CN116515983B - Typing kit, primer group and typing method for vitamin E metabolism related genes - Google Patents

Typing kit, primer group and typing method for vitamin E metabolism related genes Download PDF

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CN116515983B
CN116515983B CN202211705228.1A CN202211705228A CN116515983B CN 116515983 B CN116515983 B CN 116515983B CN 202211705228 A CN202211705228 A CN 202211705228A CN 116515983 B CN116515983 B CN 116515983B
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CN116515983A (en
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周坤
贾永娟
丛芹芹
武舒佳
倪君君
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Suzhou Hehe Medical Test Co ltd
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Abstract

The invention relates to the field of gene detection, in particular to the field of C12N15/11, and more particularly relates to a typing kit, a primer group and a typing method for non-diagnostic purposes of vitamin E metabolism related genes. A PCR amplification primer group of vitamin E metabolism related genes has a nucleotide sequence shown in SEQ ID NO:1-SEQ ID NO:12, a single base extension primer group of vitamin E metabolism related gene, the nucleotide sequence of which is shown in SEQ ID NO:13-SEQ ID NO:36, at least one of the following. The parting method comprises the following steps: the PCR amplification primer is subjected to PCR amplification reaction, alkaline phosphatase digestion, single-base extension reaction, desalting treatment and high performance liquid chromatography-mass spectrometry combined detection. The kit and the detection method can improve the typing detection efficiency of the vitamin E metabolism related genes, are simple, convenient and feasible, have high detection flux and high automation degree.

Description

Typing kit, primer group and typing method for vitamin E metabolism related genes
Technical Field
The invention relates to the field of gene detection, in particular to the field of C12N15/11, and more particularly relates to a kit, a primer group and a typing method for a vitamin E metabolism related gene.
Background
The current common method for determining vitamin E gene is multiplex PCR-sequencing, and a time-of-flight mass spectrometry detection method is also adopted. However, the average cost of detecting a small number of sites using time-of-flight mass spectrometry has also been reported to be relatively high. Currently, PCR sequencing is often used to detect gene polymorphisms at multiple genes/sites. In the detection process, the existing detection method can only detect the gene polymorphism type of one or a few genes/loci at a time, so that the detection method has the problems of long detection period and low flux, and finally has low detection efficiency. For example, chinese patent CN 110511985A provides a method for detecting mutation sites of vitamin E absorption related genes, wherein the detected genes and the detected sites are CYP4F2-rs2108622 and ZPR1-rs964184; chinese patent CN 110184335A provides a method for detecting mutation sites of vitamin E metabolism related genes, the detection genes and sites are CYP4F2 genes, ZPR1 genes.
In addition, the multiplex PCR-sequencing method has the problems of high cost of multi-site detection, time-consuming analysis and the like, although the detection sites are more, and is not suitable for high-throughput sample detection.
Therefore, it is urgently needed to find a typing kit and a typing method for genes related to vitamin E metabolism, which are simple, convenient and easy to implement, high in detection throughput and high in automation degree.
Disclosure of Invention
Aiming at some problems existing in the prior art, the first aspect of the invention provides a PCR amplification primer group of vitamin E metabolism related genes, the nucleotide sequence of which is shown as SEQ ID NO:1-SEQ ID NO:12, at least one of the following.
Preferably, the target product length of the PCR amplification primer set ranges from 200 bp to 700bp.
The nucleotide sequences of the PCR amplification primer set are specifically shown in Table 1.
TABLE 1
In a second aspect, the invention provides a kit for typing vitamin E metabolism-related genes, which comprises a nucleic acid amplification reagent and a single-base extension reaction reagent, wherein the nucleic acid amplification reagent comprises 6 pairs of primers.
In one embodiment, the nucleic acid amplification reagent further comprises GC-RICH solution, fastStart Taq DNA polymerase, preferably water, buffer, mgCl 2 dNTP mix, template.
The single base extension reaction reagents described herein include a set of single base extension primers, preferably 24 single base extension primers.
