CN112111568A - Kit and method for detecting mitochondrial 3243 locus genotype - Google Patents
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Abstract
A kit for detecting the genotype of a mitochondrial 3243 locus comprises a PCR primer and a Taqman-MGB probe, wherein the PCR primer is used for amplifying the 3243 locus of a mitochondrial ATP6 gene. The fluorescent quantitative PCR method is the most widely applied detection means in the field of molecular diagnosis, has high sensitivity and simple and convenient operation, and is suitable for large-scale clinical application. The minor groove binder on the MGB probe can improve the Tm value of the probe, is shorter than the ordinary Taqman probe in design, has no background fluorescence, has the capability of distinguishing the difference of one base and is very suitable for SNP typing detection.
Description
Technical Field
The invention relates to a kit and a detection method for detecting mitochondrial 3243 locus genotype, belonging to the technical field of gene detection.
Background
Leigh Syndrome (LS) is one of the most common mitochondrial diseases in infancy and childhood and is characterized by bilaterally symmetric necrotic lesions of the brainstem, basal ganglia, thalamus and spinal cord. The inheritance patterns of LS include autosomal recessive inheritance, X-linked recessive inheritance and maternal inheritance, because the oxidative phosphorylase components in mitochondria are jointly encoded by the nuclear and mitochondrial genomes. According to foreign research data, mitochondrial genome mutation accounts for about 10% of the etiology of Leigh syndrome. Among them, point mutations are most common, and few are caused by insertions and deletions of large fragments, and 19 mitochondrial gene mutations related to Leigh syndrome have been reported so far. The mutation of the mitochondrial ATP6 gene 3243T > G is proved to cause the Leigh syndrome in Chinese, and has important significance in prenatal diagnosis and screening of mitochondrial diseases.
The conventional methods for detecting gene polymorphism include direct DNA sequencing, restriction fragment length polymorphism analysis (PCR-PFLP), high resolution melting curve (HRM), gene chip, liquid chip method, and fluorescent quantitative PCR. The sequencing method is a recognized gold standard for gene mutation detection, can accurately display the possible specific mutation types, but has the disadvantages of high equipment cost, long detection time, complex operation, low sensitivity, complex result interpretation, high requirement on operators and difficulty in forming commercial diagnostic products. Restriction fragment length polymorphism analysis has many steps and low sensitivity, and the detection result often needs to be confirmed by a sequencing method, so the method is not a simple, convenient and effective method suitable for clinical detection. The high-resolution melting curve is a new tool for detecting gene mutation, genotyping and SNP detection, which is emerging in recent years, and can rapidly detect single-base mutation in a nucleic acid fragment. However, the method is difficult to screen homozygous mutation, has high false positive and false negative, harsh PCR conditions and incapability of typing, and the detection result needs to be confirmed by a sequencing method. Although the gene chip and the liquid chip can accurately distinguish specific mutation types, the gene chip and the liquid chip have high requirements on equipment, are difficult to popularize on a large scale and are mostly used in scientific research units.
The mitochondrial DNA mutation analysis of Leigh syndrome, published in the journal of the Chinese neurology, 2003, Vol.2, No. 36, No. 1, uses a probe which is a fragment of mtDNA from nucleotides 1 to 740. And the result judgment depends on the enzyme cutting result of the endonuclease Apa I, and the final judgment is carried out without carrying out DNA sequencing, so that whether the primer using the method achieves the purpose of detection cannot be judged. Therefore, it is necessary to design a kit and a method for detecting the genotype of the mitochondrial 3243 locus.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a kit and a method for detecting the genotype of a mitochondrial 3243 locus.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a kit for detecting the genotype of a mitochondrial 3243 locus comprises a PCR primer and a Taqman-MGB probe, wherein the PCR primer is used for amplifying the 3243 locus of a mitochondrial ATP6 gene;
the primer comprises: an upstream primer F: 5'-CCACACCCACCCAAGAACAG-3', respectively;
a downstream primer R: 5' -GGGTATGTTGTTAAGAAGAGGAATTGA-3;
the probe includes:
Taqman-MGB probe PA 5 '-FAM-TTTGTTAAGATGGCAGAGC-MGB-3';
the Taqman-MGB probe PG is 5 'VIC-TTGTTAAGATGGCAGGGC-MGB-3'.
The preferable technical scheme is as follows: also comprises a PCR premix solution, wherein the PCR premix solution contains PCR buffer solution, Taq DNA polymerase and MgCl2And dNTPs.
