CN116179669B

CN116179669B - Typing kit, primer and typing method for vitamin B12 metabolism related genes

Info

Publication number: CN116179669B
Application number: CN202310008851.XA
Authority: CN
Inventors: 贾永娟; 周坤; 丛芹芹; 武舒佳; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2023-01-04
Filing date: 2023-01-04
Publication date: 2023-10-24
Anticipated expiration: 2043-01-04
Also published as: CN116179669A

Abstract

The invention relates to the technical field of gene detection, in particular to a typing kit, a primer and a typing method for genes related to vitamin B12 metabolism. The typing kit at least comprises a nucleic acid amplification reagent and a single base extension reaction reagent; the nucleic acid amplification reagent contains PCR amplification primers shown as SEQ ID NO. 1-SEQ ID NO. 60; the single base extension reaction reagent contains single base extension primers shown as SEQ ID NO. 61-SEQ ID NO. 101. 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 B12 metabolism related genes, and are simple, convenient, feasible, high in accuracy and high in automation degree.

Description

Typing kit, primer and typing method for vitamin B12 metabolism related genes

Technical Field

The invention relates to the technical field of gene detection, in particular to a typing kit, a primer and a typing method for genes related to vitamin B12 metabolism.

Background

Vitamin B12 is an essential nutrient for all human tissues and is critical for the release of energy from food. Vitamin B12 in nature is synthesized by microorganisms, higher animals and plants cannot produce vitamin B12, so the vitamin B12 obtained by human bodies mainly comes from animal foods, wherein animal viscera, meat and eggs are rich sources of the vitamin B12, the requirement of the human bodies on the vitamin B12 is very small, and the daily requirement of the human bodies is about 12 mug (1/1000 mg). Vitamin B12 has mainly two physiological functions: firstly, the polypeptide is taken as a cofactor of methyltransferase, participates in the synthesis of methionine, thymine and the like, and influences the growth and development of infants when lacking; secondly, the transfer and storage of folic acid in cells are protected, when vitamin B12 is deficient, the folic acid content of human erythrocytes is low, and the folic acid stored in the liver is reduced.

The absorption of vitamin B12 by the human body involves the involvement of a number of genes. Vitamin B12 first binds to the protein encoded by the GIF gene, called intragastric factor, in the stomach and forms a "vitamin B12-intragastric factor complex", which prevents the destruction of vitamin B12 by the intestinal hydrolytic enzymes on the one hand, and on the other hand promotes the absorption of vitamin B12 by the ileum epithelium when the complex migrates to the ileum and binds to specific receptors of the ileum mucosa, and the absorption of vitamin B12 is impaired when the human body lacks intragastric factors, and erythropoiesis is also affected, as is possible in the case of pernicious anaemia. The "vitamin B12-intragastric factor complex" formed in the stomach is transported into the intestinal tract by the TCN1 transporter encoded by the TCN1 gene and binds to the "CUBAM complex" located on the cell membrane of the small intestine cells. The "CUBAM complex" is necessary for vitamin B12 absorption and consists of two proteins, CUbilin and Anmnionless, encoded by the CUBN and AMN genes, respectively. The Anrnionless protein exists mainly on the cell membrane of small intestine cells, the Anrnionless protein is combined with the cobilin protein, the cobilin protein is anchored on the cell membrane, when vitamin B12 passes through the small intestine, the cobilin protein is combined with vitamin B12, the Anrnionless protein helps to transfer the 'cobilin-vitamin B12 complex' into intestinal cells, and then the vitamin B12 is released into blood, and the vitamin B12 in the blood is combined with a plasma globulin called transcobalamin II coded by TCN2 genes to form a 'TCN 2-cobalamin complex', and the transcobalamin II protein mainly plays a role in transferring the vitamin B12. The CD320 gene encodes a transcobalamin receptor (TCBLR) that is located on the plasma membrane, is capable of binding to the "TCN 2-cobalamin complex" and internalizes the complex by endocytosis, whereby vitamin B12 enters the cell and participates in the process of vital activity in the body. Vitamin B12 absorption processes involve multiple genes and their apparent proteins. Thus, when a specific mutation occurs in the gene concerned, an abnormality in the functional expression of the protein is caused, thereby affecting the absorption process of vitamin B12.

The detection of the single nucleotide polymorphism (Single nucleotide polymorphism, SNP) of the vitamin B12 metabolism related genes not only can help to understand the absorption level and state of the vitamin B12 in the human body, but also can help to understand the influence of the vitamin B12 in foods on the absorption and metabolism of the human body, and can help to understand the difference of the vitamin B12 sensitivity of various foods to different human genes.

The conventional methods for detecting SNP loci include a PCR-direct sequencing method, a PCR-restriction fragment length polymorphism analysis method (PCR-RFLP), a PCR-gene chip method, a fluorescent quantitative PCR method, a high-throughput sequencing method and the like, and the techniques have wide application in genotyping and gene mutation detection.

The PCR-direct sequencing method is a recognized gold standard for detecting genotyping, but has the advantages of long detection period, low detection flux, low sensitivity, higher cost, special requirements on reagents and instruments, and difficult popularization. The PCR-restriction fragment length polymorphism analysis method has the advantages of low detection sensitivity, low flux, complicated operation steps and suitability for typing of part of SNP loci, the detection result still needs to be verified again by a first-generation sequencing method, and particularly when the sample size is large, the PCR products are extremely easy to cause cross contamination, and insufficient enzyme digestion or excessive enzyme digestion easily occur, so that false negative or false positive results are caused. The PCR-gene chip method has high flux, but the accuracy and repeatability of the detection result are poor, the sensitivity is low, the cost is high, special instruments and equipment are needed, and the operation is complex. The fluorescent quantitative PCR detection method has the advantages of high sensitivity, accurate typing, simple and quick operation, good result repeatability, easy popularization of the used instrument and the like, is a very good means for detecting SNP loci, but has limited flux, cannot conveniently and quickly meet the clinical requirements for detecting tens to hundreds of loci of multiple genes, has higher probe cost, and is mainly suitable for typing a small number of loci and large samples. High throughput sequencing, although extremely high in throughput, is not suitable for detection of gene SNP sites at cost, time consumption, personnel requirements and the like.

