CN106755525B

CN106755525B - Probe for detecting MTHFR gene mutation and application and kit thereof

Info

Publication number: CN106755525B
Application number: CN201710092235.1A
Authority: CN
Inventors: 廖玮; 莫亚勤; 林晓燕; 张晨光
Original assignee: Beijing Ezlife Sci&tech Co ltd
Current assignee: Beijing Ezlife Sci&tech Co ltd
Priority date: 2017-02-21
Filing date: 2017-02-21
Publication date: 2020-08-14
Anticipated expiration: 2037-02-21
Also published as: CN106755525A

Abstract

The invention discloses a probe for detecting MTHFR gene mutation, application thereof and a kit, and belongs to the technical field of gene detection. The probe for detecting MTHFR gene mutation comprises one or two of a detection probe shown in SEQ ID NO.1 and a capture probe shown in SEQ ID NO. 2-3. The probe can be used for detecting the C677T mutation site of the MTHFR gene and has the characteristics of high detection sensitivity, strong specificity, low false positive rate and the like.

Description

Probe for detecting MTHFR gene mutation and application and kit thereof

Technical Field

The invention relates to the technical field of gene detection, in particular to a probe for detecting MTHFR gene mutation, application thereof and a kit.

Background

MTHFR (5,10-methylene tetrahydrofolate reductase) is known as 5,10-methylenetetrahydrofolate reductase and is a key enzyme in the metabolism of folic acid and homocysteine (Hcy). At chromosome 1p 36.3. The MTHFR has a total length of 19.3kb, 12 common exons and an mRNA total length of 7,105bp, encodes a protein consisting of 657 amino acid residues, and has the main biochemical function of catalyzing the reduction of 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate. Two polymorphic types of MTHFR gene mainly exist, namely common C677T rs1801133 and A1298Crs1801131, and in addition, the A66G rs1801394 1394 locus of MTRR gene is also positioned on the folic acid metabolic cycle. However, only the polymorphism at site 677 has been recognized in relation to diseases and drug metabolism, and has been written in "clinical laboratory test items catalog of medical institutions (2013), which does not include the detection of other 2 sites, and studies suggest that the above site polymorphism is associated with various diseases.

Folic acid plays an important role in cell function, division and differentiation, methylene tetrahydrofolate reductase (MTHFR) can participate in a folate metabolism pathway, catalyze 5,10-methylene tetrahydrofolate to be converted into 5-methyl tetrahydrofolate, enable the 5-methyl tetrahydrofolate to participate in a homocysteine pathway, provide methyl for homocysteine to form methionine, further form S-adenosylmethionine (SAM), and play an important role in maintaining the normal cycle of cells and the activity of the cells. If the enzymes involved in these two pathways are defective or missing, the pathways will be blocked, resulting in increased concentrations of homocysteine in the blood and damage to the vessel wall. The conversion of homocysteine to methionine is hindered, leading to a series of pathological changes: (1) homocysteine accumulates, resulting in a reduction in methylation, which directly affects the synthesis of more than 50 important substances; (2) high concentration homocysteine can damage endothelial cells of blood vessels and become an important cardiovascular and cerebrovascular pathogenic factor; (3) the high-concentration homocysteine acts on sensitive embryonic nerve cells and can cause irreversible damage such as malformation of brain and spina bifida; (4) homocysteine has low solubility and is easy to form calculi in the urinary system.

In studies of the relationship between MTHRF gene polymorphism and birth defects, almost all focus has been on Neural Tube Defects (NTD), and although the MTHRF gene mutation frequency varies in different countries and ethnic groups, a number of studies from different regions have demonstrated that the mother MTHRF C677T mutation is a genetic risk factor for infants with childbearing neural tube defects. A study of MTHRF C677T mutations in mother, fetus, and father, respectively, showed that the maternal 677C/T homozygous mutation increased the risk of a born neural tube deficient infant by a factor of 2, and the fetal 677T/T homozygous mutation increased the risk of NTD by a factor of 1.6. Another birth defect closely related to polymorphism in the MTHRF gene is Down syndrome. The data show that 93% of chromosome 3 of patients with down syndrome are maternal, and thus maternal genetic or metabolic abnormalities are a major risk factor for developing down syndrome. Many studies in recent years have shown that mutation of the maternal MTHRF gene can lead to a decrease in MTHRF activity, hindering 5-methyltetrahydrofolate production as a methyl indirection cell, leading to DNA hypomethylation and chromosomal abnormalities. In addition, the polymorphism of the MTHFR gene C677T site is associated with the occurrence of non-syndromic cleft lip and palate.

Pregnancy related diseases include abortion, premature birth, intrauterine growth retardation of fetus, premature placental stripping, pregnancy-induced hypertension syndrome, etc. The MTHFR gene C677T mutation causes the MTHFR hemozyme activity to be reduced, the plasma homocysteine concentration to be increased, the hyperhomocysteinemia in the gestation period can cause the blood to be in a hypercoagulable state, the risk of placenta thrombosis is increased, the placenta embolism is caused, the circulation of the mother and the fetus is insufficient, and the abortion, the fetal growth restriction and the early peeling of the placenta can be caused; on the other hand, hyperhomocysteinemia can be oxidized by itself under the mediation of metal ions to generate peroxide and oxygen free radicals, damage the structure and the function of vascular endothelial cells and further cause the balance disorder of vasomotor factors of patients, thereby triggering pregnancy-hypertension. In addition, due to the lack of MTHFR activity in placenta tissues, the formation of 5-methyltetrahydrofolate as an indirect methyl donor is hindered, the metabolism of purine, pyrimidine and nucleic acid and DNA methylation are further influenced, so that the hypomethylation of biological components including DNA and protein necessary for fetal development is caused, and the hypomethylation is also one of the reasons for abortion or fetal growth restriction.

C677T homozygous mutation of MTHFR gene, resulting in enzyme activity of only 30% of wild type; heterozygous mutation, the activity of the enzyme was 70% of that of the wild type. In the Chinese population, the proportion of the wild-type gene is 27%, while the proportion of the heterozygous mutation is 44% and the proportion of the homozygous mutation is 29%. For the detection of the C677T gene of MTHFR, the absorption level of different individuals to folic acid can be found as early as possible, so that high risk groups which easily cause folic acid deficiency are screened, individualized folic acid supplementation is realized, and prenatal examination is enhanced to reduce the risk of birth defects of newborns.