The preferable technical scheme is as follows: contains a positive control substance, and the positive control substance comprises a mitochondrial ATP6 gene 3243 site AA type plasmid and a mitochondrial ATP6 gene 3243 site GG type plasmid.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for detecting a mitochondrial 3243 locus genotype comprising the steps of:
step 1: extracting DNA of a sample to be detected;
step 2: adding a PCR premix, a primer, a probe, sterile ultrapure water and the template DNA extracted in the step (1) into a PCR reaction tube to obtain a reaction system; meanwhile, establishing an AA type control tube, a GG type control tube, an AG type control tube and a blank control tube; replacing the template DNA extracted in the step 1 with an AA type plasmid at 3243 locus of mitochondrial ATP6 gene in the AA type control tube; the GG type control tube replaces the template DNA extracted in the step 1 with GG type plasmid at 3243 site of mitochondrial ATP6 gene; replacing the template DNA extracted in the step 1 by a mixture of an AA type plasmid at the 3243 site of a mitochondrial ATP6 gene and an GG type plasmid at the 3243 site of a mitochondrial ATP6 gene in the AG type control tube; the blank control tube uses sterile ultrapure water to replace the template DNA extracted in the step 1;
the primer comprises: an upstream primer F: 5'-CCACACCCACCCAAGAACAG-3', respectively;
a downstream primer R: 5' -GGGTATGTTGTTAAGAAGAGGAATTGA-3;
the probe includes:
Taqman-MGB probe PA 5 '-FAM-TTTGTTAAGATGGCAGAGC-MGB-3';
5 'VIC-TTGTTAAGATGGCAGGGC-MGB-3' of Taqman-MGB probe PG;
and step 3: after mixing the reaction system, carrying out amplification on a fluorescent real-time quantitative PCR instrument, and carrying out fluorescent PCR amplification;
and 4, step 4: and (3) judging the result according to the amplification conditions of the FAM channel and the HEX channel, judging that the sample is of an AA type if only the FAM channel has an amplification S-type curve, judging that the sample is of a GG type if only the VIC channel has an amplification S-type curve, and judging that the FAM channel and the VIC channel both have S-type curves.
The preferable technical scheme is as follows: the fluorescent PCR amplification conditions are as follows: 95 ℃/5min, 95 ℃/15sec, 60 ℃/1min, with 40 cycles in the range of 95 ℃/15sec to 60 ℃/1 min.
The preferable technical scheme is as follows: the PCR premix contains PCR buffer solution, Taq DNA polymerase and MgCl2And dNTPs.
The preferable technical scheme is as follows: mitochondrial ATP6 gene 3243 site AA-type plasmid and mitochondrial ATP6 gene 3243 site GG-type plasmid were as follows: 1 in a mass ratio to substitute for the template DNA extracted in step 1.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
the fluorescent quantitative PCR method is the most widely applied detection means in the field of molecular diagnosis, has high sensitivity and simple and convenient operation, and is suitable for large-scale clinical application. The minor groove binder on the MGB probe can improve the Tm value of the probe, is shorter than the ordinary Taqman probe in design, has no background fluorescence, has the capability of distinguishing the difference of one base and is very suitable for SNP typing detection.
Drawings
FIG. 1 is a schematic diagram of the detection step.
FIG. 2 shows the results of amplification at an annealing temperature of 56-61 ℃.
FIG. 3 shows the result of DNA template amount gradient dilution amplification of blood samples.
FIG. 4 shows the genotype detection of real samples by fluorescent quantitative PCR.
FIG. 5 shows the detection and copy number quantification of actual sample genotypes by digital PCR.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-4. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1: kit and method for detecting mitochondrial 3243 locus genotype
First, embryo biopsy procedure
Preparation of biopsy solution and operating dish
Biopsy solution: polar body (fertilization culture solution), cleavage stage embryo (cleavage culture solution), and blastocyst (blastocyst culture solution).
Biopsy operating dish: falcon351006 dish, bottom of dish labeled with patient name. 3 10 microliter biopsy droplets and 110 microliter PVP droplet were made, sealed in oil, placed at 37 ℃ with 5% CO2，5％O2，90％N2The incubator is ready for use.
Embryo culture dish after biopsy: nunclon108173 dish, using balanced culture solution (polar body-fertilization culture solution, cleavage embryo-cleavage solution, blastocyst-blastocyst) to make several (according to the number of live embryo) 35ul culture drops, sealing oil, and placing in an incubator for later use.