Therefore, it is urgently needed to find a typing kit and a typing method for genes related to vitamin B12 metabolism, which are simple, convenient, easy to implement, high in accuracy and high in automation degree.

Disclosure of Invention

In order to solve the technical problems, the invention provides a typing kit, a primer and a typing method of genes related to vitamin B12 metabolism.

The first aspect of the invention provides a typing kit for vitamin B12 metabolism related genes, wherein the typing kit at least comprises a nucleic acid amplification reagent and a single base extension reaction reagent; the nucleic acid amplification reagent contains multiplex PCR amplification primers shown as SEQ ID NO. 1-SEQ ID NO. 60; the single base extension reaction reagent contains single base extension primers shown as SEQ ID NO. 61-SEQ ID NO. 101.

The second aspect of the invention provides a vitamin B12 metabolism related genotyping primer, wherein the primer is selected from at least one group of nucleotide sequences shown as SEQ ID NO. 1-SEQ ID NO. 101; the primers also comprise reference primer pairs shown as SEQ ID NO.102 and SEQ ID NO. 103.

In a third aspect, the present invention provides a method for typing genes involved in vitamin B12 metabolism, comprising at least the steps of:

s1, performing PCR amplification reaction by using PCR amplification primers shown as SEQ ID NO. 1-SEQ ID NO.60 to obtain a specific amplification product;

s2, digestion is carried out by alkaline phosphatase, dNTPs in the specific amplification product are removed, and a digested product is obtained;

s3, performing single-base extension reaction on the digested product by using single-base extension primers shown in SEQ ID NO. 61-SEQ ID NO.101 to obtain an extension reaction product;

s4, desalting the extension reaction product to obtain a desalted product;

s5, detecting the desalted product by high performance liquid chromatography-mass spectrometry, and analyzing to obtain a typing result of the vitamin B12 metabolism related gene.

Compared with the prior art, the technical scheme provided by the invention has the following advantages:

the kit and the detection method can improve the typing detection efficiency of the vitamin B12 metabolism related genes, reduce the sample consumption, and have the technical advantages of simplicity, convenience, practicability, high accuracy and high degree of automation.

Detailed Description

In order that the above objects, features and advantages of the present disclosure may be more clearly understood, a further description of the aspects of the present invention will be provided below. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the disclosure.

In a first aspect, an embodiment of the present invention provides a typing kit for a vitamin B12 metabolism-related gene. Firstly, the embodiment of the invention screens the mutation sites of genes related to vitamin B12 metabolism, selects the mutation sites shown in table 1 in AMN genes, TCN1 genes, TCN2 genes, CUBN genes, CD320 genes and GIF genes, and the mutation sites are more closely related to the absorption and metabolism process of vitamin B12 in human body.

TABLE 1

Secondly, designing primers of the mutation sites, and providing a nucleic acid amplification reagent containing multiple PCR amplification primers shown as SEQ ID NO. 1-SEQ ID NO. 60; the single base extension reaction reagent contains single base extension primers shown as SEQ ID NO. 61-SEQ ID NO. 101. The kit provided by the embodiment of the invention can obviously improve the typing detection efficiency, reduce the sample consumption and has high accuracy. Specifically, nucleotide sequence information of the multiplex PCR amplification primers is shown in table 2:

TABLE 2

In addition to the PCR amplification primers, the nucleic acid amplification reagents also contain 10 XPCR buffer, mgCl ₂ dNTPMix, DNA polymerase, etc. Preferably, the DNA polymerase adopts Agena enzyme system DNA polymerase, so that the detection accuracy can be further improved.

In the typing kit of the embodiment of the invention, the base extension reaction reagent contains single base extension primers shown as SEQ ID NO. 61-SEQ ID NO. 101. In addition to the single-base extension primer, the base extension reagent contains 10 XPCR buffer, mgCl2, dNTP mix, DNA polymerase, etc. Specifically, the sequence information of the single base extension primer is shown in tables 3 and 4:

TABLE 3 Table 3

TABLE 4 Table 4

In addition to the nucleic acid amplification reagents and single base extension reagents, the typing kit of the embodiment of the invention further comprises alkaline phosphatase reaction solution and/or desalting reagent; wherein, alkaline phosphatase reaction liquid is used for digestion and removing dNTPs in the PCR system, and desalting reagent is used for removing K in the PCR reaction system ⁺ 、Na ⁺ 、Mg ²⁺ And plasma is prevented from interfering with the mass spectrum detection result.

Specifically, the alkaline phosphatase in the alkaline phosphatase reaction solution is at least one selected from shrimp alkaline phosphatase, calf intestinal alkaline phosphatase, E.coli alkaline phosphatase and rat alkaline phosphatase. Specifically, the concentration of alkaline phosphatase is 1.7U/. Mu.L, and alkaline phosphatase reaction solution with the concentration of 0.073U/. Mu.L can be prepared by alkaline phosphatase and buffer solution in the analysis process.

Specifically, the desalting reagent is selected from desalting Resin, desalting Resin available from Darui, guangzhou or clear Resin desalting Resin available from Agena bioscience.