Currently, there are several methods for genotyping MTHFR, each of which has its own characteristics.

1. Restriction fragment Length polymorphism polymerase chain reaction technique (PCR-RFLP)

PCR-RFLP is the most classical SNP typing method, which comprises amplifying target DNA crossing polymorphic sites by PCR, cutting PCR products by corresponding restriction endonuclease, and judging genotype according to electrophoresis bands of the cut products. The PCR-RFLP parting method has the advantages of simple operation, less required template DNA amount, no need of large-scale expensive instruments, no dangerous reagents involved in experiments and high safety. However, the method has a major disadvantage in that the genotype is erroneously judged due to false negative or false positive caused by the activity of the endonuclease, the digestion time, the improper digestion system, and the like.

2. Allele specific PCR (allele specific PCR, AS-PCR)

The basic principle of AS-PCR is to design 2 allele-specific primers (P1 for wild-type allele and P2 for mutant allele) based on the base characteristics of SNP site, whose 3' ends are complementary (or identical) to the base of SNP site, and a common primer P3 designed by conventional method is needed. The reverse was true with primers P1, P3, with amplification products in the wild type allele and no amplification products in the mutant allele, and P2, P3. After the PCR is finished, the presence or absence of the amplified product is detected by gel electrophoresis, thereby determining the genotype. The AS-PCR method avoids enzyme digestion, has simpler steps, but has higher false positive rate and stricter experimental requirements on the allele specific PCR amplification method.

Taqman probe technique

The Taqman probe technology is characterized in that a fluorescence labeled probe is added on the basis of common PCR, and the fluorescence intensity is continuously enhanced along with the continuous increase of PCR products, so that a fluorescence growth curve can be detected. The method has the main defects that the fluorescent quenching and double-end labeling technology is adopted, and the quantitative detection is influenced by the enzyme activity; the background is strong, and sometimes, highly related sequences cannot be identified; the cost of the probe labeling and the experimental instrument is high, and the popularization and the application are inconvenient.

4. High resolution melting curve method

A high resolution melting curve analysis technology (HRM) is a new technology developed on the basis of real-time fluorescence PCR, and is characterized in that saturated double-stranded DNA binding dye is added into a PCR system, a high resolution melting curve is prepared after the PCR is finished, and samples are classified according to different melting curves. The method is generally concerned because of its rapidity, high throughput, low cost of use, accurate results, and the realization of a true tube closure operation. However, the HRM technique has a very high requirement for uniformity of the temperature of the apparatus, and requires expensive special instruments and precise analysis software such as Light cycler 480 PCR instrument or Light Scanner. In addition, this method requires that the size of the amplified fragment is 400bp or less, the design of primers is limited, and the optimization of PCR conditions is complicated.

5. Direct sequencing method.

The sequencing method is a gold standard for detecting SNP, and has high accuracy. Including direct sequencing (dideoxy chain termination), pyrosequencing, microsequencing, and the like. Sequencing needs some special instruments and equipment, needs professional personnel to operate, and has high cost and long period; high requirements on the quantity, purity and specificity of PCR products, and complicated operation steps after PCR. The sequencing method still has certain difficulty in clinical detection, and is mostly used as confirmation of other analysis methods.

6. Denaturing high performance liquid chromatography.

The principle of the method is based on the difference of melting characteristics of mismatched heterozygous double-stranded DNA and perfectly matched homozygous double-stranded DNA, and the separation is carried out by a chromatographic method. As the heterozygous double-stranded DNA is mismatched at the mutation site, a Y-shaped structure is easy to form, and the binding capacity with the stationary phase of a chromatographic column is reduced, the heterozygous double-stranded DNA is preferentially eluted compared with the homozygous double-stranded DNA, and whether the mutation exists can be judged by the change of an elution peak. However, it can only determine whether there is mutation, cannot determine the position and type of SNP, requires standard sample or combined sequencing verification, and requires expensive special instruments and professional analysis software, which is one of the reasons why DHPLC has not been widely implemented. In addition, the reaction tube needs to be opened in the detection process, and pollution is easily caused.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a probe for detecting MTHFR gene mutation, which can detect the mutation of the C677T rs1801133 locus of the MTHFR gene and has the characteristics of high detection sensitivity, strong specificity, low false positive rate and the like.

The second purpose of the invention is to provide the application of the probe for detecting MTHFR gene mutation in the preparation of a kit for detecting MTHFR gene mutation.

The third purpose of the invention is to provide a kit, which contains the probe, can be used for mutation detection of the C677T rs1801133 site of the MTHFR gene, and has the characteristics of high detection sensitivity, strong specificity, low false positive rate, simple and convenient operation, short time consumption, low cost and the like.

The fourth purpose of the invention is to provide the application of the probe for detecting the MTHFR gene mutation in detecting the MTHFR gene mutation.

The invention is realized by the following steps:

a probe for detecting MTHFR gene mutation comprises one or two of a detection probe shown in SEQ ID NO.1 and a capture probe shown in SEQ ID NO.2-3, wherein an affinity substance for binding catalytic enzyme is marked at the 5 'end or the 3' end of the detection probe.

The application of the probe for detecting MTHFR gene mutation in preparing a kit for detecting MTHFR gene mutation.

A kit comprising the probe for detecting MTHFR gene mutation.

The application of the probe for detecting the MTHFR gene mutation in detecting the MTHFR gene mutation.

