CN108192965B - Method for detecting heterogeneity of mitochondrial genome A3243G locus - Google Patents

Abstract

The invention provides a method for detecting the heterogeneity of a site A3243G of a mitochondrial genome, which comprises the following steps: a) preparing a standard substance of a wild type plasmid and a mutant type plasmid containing the fragment of the mitochondrial genome at the A3243G site; b) directly taking a non-nucleic acid template as a template to be detected, carrying out Single Nucleotide Polymorphism (SNP) analysis and detection on the site A3243G by using a real-time quantitative Polymerase Chain Reaction (PCR) technology, and labeling by using a minor groove binder probe (MGB probe). The invention changes the operation steps of extracting nucleic acid DNA as a template by conventional PCR, directly takes non-nucleic acid templates such as leucocyte, urine sediment, saliva sediment, hair follicle and the like as the template and obtains good results, thereby simplifying the detection time.

Description

Method for detecting heterogeneity of mitochondrial genome A3243G locus

Technical Field

The invention relates to the field of biomedicine, in particular to a method for detecting the heterogeneity of a locus A3243G of a mitochondrial genome in different tissues of a human.

Background

Encephalomyopathy with hyperlactacidosis and stroke-like attack syndrome (MELAS) are mainly caused by mutation of adenine (a) at the 3243 th site of the human mitochondrial genome into guanine (G) (mt.3243a > G), and are the most common manifestation of human mitochondrial diseases. In addition to MELAS, mt.3243a > G site mutations also cause a variety of disease phenotypes such as mid or stroke. Research shows that mitochondrial genome mutation has the characteristic of heterogeneity, and diseases caused by mitochondrial mutation have significant threshold effect. Due to the limitations of objective conditions and economic factors, the screening and diagnosis of patients with MELAS remains at the level of clinical observation at the present stage, failing to perform extensive and accurate genetic testing of mitochondrial DNA mutations in blood/cells (or other tissues) of patients.

At present, the heterogeneity of mt.3243A > G sites cannot be rapidly and widely detected, the expression or copy number of a specific gene is quantified by the traditional qPCR technology, and the method comprises relative quantification and absolute quantification. However, in any method, it is necessary to extract nucleic acid from cells or tissues as a prerequisite, and for example, the number of gene copies is based on the nuclear DNA as a template, and the amount of mRNA expression is based on the first strand of cDNA after reverse transcription as a template, which increases the complexity of the method. Therefore, a new, simple and effective method for detecting the heterogeneity of the mitochondrial genome at the A3243G locus is yet to be provided.

Disclosure of Invention

The invention provides a novel simplified method for detecting the site heterogeneity of mitochondrial genome A3243G, which at least solves the technical problem that the method for detecting the site heterogeneity of mitochondrial genome A3243G in different tissues of human in the prior art is complex.

The invention provides a method for detecting the heterogeneity of a site A3243G of a mitochondrial genome, which comprises the following steps:

a) preparing a standard product of a wild type vector and a mutant type vector containing the fragment of the mitochondrial genome at the A3243G site;

b) directly taking a non-nucleic acid template as a template to be detected, carrying out Single Nucleotide Polymorphism (SNP) analysis and detection on the site A3243G by using a real-time quantitative Polymerase Chain Reaction (PCR) technology, and labeling by using a minor groove binder probe (MGB probe).

Optionally, the non-nucleic acid template comprises a leukocyte, a urine sediment, a saliva sediment, or a hair follicle.

Optionally, the step a) further includes:

cloning a fragment gene containing mitochondrial genome site A3243G and constructing a vector; and performing site-directed mutagenesis using an overlapping Polymerase Chain Reaction (PCR) to obtain a mutated position, and designing a mutation primer for the mutated position.

Optionally, the Polymerase Chain Reaction (PCR) technique is a quantitative polymerase chain reaction (quantitative PCR) technique labeled with minor groove binder probe (MGB probe), and/or the vector of the standard is a plasmid or a T-vector.

Alternatively, the vector of the above standard is a cloning vector propagated in E.coli.