As an improvement of the parting kit, the nucleic acid amplification reagent can be divided into a plurality of tubes for subpackaging, and each tube is divided into partial PCR amplification primers, so that the interference can be avoided and the amplification efficiency of partial primers can be improved by dividing the kit into a plurality of tubes for amplification during the PCR amplification reaction. Specifically, can divide into 3 pipes and carry out the partial shipment, the partial shipment mode is: the M1 tube contains PCR amplification primers shown as SEQ ID NO. 1-SEQ ID NO.16, and the M2 tube contains PCR amplification primers shown as SEQ ID NO. 17-SEQ ID NO. 44; the M3 tube contains PCR amplification primers shown as SEQ ID NO. 45-SEQ ID NO. 60. The split charging mode can obviously improve the amplification efficiency of each group of primers.

Specifically, in the M1 tube, the concentration of the primers shown as SEQ ID NO. 1-SEQ ID NO.6 and SEQ ID NO. 9-SEQ ID NO.16 is 0.5-10 mu mol/L; the concentration of the primers shown in SEQ ID NO. 7-SEQ ID NO.8 is 0.6-10 mu mol/L;

in the M2 tube, the concentration of the primers shown as SEQ ID NO. 17-18, SEQ ID NO. 21-30 and SEQ ID NO. 33-44 is 0.5-10 mu mol/L; the concentration of the primers shown in SEQ ID NO. 19-20 and SEQ ID NO. 31-32 is 0.6-10 mu mol/L;

in the M3 tube, the concentration of the primers shown as SEQ ID NO. 45-SEQ ID NO.53 and SEQ ID NO. 53-SEQ ID NO.60 is 0.5-10 mu mol/L; the concentration of the primer shown in SEQ ID NO. 51-SEQ ID NO.52 is 0.4-10 mu mol/L.

More preferably, the concentration of each primer in the M1 to M3 tubes is shown in Table 5.

TABLE 5

In order to match 3 tubes of the PCR amplification primer, the single-base extension reaction reagent is to divide the single-base extension primer into 3 tubes, namely S1 tube, S2 tube and S3 tube, according to the PCR amplification product: the split charging mode is as follows: the S1 tube contains single base extension primer shown as SEQ ID NO. 61-SEQ ID NO.70, and the S2 tube contains single base extension primer shown as SEQ ID NO. 71-SEQ ID NO. 86; the S3 tube contains single base extension primer shown as SEQ ID NO. 87-SEQ ID NO. 101.

Specifically, in the S1 tube, the concentrations of the primers shown in SEQ ID NO. 61-SEQ ID NO.64 and SEQ ID NO. 66-SEQ ID NO.70 are 5-15 mu mol/L, and the concentration of the primer shown in SEQ ID NO.65 is 10-20 mu mol/L; in the S2 tube, the concentration of the primers shown in SEQ ID NO. 71-SEQ ID NO.72, SEQ ID NO. 74-SEQ ID NO.78 and SEQ ID NO. 82-SEQ ID NO.86 is 5-15 mu mol/L, and the concentration of the primers shown in SEQ ID NO.73 and SEQ ID NO. 79-SEQ ID NO.81 is 25-35 mu mol/L; in the S3 tube, the concentration of the primer shown in SEQ ID NO. 87-SEQ ID NO.97 and the concentration of the primer shown in SEQ ID NO. 99-SEQ ID NO.101 are respectively 5-15 mu mol/L, and the concentration of the primer shown in SEQ ID NO.99 is 15-25 mu mol/L. More preferably, the concentration of each primer in the S1 to S3 tubes is shown in Table 6.

TABLE 6

Different concentrations of the single-base extension primer are selected in a reaction system, so that the extension efficiency of partial sites can be increased, and the mass spectrum response value is improved.

Preferably, the nucleic acid amplification reagent further comprises an N-pipe (reference primer pipe) containing a reference primer shown as SEQ ID NO.102 and SEQ ID NO.103, wherein the nucleotide sequence of the reference primer is shown in Table 7.

TABLE 7

As an improvement of the parting kit, the parting kit also comprises a homozygous mutation positive control, a negative control and a heterozygous mutation positive control, which are specifically shown in the following table 8:

TABLE 8

The second aspect of the embodiments of the present invention also relates to a primer for genotyping vitamin B12 metabolism-related genes, specifically selected from the nucleotide sequences shown as SEQ ID NO.1 to SEQ ID NO. 101. Preferably, the primers also comprise reference primer pairs shown as SEQ ID NO.102 and SEQ ID NO. 103.

The third aspect of the embodiments of the present invention also relates to a method for typing a vitamin B12 metabolic-related gene, comprising at least the steps of:

s4, desalting the extension reaction product to obtain a desalted product;

s5, detecting the desalted product by high performance liquid chromatography-mass spectrometry, and analyzing to obtain the typing result of the vitamin B12 metabolism related genes.

The method of the embodiment of the invention is mainly based on the multiplex PCR and single base extension PCR technology, and the principle is that the primer capable of specifically amplifying the target gene fragment containing the SNP locus region and the DNA polymerase system are used for carrying out PCR amplification, alkaline phosphatase digestion treatment is added after the PCR is finished, and dNTPs in the reaction liquid are removed. And then, adding related components such as an SNP locus specific extension primer, ddNTP and the like into the reaction solution, and performing single base extension reaction, wherein in the reaction process, the SNP locus specific extension primer can be specifically combined with the 5' end of the SNP locus to be detected, and extends out bases complementary with the target SNP genotype according to a base complementary pairing principle, and can obtain unused extension products according to DNA templates of different genotypes. The molecular weights of the different bases are different, and the genotype of the SNP locus to be analyzed can be determined according to the molecular weight of the extension product. The detection method provided by the embodiment of the invention can obviously improve the parting detection efficiency, reduce the sample consumption and has high accuracy.

In S1, the PCR amplification primers shown in SEQ ID NO. 1-SEQ ID NO.60 are shown in Table 2, and the reference primer shown in Table 7 is also added in the specific amplification process.