Thirdly, embryo biopsy operation steps
And opening the switch of the inverted mirror and the micromanipulation system. The biopsy needle and the fixed needle were mounted under a 4 × objective, the needle tip was moved to the center of the field of view, and fine adjustment was performed under a 20 × objective.
Perforating the transparent tape: embryo biopsy involves two basic processes, zona pellucida perforation and blastomere or polar body acquisition. The perforating method of the transparent tape comprises the following steps: laser drilling (laser Membrane-breaking apparatus, Hamilton Thorne, USA).
Pole body biopsy: fixing the pole body at the position of 12 points, punching holes (laser pulse is 200 microseconds, intensity is 100%) at the horizontal position of the right side of the pole body, horizontally entering a flat embryo biopsy needle (ID 15 mu m) along the opening of the zona pellucida, focusing to the pole body, and slowly sucking out the pole body.
Cleavage embryo biopsy: placing the blastomere to be detected at the position of 3 points, perforating the zona pellucida at the right side by laser (laser pulse is 400 microseconds, intensity is 100%), entering the embryo by an embryo biopsy needle (ID 30 microns) along the zona pellucida break, sucking the polar body or the blastomere by the biopsy needle, and slightly sucking the blastomere.
And (3) blastocyst biopsy: taking blastocysts which develop to the stage 4 on the 5 th to 7 th days after ova, placing inner cell masses at the upper and lower positions of 9 points, carrying out biopsy on trophoblast cells at the position of 3 points, and carrying out laser perforation on a zona pellucida at the position of 3 points (laser pulse is 400 microseconds, and the intensity is 100%); when the blastocyst is shrunk, sucking a small amount of trophoblast cells (about 10-15 cells) by a biopsy needle (ID 20 mu m), and slowly pulling the ovum holding needle and the biopsy needle to the two sides; the laser continues to fire (laser pulses 700 microseconds, 100% intensity) aiming at the intercellular junctions between the trophoblast cells that are pulled apart, while continuing to gently pull the blastocyst and biopsied trophoblast cells sideways until the trophoblast cells separate from the blastocyst. .
The biopsied polar body/blastomere/trophoblast cells were transferred into a PCR tube containing 4. mu.l PBS using a drawn Pasteur tube for DNA extraction and digital PCR detection, and the biopsied ovum or embryo was transferred into the corresponding culture medium for further culture.
Fourth, extracting DNA of sample to be detected
Blood sample DNA extraction: extracting by using a magnetic bead method nucleic acid extraction kit, measuring the OD value of the extracted DNA, and storing at-20 ℃.
Extracting 10 parts of blastula DNA: extracting by using a REPLI-g Singel Cell Kit, adding stop buffer, and storing at-20 ℃.
Fifthly, a kit is adopted for operation, and the kit for detecting the 3243 locus genotype of the human mitochondrial ATP6 gene and the preparation method are characterized in that: the kit comprises a primer, a probe, a PCR premix solution, a standard reference substance and sterile water.
The PCR primer and the Taqman-MGB probe are used for amplifying 3243 locus of mitochondrial ATP6 gene.
An upstream primer F: 5'-CCACACCCACCCAAGAACAG-3', respectively;
a downstream primer R: 5'-GGGTATGTTGTTAAGAAGAGGAATTGA-3'
Taqman-MGB probe PA 5 'FAM-TTTGTTAAGATGGCAGAGC-MGB-3'
5 'VIC-TTGTTAAGATGGCAGGGC-MGB-3' of Taqman-MGB probe PG;
the PCR premix contains PCR buffer solution, Taq DNA polymerase, MgCl2 and dNTPs.
The positive control product comprises a mitochondrial ATP6 gene 3243 site AA type plasmid and a mitochondrial ATP6 gene 3243 site GG type plasmid. The AA-type plasmid and the GG-type plasmid were synthesized by Nanjing King sry Biotech Ltd.
Sixthly, a fluorescent quantitative PCR amplification system and conditions:
the following reaction system is configured: adding 10 mu L of PCR premix, 1 mu L of upstream primer F (10 pmol/. mu.L), 1 mu L of downstream primer R (10 pmol/. mu.L), 0.5 mu L of Taqman-MGB probe PA (10 pmol/. mu.L), 0.5 mu L of Taqman-MGB probe PG (10 pmol/. mu.L), 1 mu L of extracted template DNA and 6 mu L of sterile ultrapure water into a PCR reaction tube by adopting a 20 mu L reaction system;
amplification conditions: 95 ℃/5min, 95 ℃/15sec, 60 ℃/1min, with 40 cycles in the range of 95 ℃/15sec to 60 ℃/1 min.