Preferably, the nucleotide sequence of the single base extension primer is shown in SEQ ID NO:13-SEQ ID NO:36, the MS molecular weight ranges from 4700 to 8600, specifically shown in tables 2-1 and 2-2.
TABLE 2-1
TABLE 2-2
Remarks: wherein VE-T3 extension primers are used to detect both the 2T > C and 2T > A sites, and VE-T17 is used to detect both the 426G > T and 426G > A sites.
In one embodiment, the typing kit further comprises an alkaline phosphatase reaction solution and/or a desalting reagent.
Preferably, the alkaline phosphatase in the alkaline phosphatase reaction solution is one or more selected from shrimp alkaline phosphatase, escherichia coli alkaline phosphatase and rat alkaline phosphatase. Specifically, the concentration of alkaline phosphatase was 1.7U/. Mu.L, and the binding buffer was used to prepare an alkaline phosphatase reaction solution having a concentration of alkaline phosphatase of 0.073U/. Mu.L.
In one embodiment, a method for typing a vitamin E metabolism-related gene comprises: the PCR amplification primer is subjected to PCR amplification reaction, alkaline phosphatase digestion, single-base extension reaction, desalting treatment and high performance liquid chromatography-mass spectrometry combined detection.
The typing method of the present application detects the sites as shown in the following table.
In one embodiment, 26 mutation sites of the vitamin E metabolism related TTPA, COMT genes are detected in two groups A, B in the typing method.
In one embodiment, in the typing method, the PCR amplification primer set includes a nucleotide sequence as set forth in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:7-SEQ ID NO:12, the initial concentration of the PCR amplification primer set is 9 to 11. Mu.M, preferably 10. Mu.M, and the solvent is water.
Preferably, the PCR amplification primer set of the A group comprises a nucleotide sequence shown as SEQ ID NO:1-SEQ ID NO: 2. SEQ ID NO:9-SEQ ID NO:12, the concentration of the PCR amplification primer group is 0.5-1 mu M; SEQ ID NO:7-SEQ ID NO:8, and the concentration of the PCR amplification primer set is 1.5-2.5 mu M.
Preferably, in the PCR amplification primer set, the nucleotide sequence is as set forth in SEQ ID NO: 1. SEQ ID NO:2 is 2-3 mu L, and the nucleotide sequence is shown in SEQ ID NO: 7. SEQ ID NO:8 is 8-12 mu L, and the nucleotide sequence is shown in SEQ ID NO: 9. SEQ ID NO:12 is 2-3 μl, more preferably, in the PCR amplification primer set, the nucleotide sequence is as set forth in SEQ ID NO: 1. SEQ ID NO:2 is 2.5 mu L, and the nucleotide sequence is shown in SEQ ID NO: 7. SEQ ID NO:8 is 10 mu L, and the nucleotide sequence is shown in SEQ ID NO: 9. SEQ ID NO:12 is 2.5. Mu.L and the volume of the solvent water is 15. Mu.L.
In one embodiment, in the typing method, the set B of said PCR amplification primer sets comprises a nucleotide sequence as set forth in SEQ ID NO:1-SEQ ID NO:6, and the concentration of the PCR amplification primer set is 0.5-1.5 mu M.
Preferably, the single base extension primer set of group a comprises SEQ ID NO: 14. SEQ ID NO:17, the concentration of which is 9-11uM; SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO:35, the concentration of the single base extension primer is 11.5-13.5uM; SEQ ID NO: 13. SEQ ID NO: 33. SEQ ID NO:36, which has a concentration of 17.5-19.5uM; SEQ ID NO:15, which has a concentration of 24 to 26. Mu.M;
further preferred, the nucleotide sequence is as set forth in SEQ ID NO:14 and SEQ ID NO:17 is 1 mul, the nucleotide sequence is shown as SEQ ID NO: 35. SEQ ID NO:34 and SEQ ID NO:32 is 1.25 mu L, the nucleotide sequence of which is shown in SEQ ID NO: 36. SEQ ID NO: 33. SEQ ID NO:13 is 1.85 mu L, the nucleotide sequence is shown as SEQ ID NO:15 is 2.5. Mu.L and the volume of the solvent water is 26.2. Mu.L.