In preparing the PCR amplification system, three amplification systems were prepared from the M1 tube, the M2 tube and the M3 tube, and the concentrations of the PCR amplification primers in the three amplification systems were as shown in Table 5. PCR amplification systems can be formulated according to methods commonly used in the art.

The procedure of PCR amplification reaction is also according to the usual procedure of PCR amplification reaction in the art, and specifically, the following procedures are selected: pre-denaturation at 90-98 ℃ for 30 seconds-10 minutes; denaturation at 90-95 ℃,10 seconds-1 minute, annealing at 50-60 ℃,10 seconds-1 minute, extension at 68-72 ℃,30 seconds-10 minutes, 25-45 cycles of amplification; extending for 5 minutes after 68-72 ℃; preferably, it is: pre-denaturation at 95 ℃ for 2-4 minutes; denaturation at 95 ℃,30 seconds, annealing at 56 ℃,30 seconds, extension at 72 ℃,1.5 minutes, 25-45 cycles of amplification, extension after 72 ℃,5 minutes.

In S2, the concentration of alkaline phosphatase was 1.7U/. Mu.L, and the concentration in the reaction solution was 0.073U/. Mu.L; the digestion conditions were: digestion treatment is carried out for 10 to 60 minutes at the temperature of between 30 and 40 ℃, heating denaturation is carried out for 5 to 30 minutes at the temperature of between 70 and 85 ℃, and termination is carried out at the temperature of between 2 and 8 ℃; preferably, it is: digestion treatment at 37 ℃ for 40 minutes, heat denaturation at 85 ℃ for 5 minutes, and termination at 4 ℃;

in S3, the single base extension primers shown in SEQ ID NO.61 to SEQ ID NO.101 are shown in Table 3. The reaction system of single base extension reaction is also divided into S1, S2 and S3 tubes according to PCR amplification products: the single-base extension primer concentrations in the three single-base extension reaction systems are shown in Table 6. The single base extension reaction system may be formulated according to methods commonly used in the art.

The single base extension reaction procedure is also performed according to the common single base extension reaction procedure in the art, and specifically, the following single base extension reaction procedures are selected: hot start at 90-98 deg.c for 30 sec-10 min; denaturation at 90-98 ℃, 4-8 seconds, annealing at 50-58 ℃, 4-8 seconds, extension at 65-85 ℃, 4-8 seconds, and amplification for 40 cycles; final extension at 68-75 deg.c for 1-10 min; preferably, it is: starting at 94 ℃ for 30 seconds; denaturation at 98 ℃,5 seconds, 52 ℃ annealing, 5 seconds, 80 ℃ extension, 5 seconds, amplification for 40 cycles; final extension at 72℃for 3 min.

In S4, the desalination method is resin desalination. Any means for removing salt ions from the solution such as chromatography and ultrafiltration can be included. By removing K in the PCR reaction system ⁺ 、Na ⁺ 、Mg ²⁺ And plasma is prevented from interfering with the mass spectrum detection result.

The specific method for adsorption comprises the following steps: to a PCR tube containing 9. Mu.L of the above reaction system, 15 to 25mg of the resin was added, followed by 40 to 90. Mu.L of distilled water. The PCR tube containing the single base extension product and the resin was fixed to a spin mixer and spun for 30 minutes at a slow speed.

In S5, the mass spectrometer is a liquid chromatography-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 LC-MS mass spectrum platform has good specificity, the lowest detection lower limit is 5ng of genome DNA, and the coincidence is 100% compared with the gold standard technology Sanger sequencing technology selected by the test.

The high performance liquid chromatography can be selected from anion exchange chromatography column with high separation degree, and DNAPac analysis column (Thermo company product) can be used for high resolution analysis and purification of synthetic and modified oligonucleotide. The parameters are as follows: 2.1X100 mm, column temperature: 40-75 ℃, preferably 60 ℃; the loading amount is 1 to 10. Mu.L, preferably 10. Mu.L.

For dnatac analytical columns, the mobile phases commonly used are: mobile phases of hexafluoroisopropanol and triethylamine. The embodiment of the invention adopts a mobile phase containing hexafluoroisopropanol and triethylamine, and specifically, the phase A is water containing hexafluoroisopropanol and triethylamine; 0.4 to 0.6 percent by volume of hexafluoroisopropanol, preferably 0.49 percent by volume of triethylamine, and 0.1 to 0.12 percent by volume of triethylamine, preferably 0.11 percent; phase B is methanol containing hexafluoroisopropanol and triethylamine; the volume percentage of hexafluoroisopropanol is 0.4-0.6%, preferably 0.49%, and the volume percentage of triethylamine is 0.1-0.12%, preferably 0.11%.

The chromatographic gradient is:

0～(0.5～1.5)min，(2％～8％)～(15％～25％)B；

(0.51～1.51)min～(3～4)min，(15％～25％)～(35％～45％)B；

(3.01～4.01)min～(5～6)min，(35％～45％)～(85％～95％)B；

(5.01～6.01)min～(6～8)min，(85％～95％)～(2％～8％)B；

(6.01～8.01)min～10min，(2％～8％)B；

the chromatographic gradient is preferably as shown in table 9:

TABLE 9

Time (min)	B％
		0	5
1	20
		3.5	45
5.5	95
		6	5
10	5

The flow rate was 0.2mL/min.