Optionally, the vector of the standard is a pEasy-Blunting vector.

Optionally, the wild-type and mutant plasmid standard samples are prepared to the same concentration, and then mixed in equal volume to prepare the standard sample stock solution, and then the standard sample stock solution is diluted 5-10 times to 6-8 concentration gradients.

Optionally, the stock solution of the standard substance is diluted by 5 times to 8 concentration gradients.

Optionally, the wild-type probe is labeled with 5' -end green fluorescent protein (VIC), the mutant-type probe is labeled with 5' -end carboxyfluorescein (FAM), and the wild-type probe and the mutant-type probe are both labeled with minor groove binder probes (MGB probes) at their 3' -ends; and/or

When the real-time quantitative Polymerase Chain Reaction (PCR) technology is used for carrying out Single Nucleotide Polymorphism (SNP) analysis and detection on the A3243G site in the step b), the used template is a non-nucleic acid template.

Optionally, the non-nucleic acid template comprises a leukocyte, a urine sediment, a saliva sediment, or a hair follicle.

The invention provides a novel simplified method for detecting the site heterogeneity of mitochondrial genome A3243G, which at least solves the technical problem that the method for detecting the site heterogeneity of mitochondrial genome A3243G in different tissues of human in the prior art is complex. The technical scheme of the invention can accurately and quickly detect the heterogeneity of the 3243 locus of the mitochondrial genome of different tissues of MELAS patients and MELAS atypical symptom or asymptomatic members. Accurate analysis of 3243 site heterogeneity of the human mitochondrial genome helps to clarify the correlation between the clinical characteristics of MELAS and the proportion of mutant gene copy number in the mitochondrial genome (level of gene mutation heterogeneity). In addition, the present invention utilizes the MGB probe method for quantification, rather than the traditional TAMRA probe. The MGB, namely minor groove combination, can increase the Tm value of the probe, so that the specificity is higher, the difference between one base in a template can be finely distinguished, in addition, wild type and mutant MGB probes are simultaneously added in a system, the simultaneous quantification of a plurality of genes is realized, and the preparation of a double-standard curve is utilized in the result analysis, so that the heterogeneity detection of mt.3243A > G sites is more accurate. In a word, the qPCR method using the MGB probe is more accurate, convenient and efficient, and can realize effective detection of heterogeneity of the mitochondrial A3243G locus in different tissues of a specific patient.

The technical scheme of the invention overcomes the difficulty of determining the detection limit when the heterogeneity of mt.3243A and G sites is detected in the prior art, because the nonspecific amplification often occurs in the conventional PCR or qPCR amplification process, and even if water is used as a template, a nonspecific amplification curve also occurs at the end of the reaction, so the definition of the fluorescence threshold of the amplification curve of the nonspecific amplification curve and the amplification curve of a low mutation rate template becomes the difficulty of the prior art, while the invention effectively detects the heterogeneity of mt.3243A and G sites, evaluates the nonspecific fluorescence threshold and the mutation rate detection limit of mt.3243A and G sites, and fully determines the accuracy and precision of the invention.

Traditional qPCR techniques quantify the expression or copy number of a particular gene, including relative and absolute quantification. However, in any method, it is necessary to extract nucleic acid from cells or tissues on the premise that the gene copy number is based on the nuclear DNA as a template, and the mRNA expression amount is based on the first strand of cDNA after reverse transcription as a template. The invention detects the heterogeneity of mt.3243A G locus, can directly take leucocyte, urine sediment or hair follicle as a template without extracting nucleic acid, effectively detects the heterogeneity of mt.3243A G locus of a patient, greatly simplifies the detection method and has unexpected effect.

The invention detects the heterogeneity of mt.3243A > G locus and utilizes the MGB probe-labeled qPCR technology, and the technology analyzes the mutation rate of a specific locus and needs probes with various labels; meanwhile, the invention changes the operation step of extracting nucleic acid DNA as a template by conventional PCR, and directly takes leucocyte, urine sediment, saliva sediment and hair follicle as the template to obtain good results, thereby simplifying the detection time; the accuracy of heterogeneity detection of mt.3243A > G sites is improved by preparing the double standard yeast; the invention establishes a strict detection limit range, namely 4-4.5% of mutation rate can be effectively detected.