Compared with the prior art, the probe for detecting MTHFR gene mutation, the application thereof and the kit have the beneficial effects that:

the probe for detecting MTHFR gene mutation provided by the invention comprises one or two of a detection probe shown in SEQ ID NO.1 and a capture probe shown in SEQ ID NO. 2-3. Wherein, the capture probe shown in SEQ ID NO.2 is a wild type capture probe and can detect wild type MTHFR gene, and the capture probe shown in SEQ ID NO.2 is a mutant type capture probe and can detect mutant type MTHFR gene. Any one capture probe is matched with the detection probe, the mutation condition of the C677T rs1801133 locus of the MTHFR gene can be detected on an EFIRM technical platform, and the detection probe has the characteristics of accurate detection result, low false positive rate, high specificity, high sensitivity, low cost, simple and convenient operation and the like, and provides a brand-new detection strategy for mutation detection of the MTHFR gene.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a diagram showing the results of detecting samples to be detected with different concentrations by using the probe for detecting MTHFR gene mutation provided in example 1;

FIG. 2 is a diagram showing the results of detecting samples to be detected with different concentrations by using the probe for detecting MTHFR gene mutation provided in example 2 according to the present invention;

FIG. 3 is a diagram showing the results of three samples tested by the probe for detecting MTHFR gene mutation provided in example 3 according to the present invention;

FIG. 4 is a graph showing the results of sequencing at C677T site of MTHFR gene in triplicate samples in example 8 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following describes the probe for detecting MTHFR gene mutation, its application and kit in the embodiment of the invention.

In one aspect, the invention provides a probe for detecting MTHFR gene mutation, which comprises one or two of a detection probe shown in SEQ ID NO.1 and a capture probe shown in SEQ ID NO.2-3, wherein the 5 'end or the 3' end of the detection probe is marked with an affinity substance for binding catalytic enzyme.

The probe is designed based on SNP site C677T rs1801133 of human MTHFR gene, and can be used for detection on an Electric Field Induced Release and Measurement (EFIRM) technical platform. The probe is obtained by long-term research and research of the inventor of the invention, and has the characteristics of good specificity, high sensitivity and the like.

Wherein, the capture probe shown in SEQ ID NO.2 is a wild-type capture probe, and can capture and fix a wild-type MTHFR gene (the rs1801133 site is C). The capture probe shown in SEQ ID NO.3 is a mutant capture probe and can be used for capturing and fixing a mutant MTHFR gene (rs1801133 site is T). Each capture probe is independently matched with a detection probe, and the mutation condition of the C677T rs1801133 locus of the MTHFR gene is detected on an EFIRM technical platform.

Wherein, the 5' ends of the wild type capture probe and the mutant type capture probe are provided with thymine-rich extension arms introduced with special sequences

(5' -TTTTATTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTA- -) for lengthening the length of the capture probe, which is helpful to improve the capture ability of the capture probe, and further improve the sensitivity and specificity of the probe.

Compared with the existing detection technology, the combination of the probe and the EFIRM technology provided by the invention has the characteristics of simple and rapid operation (the sample to be detected is simple and convenient in processing method), short detection time (the whole detection process takes about 30min, and the result can be output in half an hour), strong specificity (the signal is detected only after the dual specificity identification combination of the capture probe and the detection probe is needed), high sensitivity (a trace target sequence can be captured under the induction action of an electric field and is output through the amplification action of an electric signal), low cost (the reagent used in the detection process is simple and the equipment requirement is low) and the like.

The sequence lengths and base sequences of the two capture probes and the detection probe are obtained after long-term exploration, optimization and screening by the inventor of the invention, have stronger specificity and sensitivity, can effectively capture and fix the corresponding target sequence, and have very good specificity.

Of course, the 5 'end or 3' end of the detection probe is labeled with biotin for binding to the catalytic enzyme, and the biotin functions to bind to the streptavidin-labeled catalytic enzyme, and the current generated by the catalytic enzyme catalyzing the substrate releases the detection signal.

It will be readily appreciated that biotin may be replaced by other types of substances, such as digoxin or fluorescein isothiocyanate, etc. It is within the scope of the invention that the 5 'end or the 3' end of the detection probe is labeled with an affinity for binding to the catalytic enzyme.

On the other hand, the invention also provides application of the probe in preparing a kit for detecting MTHFR gene mutation. At present, the kit for detecting the mutation of the MTHFR gene, in particular the C677T rs1801133 locus of the MTHFR gene based on the EFIRM technology platform is basically not available. The kit fills the blank of the detection field, provides a kit for detecting the C677Trs1801133 locus of the MTHFR gene, which is more convenient, quicker, lower in cost, good in specificity and high in sensitivity, and effectively overcomes the problems in the prior detection technology. Provides a brand-new detection strategy for detecting the C677T rs1801133 locus of the MTHFR gene.

Preferably, the probes are present independently in solution, e.g., the capture probes are present in a capture probe solution comprising the capture probes and the detection probes are present in a detection probe solution comprising the detection probes. The concentration of the probe contained in each probe solution can be set according to the actual situation. Preferably, each probe solution contains 0.5 to 1.5. mu.M of probe at the final concentration.

Further, the kit provided by the present invention may further comprise a fixture for fixing the capture probe of the probe to the detection well plate. The fixture includes a conductive polymer and an ionic compound. The conductive polymer is selected from one of pyrrole, aniline and thiophene, and of course, the conductive polymer can be other conductive polymer materials. The ionic compound is selected from any one of sodium chloride and potassium chloride. The conductive polymer is positively charged, a reticular cross-linked structure is formed under the action of an electric field and is deposited at the bottom of the reaction hole, and the reticular cross-linked structure can stably fix the capture probe at the bottom, so that the stability and the capture capability of the capture probe are improved.

Further, the kit provided by the invention can also comprise catalytic enzyme, wherein the catalytic enzyme is horseradish peroxidase with a label, and preferably, the catalytic enzyme is horseradish peroxidase with a streptavidin label. Of course, the catalytic enzyme may be alkaline phosphatase with a label selected from the group consisting of digoxin antibody, fluorescein isothiocyanate antibody and streptavidin, and the label corresponds to the affinity substance and may be selected according to the type of the affinity substance on the detection probe.

When the affibody is biotin, the marker is streptavidin; when the affibody is digoxin, the marker is digoxin antibody; when the affinity substance is fluorescein isothiocyanate, the marker is fluorescein isothiocyanate antibody. So long as the affinity substance and the label correspond to each other and can bind to each other.

Further, the kit provided by the invention can also comprise a substrate, and the category of the substrate is selected according to the category of catalytic enzymes.