The conditions of the mass spectrum are shown in table 10 below:

table 10

In S5, the analysis is performed by using software adapted to the mass spectrometer, for example, thermo BioPharmaFinder software, and after setting software parameters, the genotyping result is interpreted, where the interpretation criteria are shown in table 11:

TABLE 11

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Examples of the invention are further illustrated by the following examples:

instrumentation, reagents, consumables used in the examples:

1. instrument apparatus:

biological safety cabinet: sea, HR 40-IIA 2; PCR instrument: siamese, veriti DX 96 (medical device license); low temperature high speed centrifuge: race-fly, biofuge Primo R; pipetting: eppendorf, germany, 2.5 μl, 10 μl, 100 μl, 1000 μl; single-hole electric heating constant temperature water bath kettle: HH.S11-Ni 1 of Beijing Changan Yongchuang scientific instrument factory; vortex mixer: VORTEX-5, manufactured by Haimen Ke Linbell instruments, inc.; mini centrifuge: LX-200, manufactured by Haimen Chemie, inc., of Linbell instruments, inc.; ultra-micro spectrophotometer: IMPLEN, NP80-touch, germany; electrophoresis apparatus: beijing six biotechnology limited, DYY-6C; horizontal electrophoresis tank: DYCP-31DN of Beijing six biotechnology Co., ltd; transferring and decolorizing the shaking table: TS-8, manufactured by Haimen Ke Linbell instruments, inc.; 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; vertical pressure steam sterilizer: shanghai Shen An, LDZX-50KB; 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)

DNA extraction kit: YDP304-03 of Tiangen Biochemical technology (Beijing) Co., ltd; agarose: biowst, 100g;6×loading Buffer: physical technologies for doctor of precious days (Beijing), limited (TaKaRa), 9156;100bp DNA Ladder: new England Biolabs, N3231V; absolute ethyl alcohol: the optical multiplexing technology development limited company in Tianjin is analytically pure; tris base: amresco, AR0497/500g; disodium EDTA: amresco,0105/1000g; acetic acid: the national drug group reagent company, inc., analytical grade; 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。

3. Experiment consumable:

EP tube: axygen, U.S. Pat. No. 0.2mL, 1.5mL, 2mL; pipette tip: axygen, U.S. Pat. No. 2.5. Mu.L, 20. Mu.L, 200. Mu.L, 1000. Mu.L; thermo dnappc analytical column: thermo, 2.1X100 mm; DNAPac TM PR Guard Columns: thermo,2.1 x 10mm,088925; cartridge Holder version 2: thermo,069580; inner cannula, polypropylene (with spring): agilent, 250 μl; sample bottle/cap: thermo,2mL.

Example 1

A kit having the composition shown in table 12:

table 12

Example 2 typing method

1. Extracting genomic DNA: see the standard protocol for blood cell tissue genomic DNA extraction (BJHH-SOP-PCR-QT-001).

2. PCR amplification

2.1 preparation of primer Mix, specific amplification primer Mix preparation method is shown in Table 13,

TABLE 13

The single base extension primer Mix configuration method is shown in Table 14:

TABLE 14

2.2 multiplex PCR amplification System, specifically as shown in Table 15:

table 15: multiplex PCR amplification system

Water (chromatographic grade)	0.9μL
		10 XPCR buffer	0.5μL
25mM MgCl ₂	0.4μL
		25mM dNTP mix	0.1μL
M1 pipe/M2 pipe/M3 pipe/N pipe	1μL
		5U/. Mu.L Agena enzyme system DNA polymerase	0.1μL
5 ng/. Mu.L template	2μL
		Total volume of	5μL

Note that: the three groups of M1 pipe, M2 pipe and M3 pipe amplify the sample respectively, and the three groups of control genes are amplified respectively by adopting N pipe primers and are marked as N1 pipe, N2 pipe and N3 pipe.

2.3 multiplex PCR reaction conditions, as shown in Table 16:

table 16: multiplex PCR reaction conditions

2.4 Detection of PCR products

2.4.1 preparing 2% agarose gel (0.4 g agarose with deionized water to 20mL, microwave oven dissolving, room temperature gel);

2.4.2 sampling DNA molecular weight standard 2.5. Mu.L of 100bp Ladder;

2.4.3 adding 1.0. Mu.L of 6 Xsample Buffer (Loading Buffer) to 5.0. Mu.L of each PCR amplification product, mixing uniformly, and Loading;

2.4.4 electrophoresis on 2% agarose gel with constant pressure 120V for 40min with 1 xTAE as running buffer; (50×TAE preparation method: tris 242g, na) ₂ EDTA ₂ H ₂ O37.2 g, acetic acid 57.1mL, adding about 800mL deionized water, and stirring thoroughly for dissolution; adding deionized water to fix the volume of the solution to 1L, and preserving at room temperature);

2.4.5 dyeing in GelStain-containing NaCl solution (0.1M NaCl solution plus GelRed 20. Mu.L) (1M NaCl solution preparation method: 2.92g NaCl purified water constant volume to 50mL; 200mL dye liquor per preparation, 20mL 1M NaCl solution, 20. Mu.L GelStain, 180mL DI water);

2.4.6 performing inspection and image analysis by using a gel scanning imaging system;

2.4.7 analysis of results: the positive control gel showed a bright band and the negative control had no band (except primer dimer).

3. Alkaline phosphatase digestion

3.1 digestion system is shown in Table 17: 2. Mu.L of the prepared digestion system was added to each reaction tube:

TABLE 17

3.2 digestion procedure: 40min at 37 ℃, 5min at 85 ℃ and 4 ℃.

4. Single base extension PCR

4.1 Single base extension PCR System is shown in Table 18:

TABLE 18

Reagent name	Volume (mu L)
		Water and its preparation method	0.6
iplex buffer	0.2
		Mixtures of iplex ddNTP	0.2
S1 pipe/S2 pipe/S3 pipe	0.95
		iplex enzyme	0.05
Total volume (mu L)	2

Note that: the single base extension system is configured into three groups, wherein the first group adopts an S1 tube primer pair to carry out single base extension on M1 tube and N1 tube products; the second group adopts an S2 tube primer pair to carry out single base extension on M2 tube and N2 tube products; the third set uses S3 tube primers to single base extend the M3 tube and N3 tube products.