Preferably, the single base extension primer set of group B comprises SEQ ID NO:29-SEQ ID NO:31, the concentration of the single base extension primer is 9-11uM; SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO:26, which has a concentration of 11.5 to 13.5. Mu.M; SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 25. SEQ ID NO:27-SEQ ID NO:28, which has a concentration of 17.5 to 19.5. Mu.M; SEQ ID NO: 16. SEQ ID NO:18, and the concentration of the single base extension primer is 24-26 mu M.
Further preferred, the nucleotide sequence is as set forth in SEQ ID NO:29-SEQ ID NO:31 is 1 mul, the nucleotide sequence is shown as SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, seq ID NO:21 is 1.25 mu L, the nucleotide sequence of which is shown in SEQ ID NO:28, seq ID NO:27, seq ID NO:25, SEQ ID NO:20, SEQ ID NO:19, SEQ ID NO:22 is 1.85 mu L, the nucleotide sequence of which is shown in SEQ ID NO:18, seq ID NO:16 is 2.5. Mu.L and the volume of the solvent water is 15.9. Mu.L.
The reaction conditions involved in the PCR amplification reaction in the present application can be routinely selected by those skilled in the art according to actual needs, for example: pre-denaturation at 90-100deg.C for 3-6min; then denaturation is carried out at 90-100 ℃ for 20-40s, annealing is carried out at 50-60 ℃ for 20-40s, extension is carried out at 70-80 ℃ for 1-3min, and amplification is carried out for 40-50 cycles; then extending for 5-10min at 70-80deg.C. In a preferred embodiment, the reaction conditions are: pre-denaturation at 95 ℃ for 4min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 72℃for 1.5min, amplification for 45 cycles; then, the mixture was extended at 72℃for 7 minutes.
In one embodiment, the digestion system in alkaline phosphatase digestion comprises 1-2. Mu.L of water, 0.1-0.3. Mu.L of LSAP buffer, 0.1-0.5. Mu.L of shrimp alkaline phosphatase, preferably 1.53. Mu.L of water, 0.17. Mu.L of LSAP buffer, 0.3. Mu.L of shrimp alkaline phosphatase.
The digestion conditions for alkaline phosphatase digestion in this application can be routinely selected by those skilled in the art according to actual needs, for example, digestion at 37℃for 40min, followed by heat denaturation at 85℃for 5min, followed by maintaining at 4 ℃.
In one embodiment, the group A single base extension PCR system in a single base extension reaction comprises 0.5-0.8. Mu.L of water, 0.1-0.3. Mu.L of the iplex buffer, 0.1-0.3. Mu.L of the iplex ddNTP mixture, 0.8-1.1. Mu.L of the group B extension primer mixture, 0.03-0.07. Mu.Liplex enzyme, preferably comprising: 0.6. Mu.L of water, 0.2. Mu.L of the iplex buffer, 0.2. Mu.L of the iplex ddNTP mix, 0.95. Mu.L of the set of extension primer mix, 0.05. Mu.L of the iplex enzyme;
the group B single base extension PCR system comprises 0.5-0.8. Mu.L of water, 0.1-0.3. Mu.L of Liplex buffer, 0.1-0.3. Mu.L of the iplex ddNTP mixture, 0.8-1.1. Mu.L of the group B extension primer mixture, and 0.03-0.07. Mu.L of Liplex enzyme, preferably comprising: 0.6. Mu.L of water, 0.2. Mu.L of Liplex buffer, 0.2. Mu.L of the iplex ddNTP mix, 0.95. Mu. L B set of extension primer mix, 0.05. Mu.L of the iplex enzyme.
The reaction conditions for the single base extension reactions described herein can be routinely selected by one skilled in the art according to actual needs, for example, as shown in Table 3 below.
TABLE 3 Table 3
Desalination operations herein include, but are not limited to, resin desalination, chromatographic desalination, ultrafiltration desalination, and the like. By removing K in the PCR reaction system + 、Na + 、Mg 2+ And the metal ions are prevented from interfering with the mass spectrum detection result.