When the catalytic enzyme is horseradish peroxidase, the substrate is any one of TMB (Tetramethylbenzidine ), ABTS (2,2' -Azinobis- (3-ethylbenzidine-6-sulfonate, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) and OPD (o-Phenylenediamine ), TMB, ABTS and OPD are substrates of horseradish peroxidase, and color development reaction is carried out under the catalytic action of horseradish peroxidase along with current generation, so that the release of detection signals is improved.

When the catalytic enzyme is alkaline phosphatase, the substrate is any one of a combination of BCIP (5-Bromo-4-Chloro-3-indolylphonate, 5-Bromo-4-Chloro-3-indolyl-phosphate) and NBT (Nitrotetrazolium Blue chloride, tetrazolium nitro Blue), nitrophenyl phosphate, disodium 4-nitrophenylphosphate, Naphthol AS-BI phosphate, Naphthol AS-MX-phosphate.

Further, the kit provided by the invention can also comprise a washing solution, wherein the washing solution comprises a washing solution A and a washing solution B, the washing solution A is an SSC buffer solution containing SDS, and the washing solution B is a PBS buffer solution containing Tween 20.

Further, the kit provided by the invention can also comprise a diluent, wherein the diluent is a PBS buffer solution containing casein.

Further, the kit provided by the invention can also comprise a hybridization buffer.

Furthermore, the kit provided by the invention can also comprise a detection pore plate, and the capture probe is fixed in a reaction pore of the detection pore plate. In other embodiments, the capture probes may not be immobilized in the reaction wells of the detection well plate, and the capture probes may be immobilized in the detection well plate by a corresponding method.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The probe for detecting MTHFR gene mutation provided by the embodiment comprises a wild type capture probe (SEQ ID NO.2) and a detection probe (SEQ ID NO.1), wherein Biotin (Biotin) is marked at the 5' end of the detection probe.

The base sequence of the wild-type capture probe (named MTHFR-WT-LSCP13) was as follows:

5’-TTTTATTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTAGAAATCGGCTCC-3 '(SEQ ID NO.2) wherein the underlined part is used to associate with the target region (5' -GGAG) of the wild-type MTHFR gene covering the rs1801133 site (C)CCGATTTC-3') and then capturing and fixing the target sequence.

In addition, the 1 st-77 th thymine (T) -rich region at the 5' end of the wild type capture probe is an extension arm and is used for improving the capture probability of the wild type capture probe and a wild type target sequence and improving the detection sensitivity.

The base sequence of the selected wild type target sequence (SEQ ID NO.4) covering the rs1801133 site from the wild type MTHFR gene is as follows:

5’-GGCTGACCTGAAGCACTTGAAGGAGAAGGTGTCTGCGGGAGCCGATTTCATCATCACGC-3', wherein the underlined part is the rs1801133 site (C).

The base sequence of the detection probe is as follows:

5'-ACACCTTCTCCTTCAAGTGCTTCAG-3' (SEQ ID No.1) for fully complementary binding to a target region (5'-CTGAAGCACTTGAAGGAGAAGGTGT-3') of a wild-type target sequence (SEQ ID No.4) of a wild-type MTHFR gene, enabling specific binding and detection of the wild-type target sequence.

The probe provided by the embodiment can be used for detecting the condition that the rs1801133 locus of the wild MTHFR gene is C, and has the characteristics of good specificity, high sensitivity and the like.

Example 2

The probe for detecting MTHFR gene mutation provided by the embodiment comprises a mutant capture probe (SEQ ID NO.3) and a detection probe (SEQ ID NO.1), and the base sequence and the structure of the detection probe are the same as those of the embodiment 1.

The base sequence of the mutant capture probe (named MTHFR-LSCP13) was as follows:

5’-TTTTATTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTAGAAATCGACTCC-3 '(SEQ ID NO.3) wherein the underlined part is used for comparison with the target region (5' -GGAG) of the mutant target sequence (SEQ ID NO.5) of the mutant MTHFR gene covering the rs1801133 site (T)TCGATTTC-3', the rs1801133 site (T)) is underlined to be complementarily combined, and the mutant target sequence is captured and fixed.

In addition, the thymine (T) -rich region from 1 st to 77 th positions of the 5' end of the mutant capture probe is an extension arm, so that the capture probability of the thymine (T) -rich region and a mutant target sequence (SEQ ID NO.5) is improved, and the detection sensitivity is improved.

The base sequence of the selected mutant target sequence (SEQ ID NO.5) from the mutant MTHFR gene is as follows:

5’-GGCTGACCTGAAGCACTTGAAGGAGAAGGTGTCTGCGGGAGTCGATTTCATCATCACGC-3', wherein the underlined part is the rs1801133 site (T).

The probe provided by the embodiment can be used for detecting the rs1801133 site of the mutant MTHFR gene under the condition of T, and has the characteristics of good specificity, high sensitivity and the like.

Example 3

The probe for detecting MTHFR gene mutation provided by the embodiment comprises: a wild-type capture probe, a mutant capture probe, and a detection probe.

The base sequence of the wild-type capture probe was the same as in example 1, the base sequence of the mutant-type capture probe was the same as in example 2, and the base sequence and structure of the detection probe were the same as in example 1.

The probe provided by the embodiment can detect the mutation condition of the rs1801133 locus of the MTHFR gene of the sample to be detected, can distinguish whether the sample to be detected is a wild homozygote, a mutant heterozygote or a mutant homozygote, and has the characteristics of good specificity, high sensitivity, low false positive rate and the like.

Example 4

This example provides a kit comprising a probe for detecting a mutation in the MTHFR gene as described in any one of the above examples. The kit can be used for detecting the mutation condition of the C677T rs1801133 locus of the MTHFR gene, and has the characteristics of high detection sensitivity, strong specificity, low false positive rate and the like.

Example 5

This example provides a kit comprising not only the probe for detecting a mutation in MTHFR gene described in any one of examples 1 to 3 above. Also includes horseradish peroxidase with streptavidin label, TMB, 2 XSSC buffer containing SDS, PBS buffer containing Tween20, pyrrole solution, potassium chloride solution, and hybridization buffer.