Drawings

FIG. 1 shows an alternative human mitochondrial genome 3243 site wild-type sequencing result according to an embodiment of the present invention;

FIG. 2 shows an alternative 3243 site mutant sequencing result of the human mitochondrial genome according to the embodiment of the invention;

FIG. 3 is an amplification curve of an alternative different concentration gradient standard according to embodiments of the present invention;

FIG. 4 is a standard curve of an alternative VIC-labeled probe in accordance with embodiments of the present invention;

FIG. 5 is a standard curve of an alternative FAM-labeled probe according to embodiments of the present invention;

FIG. 6 is an alternative amplification curve for qPCR detection using leukocytes as templates according to embodiments of the present invention;

FIG. 7 is an alternative amplification curve for qPCR detection using hair follicles as templates in accordance with embodiments of the present invention;

FIG. 8 is an amplification curve of an alternative qPCR assay using urine sediment as a template in accordance with embodiments of the present invention.

The following detailed description is intended to further illustrate but not limit the invention, the following example being only one preferred embodiment of the invention.

Detailed Description

Example 1

Extraction of genomic DNA from peripheral blood or tissue

1. Isolation of peripheral blood leukocytes

(1) Taking 5mL of elbow venous blood of a patient, carrying out EDTA anticoagulation, and centrifuging at 2500 rpm for 10 min;

(2) carefully remove the upper plasma, add 3 times the volume of erythrocyte lysate to the lower blood cell pellet, shake well, ice-wash for 15 min.

(3) Centrifuging at 2500 rpm for 10min, discarding the supernatant, and repeating the step 2.

(4) Centrifuging at 3000 rpm for 10min, and discarding the supernatant to obtain leukocyte.

2. Extraction of DNA from leukocytes or tissues

(1) Taking leukocyte, adding 600 μ L STE lysis buffer, 10 μ L proteinase K and 2 μ L RNase A, and digesting at 55 deg.C for 20min (tissue DNA is extracted similarly to leukocyte, and is cut into pieces with scissors before digestion);

(2) cooling the cracking liquid to room temperature, adding 600 mu L of Tris-saturated phenol, mixing uniformly, centrifuging at the temperature of 4 ℃ for 10min at 15000g (-12000 rpm), and adding the upper layer liquid into another 1.5mL centrifuge tube;

(3) adding equal volume of Tris-saturated phenol/chloroform/isoamyl alcohol (25/24/1) into the upper layer liquid, reversing and mixing evenly, centrifuging for 10min at the temperature of 4 ℃ and 15000g (-12000 rpm), and then taking the upper layer liquid and adding into another 1.5mL centrifuge tube;

(4) adding equal volume of chloroform/isoamyl alcohol (24/1) into the upper layer liquid, reversing and mixing evenly, centrifuging for 10min at the temperature of 4 ℃ at 15000g (-12000 rpm), and then taking the upper layer liquid and adding into another 1.5mL centrifuge tube;

(5) adding 0.1 volume times of 3M sodium acetate (pH 5.2) and 3 volumes of precooled absolute ethyl alcohol into the upper layer liquid, and shaking up gently to see white floccules;

(6) carefully pick up white flocculent DNA, add to 1.5mL centrifuge tube containing 1mL 75% ethanol, 15000g (-12000 rpm), centrifuge at 4 ℃ for 10min, carefully discard the supernatant, retain the white precipitate;

(7) naturally drying, and dissolving with appropriate amount of sterilized water or TE;

(8) and (3) carrying out accounting quantification and electrophoretic analysis on the completely dissolved genome sample by using a Nanodrop spectrophotometer, and diluting the working solution according to requirements and freezing and storing at-20 ℃.

Secondly, treatment of other forms of template, i.e. treatment of tissue or cells prior to extraction of nucleic acids

1. Anticoagulation and leucocyte

The method is the same as the above, and anticoagulant is directly and uniformly mixed to be used as a template; after counting leukocytes, the cells were used as templates (about 10)⁴μ L/μ L).