In one embodiment, the desalination process comprises: adding resin and distilled water, mixing, and rotating for 30-60 minutes, wherein the desalting treatment comprises: adding 15-25uL of resin into a PCR tube containing 9 uL of reaction system, adding 40-120 uL of distilled water, fixing the PCR tube containing the single base extension product and the resin onto a rotary mixer, and slowly rotating for 30-60 minutes.
In S5, the mass spectrometer is a liquid chromatography-high resolution mass spectrometer (LC-MS) detection system. And (3) carrying out liquid phase separation and mass spectrum detection on the reaction liquid subjected to desalination treatment on a machine to obtain a map. Since the molecular weights of the extension products are different, whether or not a detection peak appears at each molecular weight (mass-to-charge ratio) position is checked, and then SNP typing of the sample is judged. The liquid chromatograph-high resolution mass spectrometer mass spectrum platform has good specificity, the lowest detection lower limit is 5ng genome DNA, and the coincidence is 100% compared with the gold standard technology Sanger sequencing technology selected by the experiment.
The anion exchange chromatographic column with high separation degree can be selected in the high performance liquid chromatography condition, and the DNAPac analytical column (product of Thermo company) can be specifically selected, so that the synthesized and modified oligonucleotide can be analyzed and purified with high resolution. The parameters are as follows: 2.1X100 mm, column temperature: 60 ℃; the loading was 10. Mu.L.
Preferably, the mobile phase A phase is an aqueous solution containing hexafluoroisopropanol and triethylamine, more preferably, the volume percentage of hexafluoroisopropanol in the mobile phase is 0.4-0.6%, more preferably, the volume percentage of hexafluoroisopropanol is 0.49%; it is further preferred that the volume percentage of triethylamine in the mobile phase is 0.1-0.12%, more preferably that the volume percentage of triethylamine is 0.11%.
Preferably, phase B is a methanol solution containing hexafluoroisopropanol and triethylamine; further preferably, the volume percentage of hexafluoroisopropanol is 0.4-0.6%, more preferably 0.49%; the volume percentage of the triethylamine is 0.1-0.12%, more preferably 0.11%.
The liquid phase gradient/procedure is shown in table 4 below.
TABLE 4 Table 4
Time (min) B％
0 5
1 20
3.5 40
5.5 95
6 5
10 5
The mass spectrometry conditions are shown in table 5 below.
TABLE 5
The mutation sites of 26 TTPA and COMT genes are detected by a method of combining multiple PCR and mass spectrum detection.
Compared with the prior art, the invention has the following beneficial effects:
on the premise of meeting the detection requirement, the method adopts a nucleic acid mass spectrometry method to detect 26 mutation sites of vitamin E metabolism related genes simultaneously, so that the dosage of reagents and samples is reduced, the analysis time is shortened, and the detection flux is improved;
by adopting the single-base extension primer set and the multiplex PCR amplification primer set and combining the liquid phase-mass spectrum detection technology, the method not only can realize the base type detection of one gene/locus, but also can detect polymorphism of up to 26 polymorphic loci of a plurality of, even 2 vitamin E related genes simultaneously, and has the advantages of accurate result, high flux, high detection efficiency and the like;
the PCR amplification primer, the single-base extension primer and the amplification enzyme system are further optimized to achieve the highest extension efficiency, so that the accuracy of the base type detection result is improved.
Drawings
FIG. 1 is an amplification electrophoretogram of the different primers Pmix of comparative example 1 in the FastStart enzyme system and FastStartGC enzyme system;
FIG. 2 is an amplification electrophoretogram of comparative examples 1VE-Pmix-10A and VE-Pmix-10B in the FastStart GC enzyme line and FastStart 50% GC enzyme line;
FIG. 3 is an amplification electrophoresis chart of comparative example 1 VE-Pmix-10-E.
Detailed Description
The present invention is illustrated by the following specific embodiments, but is not limited to the specific examples given below.