It will be readily appreciated that in other embodiments, the kit may include one or more of streptavidin-labeled horseradish peroxidase, TMB, 2 xssc buffer with SDS, and PBS buffer with Tween20, pyrrole solution, potassium chloride solution, hybridization buffer.

The kit provided in this example has the same effects as example 4.

Example 6

The sensitivity of the probe for detecting MTHFR gene mutation provided in example 1 was verified.

The sensitivity of the MTHFR gene mutation detection probe provided in example 1 was verified by using the wild-type capture probe (SEQ ID NO.2) of the MTHFR gene mutation detection probe provided in example 1 as an experimental group, the capture probe (base sequence: 5'-CTGTCTATCCTGCGAAATCGGCTCC-3', 15 th to 25 th positions of which are used to bind to the target region of the target sequence (SEQ ID NO.4) to capture the wild-type target sequence, and 1 st to 14 th positions of which are also extension arms but different in sequence and length from the extension arms of the wild-type capture probe) of SEQ ID NO.6 as a control group, and the wild-type target sequence (wild-type oligonucleotide fragment) of SEQ ID NO.4 at different concentrations (100pM, 10pM, and 5pM) as a sample to be tested, as follows.

1 immobilization of Capture Probe (hereinafter referred to as CP)

1.1 preparing a mixed solution of pyrrole (pyrrole) and CP

Sequentially adding 885 mu l of ultrapure water and 100 mu l of ionic compound into 1 1.5mL centrifuge tube, uniformly mixing by vortex oscillation, and centrifuging; adding 5 μ l pyrrole (more than or equal to 98.0% from Sigma, product number W338605) of conductive polymer, vortex, shaking, mixing, and centrifuging; add 10. mu.l of 100. mu.M CP (wild type capture probe shown in SEQ ID NO.2 (MTHFR-WT-LSCP13) to the experimental group and wild type capture probe shown in SEQ ID NO.6 (MTHFR-WT-CP13) to the control group, vortex, mix well, and centrifuge for use.

1.2 immobilization of the Capture Probe

Adding 30 μ l of prepared pyrrole and CP mixed solution (4 reaction wells are respectively arranged in an experimental group and a control group and respectively added with the corresponding mixed solution according to the operation instruction on a 96-well detection pore plate (E-plate) (the structure and the working principle of which can be seen in 201620769829.2), adding samples (oligonucleotide fragments) to be detected with different concentrations in the subsequent steps as detection pores, and using the rest one pore as a blank control pore), attaching a tip close to the bottom of the pore during sample addition without contacting the bottom electrode, inclining or beating the E-plate after adding to uniformly cover the electrode surface in the pore, and immediately putting on an EFIRM instrument (the working principle and the structure of which can be seen in the application date of 2016, 8, 11, and the application number of 201610658321.X, the name of a holding structure and the name of a detector comprising the holding structure), according to the operation instruction, the electric field operation is performed.

1.3EFIRM electric field treatment

The corresponding column for the experiment was selected on the EFIRM software, with the electric field parameters set to: voltage A: 350mV, 1 s; the voltage B is 950mV for 1 s; 9 cycles were performed. And taking out the plate immediately after the electric field treatment is finished, and cleaning the E-plate.

1.4E-plate washing:

the corresponding experimental column was selected on the plate washer program, the washing program was selected (2bottom, 2top), and the washing liquid was selected as washing liquid a. After the washing, the next step, sample loading, was performed immediately. Wherein the washing reagent A is a 2 XSSC buffer containing 0.05% (mass percent) SDS.

2 hybridization of samples

2.1 hybridization buffer pretreatment

The hybridization buffer (purchased from Biotechnology engineering (Shanghai) Co., Ltd., product number: B548207) was equilibrated to room temperature.

2.2 preparing the sample to be tested

Mixing a sample to be detected with the hybridization buffer according to the volume ratio of 1:2, carrying out vortex oscillation and then centrifuging, and loading the sample for detection.

2.3 Add sample to be tested

And adding 30 mu l of mixed solution of the sample to be detected and the hybridization buffer into the corresponding reaction hole on the E-plate, and controlling the final concentration of the sample to be detected in each group of three detection holes to be 100pM, 10pM and 5pM in sequence. The blank wells of each group were loaded with only the same volume of hybridization buffer.

It should be noted that the tip is close to the bottom of the well but does not touch the bottom electrode when the sample is added, and the E-plate is tilted or flapped after the sample is added to make the liquid uniformly cover the surface of the electrode in the well, and then the E-plate is immediately put on the EFIRM to perform electric field operation.

2.4EFIRM electric field treatment

The corresponding column for the experiment was selected on the EFIRM software, with the electric field parameters set to: voltage A: 300mV, 1 s; the voltage B is 500mV for 1 s; 150 cycles were performed. And taking out the plate immediately after the electric field treatment is finished, and cleaning the E-plate.

2.5 incubation at Room temperature

Cover with E-plate lid and incubate on the bench at room temperature for 15 min.

2.6E-plate cleaning

The corresponding experimental column was selected on the plate washer program, the washing program was selected (2bottom, 2top), and the washing liquid was selected as washing liquid a. And after the cleaning is finished, immediately carrying out DP sample adding operation.

3 Detection Probe (DP) binding

3.1DP solution preparation

The diluent was taken out from a 4 ℃ refrigerator, 1 1.5mL centrifuge tube was taken, 990. mu.L of the diluent was added, 10. mu.L of 100. mu.M DP (SEQ ID NO.1, i.e., the detection probe in example 1) was added to each reaction well, vortexed, mixed well, and centrifuged for use.

Wherein the diluent is PBS buffer (pH7.4) containing 0.1% (mass/volume) casein. The casein serves to block non-specific sites to improve the sensitivity and accuracy of the detection.

3.2 sample application

And adding 30 mu l of corresponding DP solution into a corresponding hole according to the experimental design, wherein the gun head is attached to the bottom of the hole during sample addition but does not contact with a bottom electrode, after the sample addition is finished, the E-plate is inclined or flapped to enable the liquid to be uniformly covered on the surface of the electrode in the hole, and then the E-plate is immediately put on the EFIRM for electric field operation.