2. Random urine sediment

Random urine (>5mL) of the individual to be tested was collected, centrifuged at 8,000rpm/min for 15min, and then the supernatant raw urine was discarded, and the urine pellet was taken as a template.

3. Hair follicle tissue

Sampling and sampled personnel carry out strict aseptic operation, hair follicle tissues of the head of the sampled personnel are pulled out and are respectively packed in a 1.5mL centrifugal tube, and then the experiment is carried out.

4. Saliva precipitation

Rinsing with clear water 30min before sampling, collecting 2mL saliva sample 30min after 30min, centrifuging at 12,000rpm/min for 3min, discarding supernatant, and collecting saliva precipitate as template.

Third, gene cloning and vector construction

PCR amplification

The reaction system and the thermal cycling conditions of the different DNA polymerases were determined according to the respective instructions, and the following was the method of using the high fidelity polymerase Q5 from NEB:

the non-nucleic acid template may be a leukocyte, a urine sediment, a saliva sediment, or a hair follicle.

The primer sequence is shown as SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification.

The reaction condition is that the pre-denaturation is carried out for 30s at the temperature of 98 ℃; denaturation at 98 ℃ for 10s, annealing at 50-72 ℃ for 20s, extension at 72 ℃ (30s/kb), and 25-35 cycles; final extension at 72 deg.C for 2 min; storing at 4 ℃.

Recovery of PCR amplification product and digestion fragment

2.1 direct purification using the easy pure PCR purification cassette (all gold):

(1) adding 50-100 mu L of PCR product into BB with the volume 5 times of that of the PCR product, uniformly mixing, adding into a centrifugal column, standing for 1min, centrifuging at 10000 g at room temperature for 1min, and discarding the effluent;

(2) adding 650 μ L of WB solution, centrifuging at 10000 g at room temperature for 1min, and discarding the effluent;

(3) centrifuging at room temperature of 10000 g for 1-2 min, and removing residual WB solution;

(4) placing the centrifugal column into a clean centrifugal tube, adding 30-50 mu L EB (preheating at 65 ℃), standing at room temperature for 1min, centrifuging at 10000 g for 1min, and storing the eluted DNA at-20 ℃.

2.2 gel cutting and purification using agarose gel DNA recovery kit:

(1) carrying out agarose electrophoresis on the PCR product or the enzyme digestion product, cutting a single-purpose DNA band from agarose gel after the bands are obviously separated, putting the cut single-purpose DNA band into a clean centrifugal tube, and weighing and calculating the weight of the gel;

(2) adding 1 volume of Buffer PG (the gel weight is 100mg, and the volume can be regarded as 100 μ L) into the gel block, and dissolving the gel block in a water bath at 55 ℃;

(3) column balancing, adding 200 μ L Buffer PS into adsorption column (Spin Columns DM) loaded in the collection tube, centrifuging at 13000 rpm for 1min, discarding waste liquid, and placing the adsorption column back into the collection tube;

(4) adding the solution obtained in the step (2) into an adsorption column, standing at room temperature for 2min, centrifuging at 13000 rpm for 1min, discarding the waste liquid, and placing the adsorption column back into the collection pipe;

(5) adding 450 μ L Buffer PW into the adsorption column, centrifuging at 13000 rpm for 1min, discarding the waste liquid, placing the adsorption column back into the collection tube, and repeating again;

(6) centrifuging at 13000 rpm for 1min, and discarding the filtrate;

(7) placing the adsorption column in a clean centrifuge tube, adding 30-50 μ L Buffer EB (preheated at 65 ℃), standing at room temperature for 2min, centrifuging at 13000 rpm for 1min, and storing the eluted DNA at-20 ℃.

3. Ligation reaction

Ligation of pEASY-Blunting vector (all-type gold):

the linking system is as follows:

and (3) lightly mixing the reaction solution, and reacting for 10-15 min at 25 ℃. After the reaction was completed, the centrifuge tube was placed on ice.

