WO2021136123A1 - Kit and method for detecting genotype of site 8993 of human mitochondrial atp6 gene - Google Patents

Abstract

A kit for detecting a genotype of a site 8993 of a human mitochondrial ATP6 gene, comprising a PCR primer for amplifying the site 8993 of the mitochondrial ATP6 gene and a Taqman-MGB probe. A fluorescence quantitative PCR method is the most widely applied detection means in the field of molecular diagnosis. The method has high sensitivity, is easy to operate, and is suitable for large-scale clinical applications. In particular, an MGB probe technology of ABI company has a broad market in the aspect of mononucleotide typing. A minor groove binder on an MGB probe can increase the Tm value of the probe, the probe is designed to be shorter than a common Taqman probe, does not have background fluorescence, has the ability of distinguishing a difference of one base, and is suitable for SNP typing and detection.

Description

A kit and method for detecting genotype at 8993 site of human mitochondrial ATP6 gene Technical field

The invention relates to a kit and a method for detecting the genotype of human mitochondrial ATP6 gene at 8993 site, and belongs to the technical field of gene detection.

Background technique

Leigh syndrome (LS) is the most common mitochondrial disease in infancy and childhood. It is characterized by bilateral symmetrical necrotizing lesions of the brainstem, basal ganglia, thalamus and spinal cord. The inheritance of LS includes autosomal recessive inheritance, X-linked recessive inheritance and maternal inheritance. This is because the oxidative phosphorylase components in the mitochondria are coded by the nuclear genome and the mitochondrial genome. According to foreign research data, mitochondrial genome mutations account for about 10% of the etiology of Leigh syndrome. Among them, point mutations are the most common, and very few are caused by insertions and deletions of large fragments. So far, 19 mitochondrial gene mutations related to Leigh syndrome have been reported. Among them, the 8993T>G mutation of the mitochondrial ATP6 gene has been confirmed to cause Leigh syndrome in Chinese, which is of great significance in prenatal diagnosis and screening of mitochondrial diseases.

At present, the commonly used 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 fluorescence quantitative PCR Law and so on. Sequencing is the recognized gold standard for gene mutation detection, which can accurately display the specific types of mutations that may exist. However, the equipment used in the sequencing method is expensive, the detection time is slightly longer, the operation is cumbersome, the sensitivity is low, and the interpretation of the results is complicated. Operators have high requirements, and it is difficult to form commercialized diagnostic products. Restriction fragment length polymorphism analysis has many steps and low sensitivity, and the detection results often need to be confirmed by sequencing methods. It is not a simple and effective method for clinical detection. High-resolution melting curve is a new tool for detecting gene mutations, genotyping and SNP detection that has emerged in recent years. It can quickly detect single-base mutations in nucleic acid fragments. However, this method is difficult to screen for homozygous mutations, and false positives and false negatives are high. PCR conditions are harsh and cannot be typed, and the test results need to be confirmed by sequencing methods. Although gene chips and liquid-phase chips can accurately distinguish specific mutation types, they require high equipment and are difficult to promote on a large scale. They are mostly used in scientific research institutions.

"Analysis of Mitochondrial DNA Mutations in Leigh Syndrome" published in the Chinese Journal of Neurology, Vol. 36, Issue 1, February 2003, uses a probe of the 1-740th nucleotide fragment of mtDNA. And the result judgment depends on the result of endonuclease ApaI digestion, and DNA sequencing is not performed for the final judgment. Therefore, it is impossible to judge whether the primers of this method achieve the purpose of detection. Therefore, it is necessary to design a kit and detection method to detect the genotype of mitochondrial 8993 site.

Summary of the invention

In order to overcome the above-mentioned shortcomings in the prior art, the purpose of the present invention is to provide a kit and method for detecting the genotype of human mitochondrial ATP6 gene at site 8993.