3.3 incubation at Room temperature

3.4E-plate cleaning

The corresponding experimental column was selected on the plate washer program, the washing program was selected (2bottom, 2top), and the washing liquid was selected as washing liquid a. And after the cleaning is finished, immediately carrying out sample adding operation.

Binding of 4-streptavidin-labeled horse radish peroxidase (Poly-HRP) and biotin

4.1 preparation of Poly-HRP solution

The diluent was removed from the 4 ℃ freezer and 1 tube of 1.5mL centrifuge tube was added to 999. mu.l of the diluent and 1. mu.l of enzyme solution (containing Poly-HRP at a concentration of 0.5mg/mL, available from thermo fisher under the product name Pierce^TMStreptavidin Poly-HRP with a stock number of 21140 and a unit specification of 0.5mL), vortexing, shaking, mixing, and centrifuging for use.

4.2 addition of enzyme solution

30 μ l of mixed solution of the diluted solution and the enzyme solution is added to each corresponding well, and the Poly-HRP is identified and combined with the biotin on the detection probe through the labeled streptavidin.

4.3 incubation at Room temperature

4.4E-plate cleaning

The corresponding experimental column was selected on the plate washer program, the washing program was selected (3bottom, 3top), and the washing liquid was selected as washing liquid B. After the washing, the TMB sample adding operation is immediately carried out. Wherein the washing solution B is PBS buffer solution containing 0.1 percent (mass percent) Tween 20.

5 data reading

5.1 adding substrate

Mu.l of substrate was added to the reaction well, with the tip near the bottom of the well but not touching the bottom electrode. Immediately after the addition, the electric field operation is performed on the EFIRM. Wherein the substrate is TMB-containing solution (purchased from thermo fisher, product catalog number 34028, name 1-Step)^TMUltra TMB-ELISA). Adding substrate of enzyme, oxidation-reduction reaction to generate current, and detecting the current value in each hole to complete the whole detection process.

5.2EFIRM electric field readings

The corresponding column for the experiment was selected on the EFIRM software, with the electric field parameters set to: voltage A: -200mV, 60 s; the voltage B is 0mV and 0 s; 1 cycle was performed. And taking out the plate immediately after the electric field treatment is finished, and cleaning the E-plate.

The instrument automatically completes detection work, and detection data are automatically uploaded to the cloud computing platform. The results of the measurements in this example are shown in Table 1 and FIG. 1, wherein the bar graph is plotted based on the measurement data, the abscissa represents the type of the measurement group, and the ordinate represents the Current value (Current) in nanoamperes (nA) in each reaction well in each measurement group.

TABLE 1 results of the detection of samples to be tested at different concentrations using the probe for detecting MTHFR gene mutation provided in example 1

As shown in Table 1 and the results in FIG. 1, the wild-type capture probe MTHFR-WT-LSCP13 of the probe for detecting mutations in the MTHFR gene provided in example 1 has higher sensitivity than the control MTHFR-WT-CP13, and can detect a sample to be detected even at a concentration as low as 5pM, with a detection signal of 169.68nA, which is about twice as high as the control. Therefore, the wild-type capture probe shown in SEQ ID No.2 has better capture capability and can capture and fix a low-concentration target sequence by the arrangement of the extension arm, so that the probe for detecting MTHFR gene mutation provided in example 1 has higher sensitivity.

Example 7

The sensitivity of the probe for detecting MTHFR gene mutation provided in example 2 was verified.

The sensitivity of the probe for detecting MTHFR gene mutation provided in example 2 was determined using the wild-type capture probe (SEQ ID NO.3, named MTHFR-LSCP13) of the probe for detecting MTHFR gene mutation provided in example 2 as the experimental group, the capture probe (named MTHFR-CP13) shown in SEQ ID NO.7 as the control group, and the target sequences (oligonucleotide fragments) shown in SEQ ID NO.5 at different concentrations (100pM, 10pM, 5pM) as the samples to be tested, and the detection method was substantially the same as in example 6. The results of the measurements are shown in table 2 and fig. 2.

TABLE 2 results of the detection of samples to be tested at different concentrations using the probe for detecting MTHFR gene mutation provided in example 2

As shown by the results of Table 2 and FIG. 2, the detection limit of the mutant capture probe MTHFR-LSCP13 of the probe for detecting mutations in MTHFR gene provided in example 2 was lower than that of the control MTHFR-CP13, and it showed strong detection signals at concentrations as low as 5pM in the samples to be tested, and the detected current reached 107.29nA, whereas the current of the control at this concentration was only 35.67nA, which is close to that of the blank control. Therefore, it is further shown that the capture capability of the mutant capture probe (SEQ ID No.3) is improved by the arrangement of the extension arm, and the target sequence (SEQ ID No.5) with low concentration can be captured and immobilized, so that the probe for detecting MTHFR gene mutation provided in example 2 has higher sensitivity.

Example 8

The present example provides a method for detecting the mutation of site C677T rs1801133 of MTHFR gene by using the probe for detecting mutation of MTHFR gene provided in example 3, and the steps are as follows.

1 capture Probe immobilization

1.1 preparing a mixed solution of pyrrole (pyrrole) and CP

Sequentially adding 885 mu l of ultrapure water and 100 mu l of ionic compound into 1 1.5mL centrifuge tube, uniformly mixing by vortex oscillation, and centrifuging; adding 5 mul pyrrole of conductive polymer, whirling, shaking, mixing uniformly, and centrifuging; add 10. mu.l of 100. mu.M CP; and (5) centrifuging after vortex oscillation and uniform mixing for later use.

1.2 immobilization of the Capture Probe

And adding 30 mu l of prepared pyrrole and CP mixed solution into a reaction hole on a 96-hole detection pore plate (E-plate) according to the operation instruction, wherein the gun head is close to the bottom of the hole but does not contact with a bottom electrode when the sample is added, inclining or beating the E-plate after the sample is added to uniformly cover the surface of the electrode in the hole, immediately putting the E-plate on an EFIRM instrument, and performing electric field operation according to the operation instruction.

