown in SEQ ID No. 6;
preferably, the upstream primer of the specific primer pair for amplifying the MTRR gene A66G comprises the nucleotide sequence shown in SEQ ID No.7, the downstream primer comprises the nucleotide sequence shown in SEQ ID No.8, and the probe for detecting the MTRR gene A66G comprises the nucleotide sequence shown in SEQ ID No. 9.
3. The primer probe combination for detecting the polymorphism of the folate metabolism-related gene according to claim 1 or 2, wherein the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group;
preferably, the fluorophore comprises any one of FAM, VIC or CY 5;
preferably, the quencher group comprises BHQ1 or BHQ 2;
preferably, the fluorescent group of the probe for detecting the MTHFR gene C677T is FAM, and the quenching group is BHQ 1;
preferably, the fluorescent group of the probe for detecting the MTHFR gene A1298C is VIC, and the quenching group is BHQ 1;
preferably, the fluorescent group of the probe for detecting the MTRR gene A66G is CY5, and the quenching group is BHQ 2.
4. A kit for detecting polymorphism of a folate metabolism-related gene, which is characterized by comprising a primer probe combination for detecting the polymorphism of the folate metabolism-related gene according to any one of claims 1 to 3;
preferably, the final concentration of the specific primer pair is 0.006-0.3 mu mol/L, and the final concentration of the probe is 0.01-0.06 mu mol/L;
preferably, the final concentration of the upstream primer of the specific primer pair for amplifying the MTHFR gene C677T is 0.01 mu mol/L, the final concentration of the downstream primer is 0.2 mu mol/L, and the final concentration of the probe for detecting the MTHFR gene C677T is 0.05 mu mol/L;
preferably, the final concentration of the upstream primer of the specific primer pair for amplifying the MTHFR gene A1298C is 0.06 mu mol/L, the final concentration of the downstream primer is 0.006 mu mol/L, and the final concentration of the probe for detecting the MTHFR gene A1298C is 0.01 mu mol/L;
preferably, the final concentration of the upstream primer of the specific primer pair for amplifying the MTRR gene A66G is 0.3 mu mol/L, the final concentration of the downstream primer is 0.03 mu mol/L, and the final concentration of the probe for detecting the MTRR gene A66G is 0.06 mu mol/L.
5. The kit for detecting folic acid metabolism related gene polymorphism according to claim 4, characterized in that the kit for detecting folic acid metabolism related gene polymorphism further comprises dNTPs, Taq DNA polymerase, Mg2+And PCR reaction buffer;
preferably, the final concentration of the dNTPs is 0.1-0.5 mmol/L, and preferably 0.2 mmol/L;
preferably, the final concentration of the Taq DNA polymerase is 0.01-0.05U/muL, and preferably 0.025U/muL;
preferably, said Mg2+The final concentration of (b) is 1-5 mmol/L, preferably 2 mmol/L;
preferably, the PCR reaction buffer comprises Tris-HCl and KCl;
preferably, the final concentration of Tris-HCl is 2-7 mmol/L, preferably 5 mmol/L;
preferably, the final concentration of KCl is 8-12 mmol/L, and preferably 10 mmol/L.
6. A method for using the kit for detecting a folate metabolism-related gene polymorphism according to claim 4 or 5, wherein the method for using the kit comprises:
and amplifying and detecting a sample to be detected by using the kit for detecting the polymorphism of the folate metabolism-related gene, and then analyzing.
7. The method for using the kit for detecting folate metabolism-related gene polymorphism according to claim 6, wherein said amplification and detection procedure comprises:
pre-denaturation: 94-97 ℃ for 2-5 min;
and (3) cyclic extension: 94-97 ℃ for 5-15 s; 56-60 ℃ for 25-35 s; circulating for 35-40 times;
annealing and extending: 94-97 ℃ for 1-3 min; 38-42 ℃ for 0.5-2 min;
collecting signals: 40-85 ℃, wherein the heating rate is 0.01-0.06 ℃/s, preferably 0.06 ℃/s.
8. The use method of the kit for detecting a folate metabolism-related gene polymorphism according to claim 6 or 7, wherein the basis for said analysis comprises:
the Tm value of the C677T locus of the MTHFR gene is 72 +/-1.5 ℃, and the MTHFR gene is judged as T; the Tm value is 66 +/-1.5 ℃, and the sample is judged to be C;
the Tm value of the site A1298C of the MTHFR gene is 70 +/-1.5 ℃, and the MTHFR gene is judged as C; the Tm value is 64 +/-1.5 ℃, and the judgment is A;
the Tm value of the site A66G of the MTRR gene is 72 +/-1.5 ℃, and the result is judged as G; the Tm value was 66. + -. 1.5 ℃ and the result was judged as A.
9. The use method of the kit for detecting folate metabolism-related gene polymorphism according to any one of claims 6 to 8, wherein the use method comprises:
(1) the kit for detecting the polymorphism of the folate metabolism related gene is used for amplifying and detecting a sample to be detected, and the amplification and detection program comprises the following steps:
pre-denaturation: 94-97 ℃ for 2-5 min;
and (3) cyclic extension: 94-97 ℃ for 5-15 s; at the temperature of 58-62 ℃ for 25-35 s; circulating for 35-40 times;
annealing and extending: 94-97 ℃ for 1-3 min; 38-42 ℃ for 0.5-2 min;
collecting signals: the temperature is 40-85 ℃, wherein the heating rate is 0.01-0.06 ℃/s;
(2) and (3) analysis:
the Tm value of the C677T locus of the MTHFR gene is 72 +/-1.5 ℃, and the MTHFR gene is judged as T; the Tm value is 66 +/-1.5 ℃, and the sample is judged to be C;
the Tm value of the site A1298C of the MTHFR gene is 70 +/-1.5 ℃, and the MTHFR gene is judged as C; the Tm value is 64 +/-1.5 ℃, and the judgment is A;
the Tm value of the site A66G of the MTRR gene is 72 +/-1.5 ℃, and the result is judged as G; the Tm value was 66. + -. 1.5 ℃ and the result was judged as A.
10. Use of any one or a combination of at least two of the primer probe combination for detecting a polymorphism in a gene associated with folate metabolism according to any one of claims 1 to 3, the kit for detecting a polymorphism in a gene associated with folate metabolism according to claim 4 or 5, or the kit for detecting a polymorphism in a gene associated with folate metabolism according to any one of claims 6 to 9, for detecting a polymorphism in a gene associated with folate metabolism.
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