The main instrumentation, reagents used in the examples:
1. instrument apparatus:
biological safety cabinet: sea, HR 40-IIA 2; PCR instrument: race-moeid, veriti DX 96; electrophoresis apparatus: beijing six biotechnology limited, DYY-6C; horizontal electrophoresis tank: DYCP-31DN of Beijing six biotechnology Co., ltd; gel imaging system: tongbao reaching science and technology (Beijing) limited company, GI-1; microwave oven: glanshi (guangdong), P70D20TJ-03; mini centrifuge: the American Qi Co., ltd, MLX-206; rotating and evenly mixing: SCILOGEX (U.S.), MX-RL-E; high performance liquid chromatography system: thermo, vaquish; mass spectrometry: thermo, QE Plus.
2. Reagent(s)
Agarose: biowest,100g;6×loading Buffer: physical technologies for doctor of precious days (Beijing), limited (TaKaRa), 9156;100bp DNA Ladder: new England Biolabs, N3231V; deionized water: a mass spectrum stage; primer: synthesizing Huada genes; sample pretreatment reagent Pro of time-of-flight mass spectrometry detection system: agena Bioscience; methanol (mass spectrometric purity): fisher,4L; triethylamine (TEA): sigma-Aldrich, purity not less than 99.5%,73487,5mL; hexafluoroisopropanol (HFIP): sigma-Aldrich, purity not less than 99.8%,52517, 50mL; pierce TM LTQ Velos ESI Positive Ion Calibration Solution：Thermo Scientific，10mL；Pierce TM Negative Ion Calibration Solution：Thermo Scientific，10mL。
Example 1
1. Extracting genomic DNA: in this example, an initial sample of genomic DNA of the sample to be tested was obtained by sampling from peripheral blood and extracting it with a DNA extraction kit (YDP 304-03, tiangen Biochemical technology (Beijing)) from the company Limited.
2. The parting method comprises the following steps:
the 26 mutation sites of the TTPA and COMT genes are divided into two groups, which are named VE-A, VB-B respectively.
The method specifically comprises the following steps: the PCR amplification primer is subjected to PCR amplification reaction, alkaline phosphatase digestion, single-base extension reaction, desalting treatment and high performance liquid chromatography-mass spectrometry combined detection.
2.1PCR amplification
2.1.1 specific amplification primer Mix configuration method:
first group VE-a:
TABLE 6
Second group VE-B:
TABLE 7
2.1.2 Single base extension primer Mix configuration method:
first group VE-a:
TABLE 8
Second group VE-B:
TABLE 9
2.1.3 wherein the PCR amplification system was as shown in Table 10 below.
Table 10
Note that: the two groups VE-A, VE-B are amplified respectively, and correspond to VE-Pmix-A, VE-Pmix-B respectively.
2.1.4PCR amplification reaction conditions are shown in Table 11 below.
TABLE 11
2.2 alkaline phosphatase digestion:
wherein alkaline phosphatase digestion system is shown in Table 12 below, 2. Mu.L of the prepared digestion system was added to each reaction tube.
Table 12
SAP premix Volume (mu L)
Water and its preparation method 1.53
SAP buffer 0.17
Shrimp alkaline phosphatase 0.3
Total volume (mu L) 2
The digestion procedure was: 40min at 37 ℃, 5min at 85 ℃ and 4 ℃.
2.3 Single base extension reaction:
wherein, the single base extension reaction system is shown in Table 13 below, and 2. Mu.L of the prepared extension system was added to each reaction tube.
TABLE 13
Remarks: the single base extension system was divided into A, B groups, which correspond to VE-A, VE-B in the amplification system, respectively. Wherein, single base extension PCR procedure is as follows in Table 14.
TABLE 14
2.4 resin desalination:
wherein, the desalination treatment is resin desalination, and the specific steps are as follows:
to a PCR tube containing 9. Mu.L of the above reaction system, 20. Mu.L of the resin was added, and 100. Mu.L of distilled water was further added. The PCR tube containing the single base extension product and the resin was fixed to a spin mixer and spun at a slow speed for 45 minutes.