4.2 Single base extension PCR procedure is shown in Table 19:

TABLE 19

5. Resin desalination

To a PCR tube containing 9. Mu.L of the above reaction system, 15 to 25mg of the resin was added, followed by 40 to 90. Mu.L of distilled water. The PCR tube containing the single base extension product and the resin was fixed to the above mixture and rotated at a slow speed for 30 minutes.

6. LC-MS on-machine: taking the supernatant into a new PCR tube, and preparing to be on machine.

6.1 chromatographic conditions

(1) Chromatographic column and column temperature: thermo DNAPac analytical column, 2.1X100 mm,60 ℃, loading 10. Mu.L;

(2) Mobile phase and flow rate: flow rate: 0.2mL/min;

phase A: water (200 mL) +HFIP (4.211 mL) +TEA (0.23 mL)

And B phase: methanol (200 mL) +HFIP (4.211 mL) +TEA (0.23 mL)

(3) The liquid phase gradient/procedure is shown in table 9.

6.2 Mass Spectrometry conditions are shown in Table 10.

7. Analysis of results

7.1Thermo BioPharma Finder software analysis of mass spectrometry data results; the parameter settings are shown in table 20:

table 20

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7.2 determination methods are shown in Table 11.

Experimental example 1

This example is used to illustrate the accuracy of the above-described kits and detection methods.

Five normal DNA samples (sample names are RJJ, LXJ, SLP, JYX and ZHF respectively), a homozygous mutant positive control (MUT) and a negative control (WT) heterozygous mutant positive control (Con) are adopted for detection, the concentrations of the normal DNA samples and the controls are diluted to 5 ng/mu L, and the kit and the detection method are adopted for detection.

1. The liquid quality detection result is carried out by adopting the kit;

(1) The first set of mass spectral results are shown in table 22:

table 22

Sequencing results using multiplex PCR sequencing are shown in table 23;

table 23

Extension primer

LXJ

RJJ

ZHF

ZFY

ZGQ

WT

MUT

CON

VB-T-01

GG

TT#

GT*

VB-T-02

CC

TT#

CT*

VB-T-03

AA

GG#

AG*

VB-T-04

GG

AA#

GA*

VB-T-11-01

WT

MUT#

Con*

VB-T-44

CC

TT#

CT*

VB-T-45

WT

MUT#

Con*

VB-T-46

TT

CC#

TC*

VB-T-47

CC

TT#

CT*

VB-T-48

GG

GC*

CC#

GC*

GG

CC#

GC*

(2) The second set of mass spectral results are shown in table 24:

table 24

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Sequencing results using multiplex PCR sequencing are shown in table 25;

table 25

Extension primer

LXJ

RJJ

ZHF

ZFY

WT

MUT

CON

VB-T-12

GG

AA#

GA*

VB-T-13

GG

AA#

GA*

VB-T-14

CC#

CT*

TT

CC#

TC*

VB-T-15

CC

TT#

CT*

VB-T-16

GG

AA#

GA*

VB-T-17

GG

AA#

GA*

VB-T-18

GG

AA#

GA*

VB-T-19

CC

TT#

CT*

VB-T-20

GG

AA#

GA*

VB-T-21

GG

TT#

GT*

VB-T-22

CC

GG#

CG*

VB-T-23

CC

TT#

CT*

VB-T-24

CC

TT#

CT*

VB-T-25

CC

GG#

CG*

VB-T-26

AA

GG#

AG*

VB-T-43

GG

TT#

GT*

(3) The third set of mass spectral results are shown in table 26:

table 26

Sequencing results using multiplex PCR sequencing are shown in table 27;

table 27

Extension primer

LXJ

RJJ

ZHF

ZFY

WT

MUT

CON

VB-T-27

AA

GG#

AG*

VB-T-28

GG

AA#

GA*

VB-T-29

GG

AA#

GA*

VB-T-30

CC

TT#

CT*

VB-T-31

WT

AA

CC#

AC*

VB-T-32

WT

AA

CC#

AC*

VB-T-33

TT

GG#

TG*

VB-T-34

TT

CC#

TC*

VB-T-36

WT

GG

CC#

GC*

VB-T-37

TT

CC#

TC*

VB-T-38

GG

AA#

GA*

VB-T-39

WT

TT

GG#

TG*

VB-T-40

GG

AA#

GA*

VB-T-41

WT

AA

TT#

AT*

VB-T-42

GG

AA#

GA*

Remarks: genotype is marked as heterozygous mutant and genotype # is marked as homozygous mutant.

The result shows that: the genotype obtained by two detection modes of 41 sites of vitamin B metabolic genes is consistent in interpretation, which shows that the detection method of the kit has better result accuracy.

Experimental example 2

The experimental example is used for explaining the technical effect of PCR amplification primer design:

the PCR primers were designed as shown in Table 28.

Table 28

1. The DNA samples of normal persons (sample names ZHF, ZFY, RJJ, LXJ) were detected by using VB-MP-03-F/R, VB-MP-07-F/R primer pair, and the test 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 sample cannot extend out of the target product by using VB-MP-03-F/R primer pair; the genotype detected by the normal human sample of 4 wild type genotypes is CG by adopting VB-MP-07-F/R primer pair, and the result is misinterpretation.

2. The DNA samples of normal persons (sample names are ZHF, ZFY, RJJ, LXJ) are detected by using VB-MP-03-F-01/R-01 and VB-MP-07-F-01/R-01 primer pairs, and the test results are shown in Table 30:

table 30

As shown in Table 30, the genotypes detected in the normal human samples of 4 wild-type genotypes were correct using the VB-MP-03-F-01/R-01 and VB-MP-07-F-01/R-01 primer pairs.