2 detection groups are set, namely a wild type detection group and a mutant type detection group. Each detection group is provided with a positive control hole, a negative control hole, a sample 1 detection hole, a sample 2 detection hole and a sample 3 detection hole (the sample detection holes are determined according to the number of samples to be detected, and can be one or more).

Adding a wild type capture probe (SEQ ID NO.2) into each reaction hole of the wild type detection group; a mutant capture probe (SEQ ID NO.3) was added to each reaction well of the mutant test group.

1.3EFIRM electric field treatment

1.4E-plate washing:

2 hybridization of samples

2.1 hybridization buffer pretreatment

2.2 preparing the sample to be tested

Taking a sample to be tested (a sample collection mode is that the oral cavity swab is carefully taken out, the handle is held, the swabs are respectively extended into oral cavities at two sides, so that the head of the swab is fully contacted with mucous membranes at the upper and lower gums inside the left cheek/on the left side, the swab is vertically rubbed with the tooth brushing force, meanwhile, the swab is rotated, so that the head of the swab is fully contacted with the oral mucosa, the swab head is placed in a sampling tube, the PBS is added for soaking, the resuspended cells are shaken, and the cells are stored at the temperature of minus 20 ℃), the sample is taken out from a refrigerator at the temperature of minus 20 ℃, and. After complete dissolution, a sample to be detected is pretreated by a boiling method or 0.4M NaOH, then the sample to be detected and the hybrid buffer are mixed according to the volume ratio of 1:2, vortex oscillation is carried out, centrifugation is carried out, and then sample loading is carried out for detection.

2.3 Add sample to be tested

And adding 30 mu l of mixed solution of the sample to be detected and the hybridization buffer into the corresponding hole on the E-plate. (Note: the tip of the tip is close to the bottom of the well but does not touch the bottom electrode during sample addition, and the E-plate is tilted or flapped after sample addition to make the liquid uniformly cover the electrode surface in the well, and then immediately put on the EFIRM for electric field operation.)

Positive control Buffer mixed liquor containing MTHFR-WT (wild type oligonucleotide fragment, SEQ ID NO.4) is added into the positive control hole of the wild type detection group, positive control Buffer mixed liquor containing MTHFR-MT (mutant oligonucleotide fragment, SEQ ID NO.5) is added into the positive control hole of the mutant type detection group, and only Buffer is added into the negative control hole of each detection group. And adding a mixed solution of the corresponding sample to be detected (sample 1, sample 2 or sample 3) and the hybridization buffer into the sample detection hole.

2.4EFIRM electric field treatment

2.5 incubation at Room temperature

2.6E-plate cleaning

3DP binding

3.1DP solution preparation

Taking out the diluent from a refrigerator at 4 ℃, taking 1 centrifugal tube with 1.5mL, adding 990 mu l of the diluent, adding corresponding 10 mu l of DP (SEQ ID NO.1) with 100 mu M into the reaction hole of each detection group, vortexing, shaking uniformly, and centrifuging for later use. Wherein the diluent is PBS buffer (pH7.4) containing 0.1% (mass/volume) casein. The casein serves to block non-specific sites to improve the sensitivity and accuracy of the detection.

3.2 sample application

3.3 incubation at Room temperature

3.4E-plate cleaning

Binding of 4-streptavidin-labeled horse radish peroxidase (Poly-HRP) and biotin

4.1 preparation of Poly-HRP solution

The diluent was taken out from a 4 ℃ refrigerator, 1 piece of 1.5mL centrifuge tube was taken, 999. mu.l of the diluent was added, 1. mu.l of the enzyme solution (containing Poly-HRP, concentration 0.5mg/mL) was added, vortexed, shaken well, and centrifuged for use.

4.2 addition of enzyme solution

4.3 incubation at Room temperature

4.4E-plate cleaning

5 data reading

5.1 adding substrate

Mu.l of substrate was added to each corresponding well, with the tip near the bottom of the well but not touching the bottom electrode. Immediately after the addition, the electric field operation is performed on the EFIRM. Wherein the substrate is a solution containing TMB. Adding substrate of enzyme, oxidation-reduction reaction to generate current, and detecting the current value in each hole to complete the whole detection process.

5.2EFIRM electric field readings

The instrument automatically completes detection work, and detection data are automatically uploaded to the cloud computing platform. A histogram is plotted based on the detection data, with the abscissa representing the type of detection group and the ordinate representing the Current value (Current) in nanoamperes (nA) for each detection well in each detection group, and the detection results are shown in fig. 3 and table 3.

5.3 analysis of results

The quality control results show that: for the wild type and mutant detection groups, the value of a negative control needs to be less than 80nA, the value of a positive control needs to be more than 80nA, and otherwise, the result is not true. The threshold value is set to be 80 nA.

It should be noted that the method for detecting by using the probe provided in the other embodiments is basically the same as that of the present embodiment.

TABLE 3 results of triplicate samples tested using the probes for MTHFR gene mutation provided in example 3

As can be seen from Table 3 and FIG. 3 (in the figure: the ordinate is the current value nA), in the wild type detection group, the negative control value was 23.31nA and was less than 80nA, and the positive control value was 98.72nA and was more than 80nA, which satisfied the quality control requirements;

in the mutant detection group, the negative control value is 23.12nA and is less than 80nA, the positive control value is 106.20nA and is more than 80nA, and the quality control requirement is met.

The wild type detection result of the sample 1 is 477.37nA, which is more than 80nA, and the wild type detection result is positive; the mutant type detection result of the sample 1 is 23.58nA and less than 80nA, and the mutant type detection result is negative; it was confirmed that the MTHFR C677T mutation site in sample 1 was a wild-type homozygote (type CC).

The wild type detection result of the sample 2 is 461.18nA which is more than 80nA, the wild type detection result is positive, the mutant type detection result of the sample 2 is 677.92nA which is more than 80nA, and the mutant type detection result is also positive. The signals detected by both genotypes were positive, indicating that sample 2 is a MTHFR C677T mutant heterozygote (CT type).

The wild type detection result of the sample 3 is 44.45nA, which is less than 80nA, and the wild type detection result is negative; the mutant type detection result of the sample 3 is 576.55nA, which is more than 80nA, and the mutant type detection result is positive; sample 3 is identified as MTHFR C677T mutant homozygote (type TT).