2.5 mass spectrometry detection:
2.5.1 liquid phase conditions: dnappc analytical column (Thermo company product) parameters were: 2.1X100 mm, column temperature: 60 ℃; the loading was 10. Mu.L.
The mobile phase A is an aqueous solution containing hexafluoroisopropanol and triethylamine; 0.49% by volume of hexafluoroisopropanol and 0.11% by volume of triethylamine; the mobile phase B is a methanol solution containing hexafluoroisopropanol and triethylamine; the volume percentage of hexafluoroisopropanol is 0.49%, and the volume percentage of triethylamine is 0.11%.
Liquid phase gradient/procedure:
2.5.2 Mass Spectrometry parameters
Analysis of the results of the high performance liquid chromatography-mass spectrometry detection test is performed by adopting software matched with a Thermo mass spectrometer, such as Thermo BioPharma Finder software, and after software parameters are set, the genotyping result is interpreted, and the interpretation standard is shown in the following table 15.
TABLE 15
The criteria for the determination of each mutation site are shown in Table 16 below.
Table 16
The accuracy of the kit and sequencing method of example 1 was tested as follows.
Taking 3 normal human DNA samples as examples (sample names of SLP, RJJ and ZHF, respectively), detection was performed using the above-described example 1 kit and multiplex PCR-sequencing method.
The results of group a mass spectrometry are shown in table 17.
TABLE 17
The sequencing results of the sequencing using multiplex PCR are shown in table 18;
TABLE 18
Remarks: genotype is marked as heterozygous mutant.
The results of group B mass spectrometry are shown in table 19.
TABLE 19
The sequencing results of the sequencing using multiplex PCR are shown in Table 20.
Table 20
The result shows that: the genotype detected by 26 sites of vitamin E metabolic genes by adopting the kit is consistent with the genotype of a sequencing result.
Comparative example 1
In the early stage of research and development, screening experiments are carried out on PCR amplification primer sequences, a primer Pmix preparation method and an amplification enzyme system.
(1) The Primer pairs VE-MP-01-A-F/R, VE-MP-01-A-F/VE-MP-01-R, VE-MP-01-F/VE-MP-01-A-R, VE-MP-01-B-F/R in Table 21 are adopted to prepare VE Primer Mix-04, VE Primer Mix-06-A, VE Primer Mix-06-B and VE Primer Mix-07 according to the preparation method in Table 22; subsequently, DNA samples of normal persons (sample names were RJJ, LXJ, respectively) were amplified using the Fatstart enzyme system and the Fatstart GC enzyme system in Table 23, and an electrophoresis chart was obtained as shown in FIG. 1, and the results are shown in Table 24.
Table 21
The primer concentration schemes involved are shown in Table 22.
Table 22
The enzyme system referred to is shown in Table 23 in. Mu.L.
Table 23
Reaction system FastStart enzyme System FastStart GC enzyme System
Water,HPLC grade 0.8 0.3
10*PCR buffer 0.5 0.5
GC-RICH solution(5×)(vial 5) / 0.5
25mM MgCl 2 0.4 0.4
25mM dNTP mix 0.1 0.1
0.5uM primer Mix 1.1 1.1
5U/ul PCR enzyme 0.1 0.1
5ng/ul DNA 2 2
Total volume of 5 5
Table 24
Wherein, "×" indicates that no target band was detected, and the amplification was poor; "#" indicates that the detected target band is not obvious or too shallow; the sign of 'V' indicates that the obvious target strip is detected, and the amplification effect is good; "-GC" indicates that the enzyme system used was a FastStart enzyme system to which GC-RICH solution (5X) was not added, and "+GC" indicates that the enzyme system used was a FastStart enzyme system to which GC-RICH solution (5X) was added.
As shown in Table 24 and FIG. 1, none of the 4 schemes (1) detected the target band of exon4 of the TPPA gene; (2) The target band of the TTPA gene No. 1 exon can be obtained by adopting a FastStart enzyme system added with GC-RICH solution (5×), VE-Pmix-04 and VE-Pmix-07, but the band of VE-Pmix-07 is more obvious, so that the VE-MP-01-B-F/R primer group is adopted to detect the TTPA gene No. 1 exon in the later stage; (3) The apparent target band of exon1 of the TTPA gene was detected by VE-Pmix-07, but not by exon 3.