In order to achieve the above and other related purposes, the technical solution provided by the present invention is: a kit for detecting the genotype of human mitochondrial ATP6 gene at 8993 site, characterized in that it includes PCR for amplifying the 8993 site of mitochondrial ATP6 gene Primer and Taqman-MGB probe;

The PCR primers include: upstream primer F: 5'-GTTATTATCGAAACCATCAGCCT-3';

Downstream primer R: 5'-AGTAGGTGGCCTGCAGTAATGT-3';

The probe includes: Taqman-MGB probe PT: 5'VIC-CAATAGCCCGGGCC-MGB-3';

Taqman-MGB probe PG: 5'FAM-CAATAGCCCTGGCC-MGB-3'.

The preferred technical solution is that it also includes a PCR premix, which contains PCR buffer, Taq DNA polymerase, MgCl ₂ and dNTPs.

The preferred technical solution is: a positive control substance is included, and the positive control substance includes a TT-type plasmid at the 8993 site of the mitochondrial ATP6 gene and a GG-type plasmid at the 8993 site of the mitochondrial ATP6 gene.

In order to achieve the above objectives and other related objectives, the technical solution provided by the present invention is: a method for detecting the genotype of mitochondria at site 8993, which includes the following steps:

Step 1: Extract the DNA of the sample to be tested;

Step 2: Add PCR premix, primers, probes, sterile ultrapure water and template DNA extracted in step 1 to the PCR reaction tube to obtain the reaction system; at the same time establish TT type control tube, GG type control tube, and TG type Control tube and blank control tube; the TT-type control tube replaces the template DNA extracted in step 1 with the TT-type plasmid at the 8993 site of the mitochondrial ATP6 gene; the GG-type control tube replaces the GG-type plasmid at the 8993 site of the mitochondrial ATP6 gene The template DNA extracted in step 1; the TG-type control tube uses a mixture of the TT-type plasmid at the 8993 site of the mitochondrial ATP6 gene and the GG-type plasmid at the 8993 site of the mitochondrial ATP6 gene to replace the template DNA extracted in the step 1; the blank control can be used without Bacteria ultrapure water replaces the template DNA extracted in step 1;

The PCR primers include: upstream primer F: 5'-GTTATTATCGAAACCATCAGCCT-3';

Downstream primer R: 5'-AGTAGGTGGCCTGCAGTAATGT-3';

The probe includes: Taqman-MGB probe PT: 5'VIC-CAATAGCCCGGGCC-MGB-3';

Taqman-MGB probe PG: 5′FAM-CAATAGCCCTGGCC-MGB-3′;

Step 3: After mixing the reaction system, perform fluorescent PCR amplification;

Step 4: Determine the result according to the amplification of the FAM channel and the HEX channel. Only the FAM channel has an amplified S-shaped curve, and the sample is judged to be TT type, and only the VIC channel has an amplified S-shaped curve, and the sample is judged to be GG Type, FAM and VIC channels have S-shaped curves, it is judged as TG type.

Due to the application of the above technical solutions, the advantages of the present invention compared with the prior art are:

The fluorescence quantitative PCR method is the most widely used detection method in the field of molecular diagnosis. This method has high sensitivity, simple operation, and is suitable for large-scale clinical applications. In particular, ABI's MGB probe technology is more effective in single nucleotide typing. It has a broad market. The minor groove binder on the MGB probe can increase the Tm value of the probe. It is designed to be shorter than the ordinary Taqman probe, without background fluorescence, and has the ability to distinguish one base difference, which is very suitable for SNP typing detection.

Description of the drawings

Figure 1 is a schematic diagram of the detection steps.

Figure 2 is a synthetic TT type control plasmid.

Figure 3 is a synthetic GG type control plasmid.

Figure 4 is a synthetic TG type control plasmid.

Figure 5 shows the results of the dilution amplification of the amount of DNA template in the blood sample.

Detailed ways

The following specific examples illustrate the implementation of the present invention. Those familiar with the technology can easily understand the other advantages and effects of the present invention from the content disclosed in this specification.