4. Transformation of

(1) The competent cells (50. mu.L) were removed from the freezer at-80 ℃ and thawed on ice, 5. mu.L of the ligation product was added thereto and ice-cooled for 30 min;

(2) carrying out water bath heat shock at 42 ℃ for 30s, immediately placing on ice for 1-2 min, adding 700 mu L of LB liquid culture medium, and oscillating and reviving at 37 ℃ for 60 min;

(3) centrifuging at 4000 rpm for 5min, discarding 650 μ L of supernatant, blowing uniformly, spreading on LB plate containing 0.1% (V/V) ampicillin or kanamycin, and culturing at 37 deg.C for 10-15 h.

5. Colony PCR identification

(1) Picking the transformed monoclonal colonies by using a sterilized white gun head, scribing on a fresh antibiotic-containing plate, rinsing the white gun head in a prepared PCR reaction solution, and taking out;

(2) placing the PCR reaction solution on a PCR instrument for PCR reaction amplification;

(3) and (3) carrying out electrophoresis detection on the product by using 1-2% agarose, identifying a positive monoclonal colony, carrying out sequencing identification after amplification culture, and using the sequencing-free bacterial liquid for the next plasmid extraction.

6. Plasmid extraction

Plasmid DNA miniprep:

(1) adding 1-5 mL of overnight cultured bacterial liquid into a centrifuge tube, centrifuging at 13000 rpm for 30s, collecting bacterial precipitates, and discarding the supernatant as much as possible;

(2) adding 250 mu L of Buffer P1 (containing RNase A) into the centrifuge tube with the thallus precipitate, and fully and uniformly mixing by using a pipette or a vortex oscillator;

(3) adding 250 mu L of Buffer P2 into a centrifuge tube, gently turning upside down and uniformly mixing for 4-6 times, and standing for 1-2 min until the solution is clear and viscous;

(4) adding 350 mu L of Buffer N3 into a centrifuge tube, immediately and gently inverting and uniformly mixing for 8-10 times, standing for 5min, and centrifuging for 10min at 13000 rpm;

(5) transferring the supernatant obtained in the step 3 into an adsorption column (Spin Columns DM) filled in a collecting tube, centrifuging at 13000 rpm for 30s, and discarding waste liquid;

(6) adding 500 mu L Buffer PB into the adsorption column, centrifuging at 13000 rpm for 30s, and discarding waste liquid;

(7) adding 700 μ L Buffer PW into the adsorption column, centrifuging at 13000 rpm for 1min, discarding the waste liquid, and repeating again;

(8) centrifuging at 13000 rpm for 1min, and discarding the filtrate;

(9) placing the adsorption column in a clean centrifuge tube, adding 50-100 μ L Buffer EB (preheated at 65 ℃), standing at room temperature for 2min, centrifuging at 13000 rpm for 1min, carrying out accounting quantification on the eluted plasmid, and storing at-20 ℃.

PCR-RFLP identification

And (3) carrying out enzyme digestion identification on the PCR product by using Apa I, wherein an enzyme digestion reaction system is as follows:

the DNA fragment was digested at 25 ℃ for 2 hours and fractionated by 2% agarose gel electrophoresis.

Site-directed mutagenesis (overlap PCR method)

Designing corresponding mutation primers aiming at specific mutation positions, amplifying by using a conventional PCR technology to obtain upstream and downstream target fragments containing mutation sites, carrying out fusion PCR reaction after the target fragments are respectively recovered, wherein the first step reaction system comprises the following steps:

the Q5 enzyme was subjected to 10 cycles of amplification, and the second reaction was as follows:

and carrying out 33 circulation reactions on the Q5 enzyme, and carrying out vector construction after the product is recovered and purified by glue.

The site-directed mutagenesis primer is shown as SEQ ID NO: 5 and SEQ ID NO: as shown at 64.