(2) The DNA samples of normal individuals (sample names: DJL, ZFY, RJJ, respectively) were examined by using the Fatstart GC enzyme system and the Fatstart 50% GC enzyme system in Table 26, respectively, to prepare VE-Pmix-10-A and VE-Pmix-10-B according to Table 25 using the primer set of VE-MP-01-B-F/R, VE-MP-03-A-F/R, VE-MP-04-A-F/R, etc., in Table 21, and the resulting electrophoreses are shown in FIG. 2 and Table 27.
Table 25
Table 26
As is clear from Table 27 and FIG. 2, the target band of TTPA Exon1 was obtained by the FastStartGC enzyme system and the FastStart 50% GC enzyme system, and the amplification of TTPA Exon3 was obtained by changing the primer and increasing the VE-MS-03-A-F/A-F (439 bp) ratio. After TTPA Exon4 was changed to primer VE-MS-04-A-F/A-F (253 bp), the target band was seen in a hidden manner, and thus the adjustment was continued on VE-Pmix-10-B.
Table 27
Wherein, "×" indicates that no target band was detected, and the amplification was poor; "#" indicates that the detected target band is not obvious or too shallow; the sign of 'V' indicates that the obvious target strip is detected, and the amplification effect is good;
"+GC" indicates that the enzyme system used was a FastStart enzyme system to which GC-RICH solution (5X) was added; "+50% GC" indicates that the enzyme system employed was a FastStart enzyme system with 50% GC-RICH solution (5X) added.
Through a series of adjustment and screening experiments, the detection of a normal human sample (sample name is ZGQ, DJL, ZK) by using VE-Pmix-10-E, fastStart 50%GC enzyme system can obtain bands of 6 target products, and the amplification reaction system is tentatively set, and the result is shown in FIG. 3.
Comparative example 2
The typing method was the same as in example 1, except that the single base extension primers were different, and the single base extension primers involved are shown in Table 28.
Table 28
The DNA samples (sample names were DJL, JYX, LXJ, PSM, RJJ, THM) of normal persons were detected using the single base extension primer of VE-T27, and the experimental results are shown in Table 29.
Table 29
Wherein "/" indicates that no corresponding MS molecular weight is detected; the values represent the relative abundance of the corresponding MS molecular weights detected.
As shown in Table 29, the VE-T-27 primer was used, and the extension primer corresponding to the rs4680 locus of the COMT gene had more residue and the relative strength of the whole extension product was lower.
The single base extension primer of VE-T27-01 was used to detect normal human samples (sample names PH, LXJ, ZFY respectively) to obtain the experimental results shown in Table 30:
table 30
Wherein "/" indicates that no corresponding MS molecular weight is detected; the values represent the relative abundance of the corresponding MS molecular weights detected.
As shown in Table 30, the extension product was not obtained from normal human samples using the VE-T-27-01 primer, which was not available.
The single base extension primer of VE-T27-02 was used to detect normal human samples (sample names LXJ, PH, ZFY respectively) to obtain the experimental results shown in Table 31:
table 31
Wherein "/" indicates that no corresponding MS molecular weight is detected; the values represent the relative abundance of the corresponding MS molecular weights detected.
As shown in Table 31, the VE-T-27-02 primer is used to obtain the target product from normal human sample, the genotype is correctly interpreted, and the response strength is improved.
Comparative example 3
Group inspection: 26 sites were simultaneously detected using the FastStart 50% GC enzyme system and the results are shown in Table 32.
Table 32
Wherein "/" indicates that no corresponding MS molecular weight is detected; the values represent the relative abundance of the corresponding MS molecular weights detected.
As shown in Table 32, no extension products were detected by VE-T1 extension primers from 3 normal human samples; the response intensity of the extension product of VE-T7 is too low (intensity is less than 5), the extension efficiency is poor, and genotype interpretation is affected.