Please refer to Figure 1. It should be noted that the structures, proportions, sizes, etc. shown in the accompanying drawings in this specification are only used to match the content disclosed in the specification for people familiar with this technology to understand and read, and are not intended to limit the implementation of the present invention. Limited conditions, so it has no technical significance. Any structural modification, proportional relationship change or size adjustment should still fall under the present invention without affecting the effects and objectives that can be achieved by the present invention. The disclosed technical content must be within the scope of coverage. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" cited in this specification are only for ease of description, not to limit the text. The scope of implementation of the invention, the change or adjustment of its relative relationship, shall be regarded as the scope of implementation of the invention without substantial changes to the technical content.

Example 1: A kit and method for detecting genotype of human mitochondrial ATP6 gene at site 8993

1. Embryo biopsy procedure

2. Preparation of Biopsy Solution and Operation Dish

Biopsy fluid: polar body (fertilization culture fluid), cleavage stage embryo (cleavage culture fluid), blastocyst (blastocyst culture fluid)

Biopsy operation dish: Falcon351006 dish, the bottom of the dish is marked with the patient's name. Make 3 10ul biopsy drops and 1 10ul PVP drop, seal the oil, and place it in an _{incubator at 37°C, 5% CO 2} , 5% O ₂ , and 90% N _{2 for} later use.

Embryo culture dishes after biopsy: Nunclon 108173 dishes, with balanced culture medium (polar body-fertilization medium, cleavage embryo-cleavage fluid, blastocyst-blastocyst fluid) to make several (according to the number of live embryos) 35 micro Liter of culture drops, sealed with oil, and placed in an incubator for later use.

Embryo biopsy procedure

Turn on the switch of the inverted mirror and the micromanipulation system. Install the biopsy needle and the fixed needle under the 4× objective lens, move the needle tip to the center of the field of view, and perform fine-tuning under the 20× objective lens.

Zona pellucida perforation: Embryo biopsy includes two basic processes: zona pellucida perforation and blastomere or polar body acquisition. Transparent belt drilling method: laser drilling method (laser rupture instrument, American Hamilton Thorne company).

Polar body biopsy: fix the polar body at the 12 o'clock position, drill a hole in the horizontal position on the right side of the polar body (laser pulse 200 microseconds, intensity 100%), and insert the flat embryo biopsy needle (ID 15μm) along the zona pellucida breach horizontally , Adjust the focus to the polar body, and slowly suck out the polar body.

Cleavage embryo biopsy: Place the blastomere to be inspected at the 3 o'clock position, drill a hole in the zona pellucida on the right side (laser pulse 400 microseconds, intensity 100%), and the embryo biopsy needle (ID 30μm) along the zona pellucida The rupture enters the embryo, the polar body or blastomere is sucked through the biopsy needle, and the blastomere is gently sucked out.

Blastocyst biopsy: The blastocysts that have developed to stage 4 on the 5th-7th day after the egg are taken, the inner cell mass is placed at 9 o’clock up and down, the trophoblast cells at 3 o’clock are biopsy, and the zona pellucida is laser perforated at 3 o’clock Pulse 400 microseconds, intensity 100%); When the blastocyst shrinks, suck a small amount of trophoblast cells (about 10-15 cells) with the biopsy needle (ID 20μm), hold the egg needle and the biopsy needle slowly to both sides; Aim at the intercellular connections between the trophoblast cells that have been pulled apart and continuously emit lasers (laser pulse 700 microseconds, intensity 100%), while continuing to gently pull the blastocyst and biopsy trophoblast cells to both sides until the trophoblast The cell is separated from the blastocyst. .

Transfer the polar body/blastomere/trophoblast cells from the biopsy using a drawn Pasteur tube to a PCR tube containing 4μl PBS for DNA extraction and digital PCR detection, and transfer the biopsy egg or embryo into the corresponding culture medium to continue bring up.

3. Extraction of DNA from the sample to be tested

DNA extraction from blood samples: Use magnetic bead method nucleic acid extraction kit to extract the extracted DNA, measure the OD value of the extracted DNA, and store it at -20 degrees.