(1) Extracting DNA of a sample to be detected;
(2) adding 10 mu L of PCR premix, 1 mu L of upstream primer F (10 pmol/. mu.L), 1 mu L of downstream primer R (10 pmol/. mu.L), 0.5 mu L of Taqman-MGB probe PA (10 pmol/. mu.L), 0.5 mu L of Taqman-MGB probe PG (10 pmol/. mu.L), 1 mu L of extracted template DNA, 6 mu L of sterile ultrapure water into a PCR reaction tube, and adding 20 mu L of reaction system in total; amplifying two tubes of DNA of each sample to be detected in parallel, and simultaneously carrying AA type, GG type and AG type control tubes and blank control tubes; the AA type and GG type control tubes respectively use synthesized plasmid DNA to replace template DNA, and the blank control tube uses sterile ultrapure water to replace the template DNA; the AG type control tube adopts AA type and GG type plasmid DNA to replace template DNA after being mixed according to the mass ratio of 1: 1;
(3) after the reaction system is well mixed, the fluorescent PCR amplification conditions are as follows: 95 ℃/5min, 95 ℃/15sec, 56 ℃/1min, with 40 cycles in the course of 95 ℃/15sec to 60 ℃/1 min.
Fourthly, constructing a digital PCR amplification system and conditions
The following reaction system is configured: adopting a 25 mu L reaction system, adding 13 mu L ddPCR premix, 1 mu L upstream primer F (10 pmol/. mu.L), 1 mu L downstream primer R (10 pmol/. mu.L), 0.5 mu L Taqman-MGB probe PT (10 pmol/. mu.L), 0.5 mu L Taqman-MGB probe PG (10 pmol/. mu.L), 1 mu L extracted template DNA and 8 mu L sterile ultrapure water into a PCR reaction tube; using a digital PCR dedicated droplet generation plate, add 70 ul/well droplet generation oil, in a droplet generator prepared as a water-in-oil solution, and then transfer to 96-well plate.
Amplification conditions: the first stage, 50 ℃/5min, 95 ℃/5 min; in the second stage, the temperature is 95 ℃/15sec, 56-61 ℃/1min, and 45 cycles are carried out; the third stage, storage at 98 deg.C/10 min and 4 deg.C.
And judging that the sample is of an AA type when the FAM channel has an amplification S-type curve, judging that the sample is of a GG type when only the VIC channel has an amplification S-type curve, and judging that the sample is of an AG type when both the FAM channel and the VIC channel have S-type curves.
Seventhly, the genotype detection of the actual sample by the fluorescent quantitative PCR is shown in figure 4.
The fluorescent quantitative PCR can not accurately quantify the copy number of the A and G genotypes because a standard curve is not constructed, but can distinguish which genotype accounts for more than 50% through the CT value.
Eighth, the detection of actual sample genotypes and copy number quantification by digital PCR are shown in fig. 5.
The digital PCR can accurately quantify the copy numbers of the A and G genotypes without a standard curve, the copy numbers of the A and G genotypes and the A/(A + G) proportion in each blastocyst and polar body sample are shown in the table, and in addition, the copy numbers can be compared with the detection result of the fluorescent quantitative PCR, so that the detection results of the two methods are roughly classified and consistent, and the established two methods can effectively and accurately quantify the copy numbers of the A and G genotypes of the mtDNA 3243 site in the blood, the blastocyst and the polar body sample.
Example 2: kit and method for detecting mitochondrial 3243 locus genotype
A kit for detecting the genotype of a mitochondrial 3243 locus comprises a PCR primer and a Taqman-MGB probe, wherein the PCR primer is used for amplifying the 3243 locus of a mitochondrial ATP6 gene;
the primer comprises: an upstream primer F: 5'-CCACACCCACCCAAGAACAG-3', respectively;
a downstream primer R: 5' -GGGTATGTTGTTAAGAAGAGGAATTGA-3;
the probe includes:
Taqman-MGB probe PA 5 '-FAM-TTTGTTAAGATGGCAGAGC-MGB-3';
the Taqman-MGB probe PG is 5 'VIC-TTGTTAAGATGGCAGGGC-MGB-3'.
The preferred embodiment is: also comprises a PCR premix solution, wherein the PCR premix solution contains PCR buffer solution, Taq DNA polymerase and MgCl2And dNTPs. PCR buffer, TaqDNA polymerase, dNTPs and MgCl2Purchased from Tiangen Biochemical technology Co., Ltd. (Beijing), 10xPCR buffer, 5U/μ l TaqDNA polymerase content, 2.5mM dNTPs content, MgCl2The content was 1.5 mM.