After multiple adjustments, A, B grouping detection is finally carried out by adopting the kit method of the scheme, genotypes of 26 sites are correct, the effect is good, and the data result is the same as that of the example 1.

Claims (3)

1. The kit for typing the vitamin E metabolism related genes is characterized by comprising a nucleic acid amplification reagent and a single base extension reaction reagent, wherein the nucleic acid amplification reagent comprises 6 pairs of primers, and the nucleic acid amplification reagent also comprises GC-RICH solution and FastStart Taq DNA polymerase;
the nucleotide sequences of the 6 pairs of primers are shown in SEQ ID NO:1-SEQ ID NO:12, the length range of the target product of the primer is 200-700bp;
the single base extension reaction reagent comprises 24 single base extension primers, and the nucleotide sequence of each single base extension primer is shown as SEQ ID NO:13-SEQ ID NO: shown at 36;
dividing 26 mutation sites of TTPA and COMT genes related to vitamin E metabolism into A, B groups for detection, wherein in the detection process, a group A PCR amplification primer group is used in the detection of group A, a group A single base extension primer group is used in the detection of group B, and a group B PCR amplification primer group and a group B single base extension primer group are used in the detection of group B;
the PCR amplification primer group of the A group comprises a nucleotide sequence shown as SEQ ID NO:1-SEQ ID NO: 2. SEQ ID NO:9-SEQ ID NO:12, the concentration of the PCR amplification primer group is 0.5-1 mu M; SEQ ID NO:7-SEQ ID NO:8, wherein the concentration of the PCR amplification primer group is 1.5-2.5 mu M; the PCR amplification primer group of the B group comprises a nucleotide sequence shown as SEQ ID NO:1-SEQ ID NO:6, the concentration of the PCR amplification primer group is 0.5-1.5 mu M;
the single base extension primer set of the A group comprises SEQ ID NO: 14. SEQ ID NO:17, the concentration of which is 9-11 mu M; SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO:35, the concentration of the single base extension primer is 11.5-13.5 mu M; SEQ ID NO: 13. SEQ ID NO: 33. SEQ ID NO:36, the concentration of which is 17.5-19.5 mu M; SEQ ID NO:15, which has a concentration of 24 to 26. Mu.M; the single base extension primer set of the B group comprises SEQ ID NO:29-SEQ ID NO:31, the concentration of the single base extension primer is 9-11uM; SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO:26, which has a concentration of 11.5 to 13.5. Mu.M; SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 25. SEQ ID NO:27-SEQ ID NO:28, which has a concentration of 17.5 to 19.5. Mu.M; SEQ ID NO: 16. SEQ ID NO:18, and the concentration of the single base extension primer is 24-26 mu M.
2. The kit for typing of vitamin E metabolism-related genes according to claim 1, wherein the single base extension primer has an MS molecular weight ranging from 4700 to 8600.
3. The kit for typing of vitamin E metabolism-related genes according to claim 2, wherein the kit for typing further comprises an alkaline phosphatase reaction solution and/or a desalting reagent.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106222247A (en) * 2016-07-01 2016-12-14 长春恒晨生物科技有限责任公司 COMT site relevant primer and this single nucleotide polymorphisms detection kit
CN110257503A (en) * 2019-06-28 2019-09-20 北京和合医学诊断技术股份有限公司 Detect the primer sets and multi-PCR detection method of vitamin E metabolic gene TTPA
CN114438200A (en) * 2022-04-12 2022-05-06 北京和合医学诊断技术股份有限公司 Typing kit, primers and typing method for vitamin K metabolism related genes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106222247A (en) * 2016-07-01 2016-12-14 长春恒晨生物科技有限责任公司 COMT site relevant primer and this single nucleotide polymorphisms detection kit
CN110257503A (en) * 2019-06-28 2019-09-20 北京和合医学诊断技术股份有限公司 Detect the primer sets and multi-PCR detection method of vitamin E metabolic gene TTPA
CN114438200A (en) * 2022-04-12 2022-05-06 北京和合医学诊断技术股份有限公司 Typing kit, primers and typing method for vitamin K metabolism related genes

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