The constructed vector is used for PCR-RFLP and sequencing identification, the sequencing result of the 3243 locus wild type sequence of the human mitochondrial genome is shown in figure 1, and the 3243 locus wild type sequence of the human mitochondrial genome is shown in SEQ ID NO:1 is shown in the specification; the sequencing result of the 3243 site mutant of the human mitochondrial genome is shown in figure 2, and the 3243 site mutant sequence of the human mitochondrial genome is shown as SEQ ID NO: 2, respectively.

Fifth, preparation of standard substance

(1) Diluting wild type plasmids and mutant plasmids containing 3243 site fragments of mitochondrial genome into the same concentration;

(2) 5 mu g of plasmid is respectively taken to be mixed in equal mass, and the mixture is used as stock solution of a standard substance and marked as No. 1;

(3) taking 7 Ep tubes with the volume of 0.2mL, respectively marking the Ep tubes as No. 2, No. 3, No. 4, No. 5, No. 6, No. 7 and No. 8, and adding 40 mu L of deionized water into each tube;

(4) sucking 10 mu L of the stock solution of the standard substance in the step 2, adding the stock solution of the standard substance into a No. 2 tube, after oscillating and mixing uniformly, sucking 10 mu L of the stock solution of the standard substance, adding the stock solution of the standard substance into a No. 3 tube, and so on, and diluting the standard substance by 5 times until reaching a No. 8 tube;

(5) 8 tubes of standard were carefully stored under the same conditions.

The above standard relates to the calculation of plasmid copy number, which is calculated as follows:

copy number (copies/mL) ═ plasmid concentration (g/mL) × avogalois constant/plasmid molecular mass;

plasmid molecular mass-average molecular mass of one base pair X number of bases

Sixthly, carrying out SNP analysis and detection by real-time quantitative PCR technology

1. Quantitative PCR (qPCR)

The reaction system is as follows:

the template in the reaction system is tissue or cell, and may be leucocyte, urine sediment, saliva sediment or hair follicle.

Selecting a TaqMan probe method during program design, and selectively adding the required fluorescence under the reaction condition of pre-denaturation at 94 ℃ for 30 s; at 94 ℃ for 5s and at 60 ℃ for 30s, for 40 cycles. Each sample was replicated in 3 wells. The fluorescence quantitative PCR instrument automatically obtains the threshold value cycle number (Ct) of the wild type and the mutant type according to different fluorescence labeling intensities, calculates the difference (delta Ct), then carries out standardization with the DNA solution with the known mutation rate of 50 percent to obtain the delta Ct, and the ratio of the mutation type in the stock solution to the wild type DNA copy number is 2^-ΔΔCtFrom this, the ratio of mutant heterogeneity can be derived.

The quantitative primer of the 3243 site of the mitochondrial genome is shown as SEQ ID NO 7 and SEQ ID NO 8 in the sequence table, and the probe sequence is shown as SEQ ID NO 9 and SEQ ID NO 10 in the sequence table.

Dose-dependent validation of qPCR probes

In order to more truly reflect the combination efficiency of wild type and mutant type probes, the qPCR probe used in the invention is subjected to dose-dependent verification, namely the standard curve is constructed, the detection method comprises the steps of taking prepared 8 concentration gradient standard products as templates, carrying out quantitative PCR experiments by using the probes to be verified, establishing the standard curves of gene copy number (Log value) and corresponding Ct value by taking the amplification curves of the different concentration gradient standard products as shown in figure 3, establishing the standard curves of gene copy number (Log value) and corresponding Ct value by taking the standard curve of VIC labeled probe as shown in figure 4, taking the standard curve of FAM labeled probe as shown in figure 5, and passing through R to obtain the standard curve²The values reflect the effectiveness of the different probes. Experimental analysis of non-nucleic acid templates

(1) Taking healthy human white blood cells as a template directly, adding 4 muL, 3 muL, 2 muL and 1 muL of white blood cells into a 10 muL amplification system respectively for qPCR detection (after cell counting, the number of the white blood cells of a sample to be detected is 3-7 multiplied by 10)⁴μ L/μ L);

(2) taking random urine sediment (from 5mL of original urine) of healthy people as a template directly, and adding 4 muL, 3 muL, 2 muL and 1 muL of random urine sediment into a 10 muL amplification system respectively to carry out qPCR detection;