The preferred embodiment is: contains a positive control substance, and the positive control substance comprises a mitochondrial ATP6 gene 3243 site AA type plasmid and a mitochondrial ATP6 gene 3243 site GG type plasmid.
A method for detecting a mitochondrial 3243 locus genotype comprising the steps of:
step 1: extracting DNA of a sample to be detected;
step 2: adding a PCR premix, a primer, a probe, sterile ultrapure water and the template DNA extracted in the step (1) into a PCR reaction tube to obtain a reaction system; meanwhile, establishing an AA type control tube, a GG type control tube, an AG type control tube and a blank control tube; replacing the template DNA extracted in the step 1 with an AA type plasmid at 3243 locus of mitochondrial ATP6 gene in the AA type control tube; the GG type control tube replaces the template DNA extracted in the step 1 with GG type plasmid at 3243 site of mitochondrial ATP6 gene; replacing the template DNA extracted in the step 1 by a mixture of an AA type plasmid at the 3243 site of a mitochondrial ATP6 gene and an GG type plasmid at the 3243 site of a mitochondrial ATP6 gene in the AG type control tube; the blank control tube uses sterile ultrapure water to replace the template DNA extracted in the step 1;
the primer comprises: an upstream primer F: 5'-CCACACCCACCCAAGAACAG-3', respectively;
a downstream primer R: 5' -GGGTATGTTGTTAAGAAGAGGAATTGA-3;
the probe includes:
Taqman-MGB probe PA 5 '-FAM-TTTGTTAAGATGGCAGAGC-MGB-3';
5 'VIC-TTGTTAAGATGGCAGGGC-MGB-3' of Taqman-MGB probe PG;
and step 3: after mixing the reaction system, carrying out amplification on a fluorescent real-time quantitative PCR instrument, and carrying out fluorescent PCR amplification;
and 4, step 4: and (3) judging the result according to the amplification conditions of the FAM channel and the HEX channel, judging that the sample is of an AA type if only the FAM channel has an amplification S-type curve, judging that the sample is of a GG type if only the VIC channel has an amplification S-type curve, and judging that the FAM channel and the VIC channel both have S-type curves.
The preferred embodiment is: the fluorescent PCR amplification conditions are as follows: 95 ℃/5min, 95 ℃/15sec, 60 ℃/1min, with 40 cycles in the range of 95 ℃/15sec to 60 ℃/1 min.
The preferred embodiment is: the PCR premix contains PCR buffer solution, Taq DNA polymerase and MgCl2And dNTPs.
The preferred embodiment is: mitochondrial ATP6 gene 3243 site AA-type plasmid and mitochondrial ATP6 gene 3243 site GG-type plasmid were as follows: 1 in a mass ratio to substitute for the template DNA extracted in step 1.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
<110> first subsidiary hospital of medical university in Anhui province, Hefei customs technical center
<120> kit and method for detecting mitochondrial 3243 locus genotype
<140> 2019114207185
<141> 2019-12-31
<160> 4
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
ccacacccac ccaagaacag 20
<210> 2
<211> 27
<212> DNA
<213> Artificial sequence
<400> 2
gggtatgttg ttaagaagag gaattga 27
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence
<400> 3
tttgttaaga tggcagagc 19
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence
<400> 4
ttgttaagat ggcagggc 18
Claims (7)
1. A kit for detecting the genotype of a mitochondrial 3243 locus, which is characterized in that: comprises a PCR primer and a Taqman-MGB probe for amplifying 3243 locus of mitochondrial ATP6 gene;
the primer comprises: an upstream primer F: 5'-CCACACCCACCCAAGAACAG-3', respectively;
a downstream primer R: 5' -GGGTATGTTGTTAAGAAGAGGAATTGA-3;
the probe includes:
Taqman-MGB probe PA 5 '-FAM-TTTGTTAAGATGGCAGAGC-MGB-3';
the Taqman-MGB probe PG is 5 'VIC-TTGTTAAGATGGCAGGGC-MGB-3'.
2. The kit for detecting the genotype of mitochondrial 3243 site according to claim 1, characterized in that: also comprises a PCR premix solution, wherein the PCR premix solution contains PCR buffer solution, Taq DNA polymerase and MgCl2And dNTPs.