In addition, the results of the three samples also show that the wild-type capture probe (SEQ ID NO.2) and the mutant-type capture probe (SEQ ID NO.3) of the probe provided in example 3 have better specificity. Because the base sequences of the two have only one base difference, but each base sequence only specifically binds to the complementary target sequence, for example, the mutant type detection result of the sample 1 is negative (23.58nA), which indicates that the mutant type capture probe (SEQ ID NO.3) does not bind to the target sequence shown in SEQ ID NO.4, and indicates that the specificity of the mutant type capture probe is good and the false positive rate is low; for example, the wild-type detection result of the sample 3 is negative (44.45nA), which indicates that the wild-type capture probe (SEQ ID NO.2) is not combined with the target sequence shown in SEQ ID NO.5, and this also indicates that the wild-type capture probe has good specificity and low false positive rate.

In addition, in this example, sequencing primers were also used

An upstream primer: 5'-ATCCCTCGCCTTGAACAG-3', and a downstream primer:

5'-TCACCTGGATGGGAAAGA-3', the mutation of the MTHFR gene at the C677T site of the MTHFR genes of the sample 1, the sample 2 and the sample 3 is detected by sequencing to verify the accuracy of the detection result, and the sequencing result for the C677T site region is shown in FIG. 4.

The results in FIG. 4 show (in the figure, a represents sample 1, b represents sample 2, C represents sample 3, and the arrow indicates the mutation site to be detected), sample 1 is a wild type homozygote, sample 2 is a mutant heterozygote at site C677T rs1801133, and sample 3 is a mutant homozygote at site C677Trs 1801133. This indicates that the detection of the mutation site of MTHFR gene C677T using the MTHFR gene mutation probe provided in example 3 is accurate and reliable.

In conclusion, the probe for detecting the mutation of the MTHFR gene provided by the invention is designed based on the C677Trs1801133 site of the human MTHFR gene, the capture capability of the capture probe is improved by introducing extension arm modification of a special sequence at the 5' end of the capture probe, the detection sensitivity of the capture probe is further improved, and then a complementary binding region (target region) is selected as the binding region of the capture probe according to the sequence near the C677T rs1801133 site, so that the detection specificity of the capture probe is improved, and the false positive rate of the capture probe is reduced; in addition, the probe for detecting the MTHFR gene mutation provided by the invention can detect whether the MTHFR gene is mutated at the C677Trs1801133 site or not, and can also detect whether the mutant genotype is heterozygous or homozygous. When the probe provided by the invention is used for detection on an EFIRM technical platform, the probe has the characteristics of simplicity, convenience and rapidness in operation, short time consumption, low cost and the like.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Beijing Yihuo Biotechnology Ltd

<120> probe for detecting MTHFR gene mutation, application thereof and kit

<160>7

<170>PatentIn version 3.5

<210>1

<211>25

<212>DNA

<213> Artificial sequence

<400>1

acaccttctc cttcaagtgc ttcag 25

<210>2

<211>89

<212>DNA

<213> Artificial sequence

<400>2

ttttattttt tttttttttt ttattttttt tttttttttt attttttttt ttttttttat 60

tttttttttt ttttttagaa atcggctcc 89

<210>3

<211>89

<212>DNA

<213> Artificial sequence

<400>3

ttttattttt tttttttttt ttattttttt tttttttttt attttttttt ttttttttat 60

tttttttttt ttttttagaa atcgactcc 89

<210>4

<211>59

<212>DNA

<213> Intelligent (Homo sapiens)

<400>4

ggctgacctg aagcacttga aggagaaggt gtctgcggga gccgatttca tcatcacgc 59

<210>5

<211>59

<212>DNA

<213> Intelligent (Homo sapiens)

<400>5

ggctgacctg aagcacttga aggagaaggt gtctgcggga gtcgatttca tcatcacgc 59

<210>6

<211>25

<212>DNA

<213> Artificial sequence

<400>6

ctgtctatcc tgcgaaatcg gctcc 25

<210>7

<211>25

<212>DNA

<213> Artificial sequence

<400>7

ctgtttatcc tgcgaaatcg actcc 25

Claims

1. A probe for detecting MTHFR gene mutation is characterized by comprising a detection probe shown in SEQ ID NO.1 and a capture probe shown in SEQ ID NO.2-3, wherein an affinity substance for binding catalytic enzyme is marked at the 5 'end or the 3' end of the detection probe.

2. The probe for detecting MTHFR gene mutation of claim 1, wherein the affinity substance is any one of digoxin, fluorescein isothiocyanate, and biotin.

3. Use of the probe for detecting MTHFR gene mutation according to claim 1 or 2 in the preparation of a kit for detecting MTHFR gene mutation.

4. A kit comprising the probe for detecting a mutation in MTHFR gene according to claim 1 or 2.

5. The kit of claim 4, further comprising an anchor for immobilizing the capture probes of the probes to a detection well plate, the anchor comprising a conductive polymer and an ionic compound;

the conductive polymer is any one selected from pyrrole, aniline and thiophene;

the ionic compound is any one selected from sodium chloride and potassium chloride.

6. The kit of claim 4, further comprising the catalytic enzyme, wherein the catalytic enzyme is horseradish peroxidase or alkaline phosphatase with a label for binding to the affibody, and wherein the label is one of a digoxin antibody, a fluorescein isothiocyanate antibody, and streptavidin.

7. The kit of claim 6, further comprising a substrate for the catalytic enzyme;

when the catalytic enzyme is the horseradish peroxidase, the substrate is any one of TMB, ABTS and OPD;

when the catalytic enzyme is the alkaline phosphatase, the substrate is any one of a combination of BCIP and NBT, p-nitrophenylphosphate, disodium 4-nitrophenylphosphate, naphthol AS-BI phosphate and naphthol-AS-MX-phosphate.

8. The kit of claim 4, further comprising a wash solution A and a wash solution B, wherein the wash solution A is an SSC buffer containing SDS and the wash solution B is a PBS buffer containing Tween 20.

9. The kit of claim 4, further comprising a detection well plate, wherein the capture probes are immobilized within reaction wells of the detection well plate.