(3) directly taking healthy human hair follicle tissues as templates, and respectively adding 4, 3, 2 and 1 hair follicle tissues in a 10 mu L amplification system to carry out qPCR detection;

the results show that:

(1) in a 10 mu L amplification system, taking white blood cells (1 mu L-4 mu L) or random urine sediment (1 mu L-3 mu L) as a template to carry out qPCR detection, wherein an amplification curve with good quality can be obtained, the corresponding Ct value is also in a proper ideal range, the amplification curve of the qPCR detection by taking the white blood cells as the template is shown in figure 6, the amplification curve of the qPCR detection by taking the urine sediment as the template is shown in figure 8, wherein the variation in batches is more than or equal to 0.27 and less than or equal to 7.53 in CV batches;

(2) in a 10 mu L amplification system, qPCR detection is carried out by taking hair follicle tissues (1-4 roots) as a template, an amplification curve is relatively intact, the amplification curve of the qPCR detection by taking the hair follicles as the template is shown in figure 7, and the corresponding Ct value is also in a proper ideal range.

It can be seen from the above embodiments that the technical solution of the present invention changes the operation steps of conventional PCR extraction of nucleic acid DNA as a template, and directly uses leukocytes, urine sediment or hair follicles as a template to obtain a good result, and particularly, directly simplifies the template of a reaction system in SNP analysis and detection by real-time quantitative PCR technology into a non-nucleic acid template, which can be leukocytes, urine sediment or hair follicles, thereby obtaining unexpected effects and using nucleic acid as a template, and greatly simplifying the detection duration.

Sequence listing

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Claims (8)

1. A method for detecting heterogeneity of the mitochondrial genome at position a3243G for non-disease diagnostic purposes, the method comprising:

the method comprises the following steps:

a) preparing a standard product of a wild type vector and a mutant vector containing the fragment of the mitochondrial genome at the A3243G site;

b) directly taking a non-nucleic acid template as a template to be detected, carrying out Single Nucleotide Polymorphism (SNP) analysis and detection on the site A3243G by using a real-time quantitative Polymerase Chain Reaction (PCR) technology, and labeling by using a minor groove binder probe (MGB probe);

the non-nucleic acid template comprises a leukocyte, a urine sediment, or a hair follicle.

2. The method according to claim 1, wherein the step a) further comprises:

cloning a fragment gene containing mitochondrial genome site A3243G and constructing a vector; and carrying out site-directed mutagenesis by using an overlapping polymerase chain reaction to obtain a mutation position, and designing a mutation primer of the mutation position.

3. The method of claim 1, wherein the polymerase chain reaction technique is a quantitative polymerase chain reaction technique labeled with minor groove binder probe (MGB probe), and/or the vector of the standard is a plasmid or a T-vector.

4. The method according to claim 3, wherein the vector for the standard is a cloning vector propagated in Escherichia coli.

5. The method according to claim 4, wherein the vector for the standard is pEasy-Blunting vector.

6. The method according to claim 1, wherein the standards for the wild-type and the mutant-type vectors are prepared to have the same concentration, and then mixed in equal volumes to prepare the stock solution of the standard, and then the stock solution of the standard is diluted 5 to 10-fold to 6 to 8 concentration gradients.

7. The method of claim 6, wherein the stock solution of the standard is diluted 5-fold to 8 concentration gradients.

8. The method according to claim 1, wherein the wild-type probe is labeled with 5' -end green fluorescent protein (VIC), the mutant-type probe is labeled with 5' -end carboxyfluorescein (FAM), and the wild-type probe and the mutant-type probe are each labeled with a minor groove binder probe (MGB probe) at their 3' -ends; and/or

When the real-time quantitative polymerase chain reaction technology is used for carrying out Single Nucleotide Polymorphism (SNP) analysis and detection on the A3243G locus in the step b), the used template is a non-nucleic acid template;

the non-nucleic acid template comprises a leukocyte, a urine sediment, or a hair follicle.

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