3. The kit for detecting the genotype of mitochondrial 3243 site according to claim 1, characterized in that: contains a positive control substance, and the positive control substance comprises a mitochondrial ATP6 gene 3243 site AA type plasmid and a mitochondrial ATP6 gene 3243 site GG type plasmid.
4. A method for detecting a genotype at a mitochondrial 3243 locus, comprising: comprises the following steps:
step 1: extracting DNA of a sample to be detected;
step 2: adding a PCR premix, a primer, a probe, sterile ultrapure water and the template DNA extracted in the step (1) into a PCR reaction tube to obtain a reaction system; meanwhile, establishing an AA type control tube, a GG type control tube, an AG type control tube and a blank control tube; replacing the template DNA extracted in the step 1 with an AA type plasmid at 3243 locus of mitochondrial ATP6 gene in the AA type control tube; the GG type control tube replaces the template DNA extracted in the step 1 with GG type plasmid at 3243 site of mitochondrial ATP6 gene; replacing the template DNA extracted in the step 1 by a mixture of an AA type plasmid at the 3243 site of a mitochondrial ATP6 gene and an GG type plasmid at the 3243 site of a mitochondrial ATP6 gene in the AG type control tube; the blank control tube uses sterile ultrapure water to replace the template DNA extracted in the step 1;
the primer comprises: an upstream primer F: 5'-CCACACCCACCCAAGAACAG-3', respectively;
a downstream primer R: 5' -GGGTATGTTGTTAAGAAGAGGAATTGA-3;
the probe includes:
Taqman-MGB probe PA 5 '-FAM-TTTGTTAAGATGGCAGAGC-MGB-3';
5 'VIC-TTGTTAAGATGGCAGGGC-MGB-3' of Taqman-MGB probe PG;
and step 3: after mixing the reaction system, carrying out amplification on a fluorescent real-time quantitative PCR instrument, and carrying out fluorescent PCR amplification;
and 4, step 4: and (3) judging the result according to the amplification conditions of the FAM channel and the HEX channel, judging that the sample is of an AA type if only the FAM channel has an amplification S-type curve, judging that the sample is of a GG type if only the VIC channel has an amplification S-type curve, and judging that the sample is of an AG type if both the FAM channel and the VIC channel have S-type curves.
5. The method for detecting the genotype of mitochondrial 3243 site according to claim 4, wherein: the fluorescent PCR amplification conditions are as follows: 95 ℃/5min, 95 ℃/15sec, 60 ℃/1min, with 40 cycles in the range of 95 ℃/15sec to 60 ℃/1 min.
6. The method for detecting the genotype of mitochondrial 3243 site according to claim 4, wherein: the PCR premix contains PCR buffer solution, Taq DNA polymerase and MgCl2And dNTPs.
7. The method for detecting the genotype of mitochondrial 3243 site according to claim 4, wherein: mitochondrial ATP6 gene 3243 site AA-type plasmid and mitochondrial ATP6 gene 3243 site GG-type plasmid were as follows: 1 in a mass ratio to substitute for the template DNA extracted in step 1.
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| CN114085903A (en) * | 2022-01-19 | 2022-02-25 | 广州嘉检医学检测有限公司 | Primer pair probe combination product for detecting mitochondria 3243A & gtG mutation, kit and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1806052A (en) * | 2003-04-16 | 2006-07-19 | 爱科来株式会社 | Method of detecting or quantitatively determining mitochondrial DNA 3243 variation, and kit therefor |
| CN107267614A (en) * | 2017-06-28 | 2017-10-20 | 浙江大学 | The fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation and its application |
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| CN1806052A (en) * | 2003-04-16 | 2006-07-19 | 爱科来株式会社 | Method of detecting or quantitatively determining mitochondrial DNA 3243 variation, and kit therefor |
| CN107267614A (en) * | 2017-06-28 | 2017-10-20 | 浙江大学 | The fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation and its application |
| CN107287302A (en) * | 2017-06-28 | 2017-10-24 | 浙江大学 | The fluorescent quantificationally PCR detecting kit of chondriosome deafness T12201C mutation and its application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114085903A (en) * | 2022-01-19 | 2022-02-25 | 广州嘉检医学检测有限公司 | Primer pair probe combination product for detecting mitochondria 3243A & gtG mutation, kit and detection method thereof |
| CN114085903B (en) * | 2022-01-19 | 2022-04-26 | 广州嘉检医学检测有限公司 | Primer pair probe combination product for detecting mitochondria 3243A & gtG mutation, kit and detection method thereof |
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