DNA extraction of 6 blastocyst cells and 4 polar body cells: Use REPLI-g Singel Cell Kit to extract, and store at -20°C after adding stop buffer.

Kit: including primers, probes, PCR master mix, standard reference substance, sterile water.

Said PCR primer and Taqman-MGB probe for amplifying the 8993 site of mitochondrial ATP6 gene.

Upstream primer F: 5'-GTTATTATCGAAACCATCAGCCT-3';

Downstream primer R: 5'-AGTAGGTGGCCTGCAGTAATGT-3';

Taqman-MGB probe PT: 5'VIC-CAATAGCCCGGGCC-MGB-3';

Taqman-MGB probe PG: 5'FAM-CAATAGCCCTGGCC-MGB-3';

The PCR premix contains PCR buffer, Taq DNA polymerase, MgCl ₂ , and dNTPs. PCR buffer, Taq DNA polymerase, dNTPs and MgCl2 were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. PCR buffer was 10xPCR buffer, Taq DNA polymerase content 5U/μl, dNTPs content 2.5mM, MgCl ₂ content 1.5mM

The positive control substance includes the TT type plasmid at the 8993 site of the mitochondrial ATP6 gene and the GG type plasmid at the 8993 site of the mitochondrial ATP6 gene. TT type plasmid, GG type plasmid and TG type positive control plasmid were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

4. Fluorescence quantitative PCR amplification system and conditions:

Configure the following reaction system: using a 20μL reaction system, add 10μL of PCR premix to the PCR reaction tube, upstream primer F (10pmol/μL) 1μL, downstream primer R (10pmol/μL) 1μL, Taqman-MGB probe PT (10pmol/ μL)0.5μL, Taqman-MGB probe PG (10pmol/μL) 0.5μL, extracted template DNA 1μL, sterile ultrapure water 6μL;

Amplification conditions: 95°C/5min, 95°C/15sec, 60°C/1min, and 40 cycles of 95°C/15sec to 60°C/1min.

5. Construction of digital PCR amplification system and conditions

Configure the following reaction system: using a 25μL reaction system, add 13μL of ddPCR premix to the PCR reaction tube, upstream primer F (10pmol/μL) 1μL, downstream primer R (10pmol/μL) 1μL, Taqman-MGB probe PT (10pmol/ μL) 0.5μL, Taqman-MGB probe PG (10pmol/μL) 0.5μL, extracted template DNA 1μL, sterile ultrapure water 8μL; use digital PCR special droplet generator plate, add 70ul/well droplet generator oil , Prepared into an oil-in-oil aqueous solution in the droplet generator, and then transferred to a 96-well plate.

Amplification conditions: the first stage, 50℃/5min, 95℃/5min; the second stage, 95℃/15sec, 60℃/1min, 45 cycles; the third stage, 98℃/10min, 4℃ storage . Explore the amount of DNA template and channel amplification:

Two tubes of blood samples were used to explore and optimize the digital PCR amplification conditions, A04, B04 and C04, D04, the latter DNA template concentration was diluted 10 times, E04, F04 only put FAM labeled probe, G04, H04 only put VIC The labeled probes, the amplification results are as follows:

Only the FAM channel has an amplified S-shaped curve, the sample is judged to be TT type, only the VIC channel has an amplified S-shaped curve, and the sample is judged to be GG type, and both FAM and VIC channels have an S-shaped curve, it is judged as TG type.

It can be seen that after the template concentration is diluted by 10 times, a more suitable copy number range can be obtained. The two probes are amplified separately, and the degree of mutual interference is not high in the unified system, so it is suitable for use as a typing probe.

6. Fluorescence quantitative PCR for genotype detection of actual samples

Fluorescence quantitative PCR cannot accurately quantify the copy number of T and G genotypes because it has not constructed a standard curve, but the CT value can distinguish which genotype accounts for more than 50%.