Comparative example 3: single base extension primer screening

The design of PCR single base primers is shown in Table 31.

Table 31

1. The single base extension primer VB-T-11 was used to detect normal human samples and control gene samples (sample name RJJ, LXJ, ZHF, WT, MUT, CON), and the test results are shown in Table 32:

table 32

As shown in Table 32, the result of the experiment shows that the correct extension product can be obtained for the wild type genotype sample and the control gene by using VB-T-11 primer pair, the genotype is correct, but the genotype of the homozygous mutant control gene sample is CG and the genotype is misinterpretation.

2. The single base extension primer VB-T-11-1 was used to detect normal human samples and control gene samples (sample name RJJ, LXJ, ZHF, WT, MUT, CON), and test results are shown in Table 33:

table 33

As shown in Table 33, the VB-T-11-01 primer pair was used to obtain correct extension products for both the wild-type genotype samples and the three genotype control samples, and the genotype was correctly read.

Comparative example 4: primer concentration screening

The difference between this comparative example and the example is that the concentrations of the second set of single base extension primers VB-T-14, VB-T-20, VB-T-21, VB-T-22 are different. In this comparative example, the concentration ratios of the second set of extension primers were 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1. in this comparative example, the extension products of VB-T-14, VB-T-20, VB-T-21 and VB-T-22 were not detected, or the relative response was low, and genotype interpretation was not performed.

In the examples, the concentration of VB-T-14, VB-T-20, VB-T-21, VB-T-22 in the second set of extension primers was increased to 3 relative to the other primers in the same set, the ratio of the concentration in the second set being 1:1:3:1:1:1:1:1:3:3:3:1:1:1:1:1, the result is correctly interpreted.

Example 5: enzyme system screening

For the purpose of illustrating comparison of the effect of enzyme system selection (KOD enzyme system, agena enzyme system). The preparation system is as follows:

watch 34

Amplification reaction was performed on SNP loci of TCN1 gene and TCN2 gene of normal human sample (ZHF, ZFY, RJJ, LXJ, SLP) by KOD FX enzyme system, and test results are shown in Table 35:

table 35

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As shown in Table 35, the KOD FX enzyme system was used to obtain neither an extension product in the normal human sample portion nor an extension product having too low a response intensity to be able to accurately interpret the genotype.

The amplification reaction is carried out on SNP loci of TCN1 gene and TCN2 gene of a normal human sample (ZHF, ZFY, RJJ, LXJ, SLP) by using an Agena enzyme system, and test results are shown in Table 36:

table 36

As shown in Table 36, the samples of normal persons all obtained the extension products with higher response intensity by using the Agena enzyme system, and the genotype was correctly interpreted.

The foregoing is merely a specific embodiment of the disclosure to enable one skilled in the art to understand or practice the disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Thus, the present disclosure is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kit for typing genes related to vitamin B12 metabolism, which is characterized by comprising at least a nucleic acid amplification reagent and a single base extension reaction reagent;

the nucleic acid amplification reagent contains multiple PCR amplification primers shown as SEQ ID NO. 1-SEQ ID NO. 60; the nucleic acid amplification reagent comprises an M1 tube, an M2 tube and an M3 tube: the M1 tube contains PCR amplification primers shown as SEQ ID NO. 1-SEQ ID NO.16, and the M2 tube contains PCR amplification primers shown as SEQ ID NO. 17-SEQ ID NO. 44; the M3 tube contains PCR amplification primers shown as SEQ ID NO. 45-SEQ ID NO. 60; the single-base extension reaction reagent contains a single-base extension primer shown as SEQ ID NO. 61-SEQ ID NO. 101; the single base extension reaction reagent comprises an S1 pipe, an S2 pipe and an S3 pipe: the S1 tube contains a single-base extension primer shown as SEQ ID NO. 61-SEQ ID NO.70, and the S2 tube contains a single-base extension primer shown as SEQ ID NO. 71-SEQ ID NO. 86; the S3 tube contains a single base extension primer shown as SEQ ID NO. 90-SEQ ID NO. 101.

2. The typing kit according to claim 1, further comprising an alkaline phosphatase reaction solution and/or a desalting reagent;

the alkaline phosphatase in the alkaline phosphatase reaction solution is at least one selected from shrimp alkaline phosphatase, calf intestinal alkaline phosphatase, escherichia coli alkaline phosphatase and rat alkaline phosphatase; and/or

The desalting reagent is selected from desalting resins; and/or

The nucleic acid amplification reagent also contains Agena enzyme system DNA polymerase.

3. The typing kit according to claim 1, further comprising a homozygous mutant positive control, a negative control and a heterozygous mutant positive control;

the homozygous mutant positive reference substance is a reference gene of a DNA fragment containing all mutant sites;

the negative control is a reference gene containing DNA fragments of all wild type sites;

the heterozygous mutation positive reference substance is the homozygous mutation positive reference substance and the negative reference substance with the molar ratio of 1:1.