7. Digital PCR detects the genotype of the actual sample and quantifies the copy number

Digital PCR can accurately quantify the copy numbers of T and G genotypes without a standard curve. The copy numbers of T and G genotypes and the T/(T+G) ratio in each blastocyst and polar body sample are shown in the above table. Compared with the results of fluorescence quantitative PCR, it can be seen that the detection results of the two methods are roughly the same, indicating that the two methods established can effectively detect the copy numbers of T and G genotypes at 8993 sites of mtDNA in blood, blastocysts and polar body samples. Perform accurate quantification.

Example 2: A kit and method for detecting genotype of human mitochondrial ATP6 gene at site 8993

A kit for detecting the genotype of human mitochondrial ATP6 gene at 8993 site, including PCR primers and Taqman-MGB probe for amplifying the 8993 site of mitochondrial ATP6 gene;

The PCR primers include: upstream primer F: 5'-GTTATTATCGAAACCATCAGCCT-3';

Downstream primer R: 5'-AGTAGGTGGCCTGCAGTAATGT-3';

The probe includes: Taqman-MGB probe PT: 5'VIC-CAATAGCCCGGGCC-MGB-3';

Taqman-MGB probe PG: 5'FAM-CAATAGCCCTGGCC-MGB-3'.

A preferred embodiment is that it further comprises a PCR premix, which contains PCR buffer, Taq DNA polymerase, MgCl ₂ and dNTPs.

A preferred embodiment is: a positive control substance is included, and the positive control substance includes a TT-type plasmid at the 8993 site of the mitochondrial ATP6 gene, and a GG-type plasmid at the 8993 site of the mitochondrial ATP6 gene.

A method for detecting the genotype of mitochondria at 8993 site, including the following steps:

Step 1: Extract the DNA of the sample to be tested;

Step 2: Add PCR premix, primers, probes, sterile ultrapure water and template DNA extracted in step 1 to the PCR reaction tube to obtain the reaction system; at the same time establish TT type control tube, GG type control tube, and TG type Control tube and blank control tube; the TT-type control tube replaces the template DNA extracted in step 1 with the TT-type plasmid at the 8993 site of the mitochondrial ATP6 gene; the GG-type control tube replaces the GG-type plasmid at the 8993 site of the mitochondrial ATP6 gene The template DNA extracted in step 1; the TG-type control tube uses a mixture of the TT-type plasmid at the 8993 site of the mitochondrial ATP6 gene and the GG-type plasmid at the 8993 site of the mitochondrial ATP6 gene to replace the template DNA extracted in the step 1; the blank control can be used without Bacteria ultrapure water replaces the template DNA extracted in step 1;

The PCR primers include: upstream primer F: 5'-GTTATTATCGAAACCATCAGCCT-3';

Downstream primer R: 5'-AGTAGGTGGCCTGCAGTAATGT-3';

The probe includes: Taqman-MGB probe PT: 5'VIC-CAATAGCCCGGGCC-MGB-3';

Taqman-MGB probe PG: 5′FAM-CAATAGCCCTGGCC-MGB-3′;

Step 3: After mixing the reaction system, perform fluorescent PCR amplification;

Step 4: Determine the result according to the amplification of the FAM channel and the HEX channel. Only the FAM channel has an amplified S-shaped curve, and the sample is judged to be TT type, and only the VIC channel has an amplified S-shaped curve, and the sample is judged to be GG Type, FAM and VIC channels have S-shaped curves, it is judged as TG type.

A preferred embodiment is: the fluorescent PCR amplification conditions: 95°C/5min, 95°C/15sec, 60°C/1min, and 40 cycles of 95°C/15sec to 60°C/1min.

A preferred embodiment is: the PCR premix, which contains PCR buffer, Taq DNA polymerase, MgCl ₂ and dNTPs.

A preferred embodiment is: the TT-type plasmid at 8993 site of the mitochondrial ATP6 gene and the GG type plasmid at the 8993 site of the mitochondrial ATP6 gene in a mass ratio of 1:1 replace the template DNA extracted in step 1.

The above-mentioned embodiments only exemplarily illustrate the principles and effects of the present invention, but are not used to limit the present invention. Anyone familiar with this technology can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (7)

1. A kit for detecting the genotype of human mitochondrial ATP6 gene at 8993 site, characterized in that it comprises PCR primers and Taqman-MGB probe for amplifying the 8993 site of mitochondrial ATP6 gene;

   The PCR primers include: upstream primer F: 5'-GTTATTATCGAAACCATCAGCCT-3';

   Downstream primer R: 5'-AGTAGGTGGCCTGCAGTAATGT-3';

   The probe includes: Taqman-MGB probe PT: 5'VIC-CAATAGCCCGGGCC-MGB-3';

   Taqman-MGB probe PG: 5'FAM-CAATAGCCCTGGCC-MGB-3'.
2. The kit for detecting mitochondrial 8993 genotype according to claim 1, characterized in that it further comprises a PCR premix containing PCR buffer, Taq DNA polymerase, MgCl ₂ and dNTPs.
3. The kit for detecting the genotype of mitochondrial 8993 site according to claim 1, characterized in that it comprises a positive control substance, the positive control substance includes the TT type plasmid at the 8993 site of the mitochondrial ATP6 gene, and the GG type at the 8993 site of the mitochondrial ATP6 gene Plasmid.
4. A method for detecting genotype at 8993 site of mitochondria, which is characterized in that it comprises the following steps:

   Step 1: Extract the DNA of the sample to be tested;

   Step 2: Add PCR premix, primers, probes, sterile ultrapure water and template DNA extracted in step 1 to the PCR reaction tube to obtain the reaction system; at the same time establish TT type control tube, GG type control tube, and TG type Control tube and blank control tube; the TT-type control tube replaces the template DNA extracted in step 1 with the TT-type plasmid at the 8993 site of the mitochondrial ATP6 gene; the GG-type control tube replaces the GG-type plasmid at the 8993 site of the mitochondrial ATP6 gene The template DNA extracted in step 1; the TG-type control tube uses a mixture of the TT-type plasmid at the 8993 site of the mitochondrial ATP6 gene and the GG-type plasmid at the 8993 site of the mitochondrial ATP6 gene to replace the template DNA extracted in the step 1; the blank control can be used without Bacteria ultrapure water replaces the template DNA extracted in step 1;

   The PCR primers include: upstream primer F: 5'-GTTATTATCGAAACCATCAGCCT-3';

   Downstream primer R: 5'-AGTAGGTGGCCTGCAGTAATGT-3';

   The probe includes: Taqman-MGB probe PT: 5'VIC-CAATAGCCCGGGCC-MGB-3';

   Taqman-MGB probe PG: 5'FAM-CAATAGCCCTGGCC-MGB-3';

   Step 3: After mixing the reaction system, perform fluorescent PCR amplification;

   Step 4: Determine the result according to the amplification of the FAM channel and the HEX channel. Only the FAM channel has an amplified S-shaped curve, and the sample is judged to be TT type, and only the VIC channel has an amplified S-shaped curve, and the sample is judged to be GG Type, FAM and VIC channels have S-shaped curves, it is judged as TG type.
5. The method for detecting the genotype of mitochondria at 8993 locus according to claim 4, characterized in that the fluorescent PCR amplification conditions: 95°C/5min, 95°C/15sec, 60°C/1min, wherein 95°C/15sec to The 60°C/1min process carries out 40 cycles.
6. The method for detecting the genotype at 8993 site of mitochondria according to claim 4, wherein the PCR premix contains PCR buffer, Taq DNA polymerase, MgCl ₂ and dNTPs.
7. The method for detecting the genotype of mitochondria at 8993 site according to claim 4, characterized in that: the TT type plasmid at the 8993 site of the mitochondrial ATP6 gene and the GG type plasmid at the 8993 site of the mitochondrial ATP6 gene are a mixture of 1:1 mass ratio. Replace the template DNA extracted in step 1.

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