4. A typing kit according to claim 1, wherein,

in the M1 tube, the concentrations of the primers shown as SEQ ID NO. 1-SEQ ID NO.6 and SEQ ID NO. 9-SEQ ID NO.16 are all 0.5-10 mu mol/L; the concentration of the primers shown in SEQ ID NO. 7-SEQ ID NO.8 is 0.6-10 mu mol/L;

in the M2 tube, the concentration of the primers shown as SEQ ID NO. 17-18, SEQ ID NO. 21-30 and SEQ ID NO. 33-44 is 0.5-10 mu mol/L; the concentrations of the primers shown in SEQ ID NO. 19-20 and SEQ ID NO. 31-32 are all 0.6-10 mu mol/L;

in the M3 tube, the concentration of the primers shown as SEQ ID NO. 45-SEQ ID NO.53 and SEQ ID NO. 53-SEQ ID NO.60 is 0.5-10 mu mol/L; the concentration of the primers shown in SEQ ID NO. 51-SEQ ID NO.52 is 0.4-10 mu mol/L;

in the S1 tube, the concentrations of the primers shown in SEQ ID NO. 61-SEQ ID NO.64, SEQ ID NO. 66-SEQ ID NO.70 are 5-15 [ mu ] mol/L, and the concentration of the primer shown in SEQ ID NO.65 is 10-20 [ mu ] mol/L;

in the S2 tube, the concentrations of the primers shown by SEQ ID NO. 71-72, SEQ ID NO. 74-78 and SEQ ID NO. 82-86 are 5-15 [ mu ] mol/L, and the concentrations of the primers shown by SEQ ID NO.73, SEQ ID NO. 79-81 are 25-35 [ mu ] mol/L;

in the S3 tube, the concentrations of the primers shown in SEQ ID NO. 87-SEQ ID NO.97, SEQ ID NO. 99-SEQ ID NO.101 are 5-15 [ mu ] mol/L, and the concentration of the primer shown in SEQ ID NO.98 is 15-25 [ mu ] mol/L.

5. A typing kit according to claim 1, wherein,

the nucleic acid amplification reagent also comprises an N pipe, wherein the N pipe contains reference primers shown as SEQ ID NO.102 and SEQ ID NO. 103.

6. The primer for genotyping related vitamin B12 metabolism is characterized by comprising a nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 101;

the primer comprises a PCR amplification primer and a single base extension primer;

the PCR amplification primers are divided into three groups: the first group contains PCR amplification primers shown as SEQ ID NO. 1-SEQ ID NO.16, the second group contains PCR amplification primers shown as SEQ ID NO. 17-SEQ ID NO.44, and the third group contains PCR amplification primers shown as SEQ ID NO. 45-SEQ ID NO. 60;

the single base extension primers are divided into three groups: the first group contains single-base extension primers shown as SEQ ID NO. 61-SEQ ID NO.70, the second group contains single-base extension primers shown as SEQ ID NO. 71-SEQ ID NO.86, and the third group contains single-base extension primers shown as SEQ ID NO. 90-SEQ ID NO. 101;

the primer also comprises a reference primer pair shown as SEQ ID NO.102 and SEQ ID NO. 103.

7. A method for typing a vitamin B12 metabolism-related gene for non-diagnostic purposes, comprising at least the steps of:

s1, respectively carrying out PCR amplification reaction by using an M1 tube, an M2 tube and an M3 tube in the kit according to claim 1 to obtain specific amplification products; s2, digestion is carried out by alkaline phosphatase, dNTPs in the specific amplification product are removed, and a digested product is obtained;

s3, performing single base extension reaction on the digested product by using an S1 pipe, an S2 pipe and an S3 pipe in the kit according to claim 1 to obtain an extension reaction product, wherein the extension reaction product is specifically: the single base extension system is configured into three groups, wherein the first group adopts an S1 tube primer pair to carry out single base extension reaction on the digested product in an M1 tube; the second group adopts an S2 tube primer pair to carry out single base extension reaction on the digested product in an M2 tube; the third group uses S3 tube primer to make single base extension reaction on the digested product in M3 tube;

s4, desalting the extension reaction product to obtain a desalted product;

8. A typing method according to claim 7, wherein,

the PCR amplification reaction is carried out by the following steps: pre-denaturation at 90-98 ℃ for 30 seconds-10 minutes; denaturation at 90-95 ℃, annealing at 50-60 ℃, annealing for 10 seconds-1 minute, extension at 68-72 ℃, extension for 30 seconds-10 minutes, and amplification for 25-45 cycles; extending for 5 minutes at 68-72 ℃;

the digestion conditions were: digestion treatment is carried out for 20-60 minutes at 30-40 ℃, heating denaturation is carried out for 5-20 minutes at 75-85 ℃, and termination is carried out at 2-8 ℃;

the single base extension reaction is performed by the following steps: hot start at 90-98 ℃ for 30 seconds-10 minutes; denaturation at 90-98 ℃, 4-8 seconds, 50-58 ℃ annealing, 4-8 seconds, 65-85 ℃ extension, 4-8 seconds, and amplification for 40 cycles; and finally extending at 68-75 ℃ for 1-10 minutes.

9. A typing method according to claim 7, wherein,

in S5, in the high performance liquid chromatography:

mobile phase: phase A is water containing hexafluoroisopropanol and triethylamine; 0.4-0.6% of hexafluoroisopropanol and 0.1-0.12% of triethylamine;

phase B is methanol containing hexafluoroisopropanol and triethylamine; the volume percentage of hexafluoroisopropanol is 0.4-0.6%, and the volume percentage of triethylamine is 0.1-0.12%.

10. The typing method according to claim 7 or 9, wherein in S5, the high performance liquid chromatography:

the chromatographic gradient is:

0 ~（0.5~1.5） min，（2%~8%）~（15%~25%）B；

（0.51~1.51）~（3~4） min，（15%~25%）~（35%~45%）B；

（3.01~4.01）~（5~6）min，（35%~45%）~（85%~95%）B；

（5.01~6.01）~（6~8）min，（85%~95%）~（2%~8%）B；

（6.01~8.01）~ 10 min，（2%~8%）B；

the flow rate is 0.1-0.5 mL/min;

the chromatographic column adopts DNAPac analysis column, 2.1X100 mm, and the column temperature is: 40-75 ℃;

the loading amount is 1-10 mu L;

in S5, the conditions of the mass spectrum are: sheath gas 35 Arb; auxiliary gas 10 Arb; spray voltage 3400V; the temperature of the ion transmission tube is 320 ℃; the resolution is 140000.