WO2023108865A1 - Primer pair, kit and detection method for detecting mitochondrial loop gene mutation - Google Patents

Abstract

Provided are a primer pair, kit and detection method for detecting mitochondrial loop gene mutation. The primer pair is a primer pair M4 or a primer pair M5, wherein the primer pair M4 is: M4-F: AAGTGAACTGTATCCGACATCTGGTTCC, and M4-R: TAGCTACCCCCAAGTGTTATGGGC; and the primer pair M5 is: M5-F: ATAAAGCCTAAATAGCCCACACGTTCC, and M5-R: GACCCTGAAGTAGGAACCAGATGTCG. A sequencing library is prepared by means of extracting DNA, performing long-fragment amplification on mtDNA using the primer pair, and performing fragmentation, terminal repair, linker linking, purification and PCR enrichment on the amplified product; quantification and quality control are performed on the sequencing library; the quantified sequencing library is subjected to high-throughput sequencing using a gene sequencer to obtain sequencing data; and the sequencing data is analyzed using bioinformation analysis software to obtain mtDNA mutation-related information.

Description

Primer pair, kit and detection method for detecting mitochondrial loop gene mutation technical field

The invention relates to the field of molecular biology, in particular to a pair of primers, a kit and a detection method for detecting mutations of mitochondrial ring genes.

Background technique

Mitochondria are important sites for energy metabolism, and mutations in the mitochondrial genome or genes encoding mitochondrial proteins on the nuclear genome may lead to disease. Three typical phenotypes caused by mtDNA deletion are Kearns-Sayre syndrome (KSS), Pearson syndrome and progressive external ophthalmoplegia (PEO), and very rare Leigh syndrome. However, in clinical practice, patients with mitochondrial deficiency diseases often show incomplete or overlapping clinical phenotypes, and cannot be classified as any typical syndrome, which makes the clinical diagnosis and treatment of such patients very challenging. It is of great clinical significance to understand the phenotypic characteristics, molecular etiology and disease diagnosis and treatment methods of diseases involving multiple systems of mitochondrial deletion in detail. .

Contents of the invention

Aiming at the deficiencies in the above-mentioned prior art, the problem to be solved in the present invention is: how to provide a primer pair, a kit and a detection method for detecting the mutation of the mitochondrial ring gene, so as to solve the inconvenience and detection of the mutation of the mitochondrial ring gene at present. difficult question.

In order to solve the above problems, the present invention adopts the following technical solutions:

A pair of primers for detecting the mutation of the mitochondrial ring gene, the primer pair is the primer pair M4 or the primer pair M5;

The primer pair M4 is:

M4-F: AAGTGAACTGTATCCGACATCTGGTTCC,

M4-R: TAGCTACCCCCAAGTGTTATGGGC;

The primer pair M5 is:

M5-F: ATAAAGCCTAAATAGCCCACACGTTCC,

M5-R: GACCCTGAAGTAGGAACCAGATGTCG.

The present invention also provides a kit for detecting the mutation of the mitochondrial loop gene, including the above-mentioned primer pair.

Specifically, the kit includes: 2 parts by volume of PCR amplification solution, 4 parts by volume of 2.5M dNTP, 4 parts by volume of 5M betaine, 0.2 parts by volume of TaKaRa LA Taq HS, 7.8 parts by volume of nuclease-free Purified water and 1 part by volume of the primer pair described in claim 1.

The present invention also provides a non-disease diagnosis detection method for detecting mitochondrial loop gene mutation, comprising:

(1) DNA is extracted, and the above-mentioned primer pair is used to amplify long fragments of mtDNA;

(2) Fragmentation, end repair, adapter connection, purification and PCR enrichment of the amplified product in the step (1) to prepare a sequencing library, quantification and quality control of the sequencing library, the quantitative sequencing library Using a gene sequencer for high-throughput sequencing to obtain sequencing data;

(3) Using biological information analysis software to analyze the sequencing data to obtain information related to mtDNA mutations.

Further, after the step (1) and before the step (2), the product amplified in the step (1) is subjected to quality inspection, and a specificity is selected from the products amplified by the primer pair M4 and M5. The step (2) is preferably carried out.

Further, the quality inspection includes: using 0.8% agarose gel to perform quality inspection on the products amplified in the step (1), so that each sample has at least one comparison between the products amplified by the primer pair M4 and M5 Marker can observe a single specific band in the target area, if not, repeat the step (1).

Further, the conditions for carrying out long-fragment amplification of mtDNA in the step (1) are:

Further, performing quantification and quality control on the sequencing library in the step (2) includes: the concentration of the sequencing library is greater than 5 ng/μL, the volume is greater than 10 ul, and the fragment length of the sequencing library is 200-500 bp.

The present invention also provides the application of the above-mentioned primer pair for detecting the mutation of the mitochondrial ring gene in the preparation of a reagent for detecting the mutation of the mitochondrial ring gene.

The present invention also provides the application of the above-mentioned kit for detecting mitochondrial ring gene mutations in the preparation of reagents for detecting mitochondrial ring gene mutations.

The beneficial effect of the present invention is that: the primer pair, kit and detection method for detecting the mutation of the mitochondrial ring gene provided by the present invention can quickly detect the mutation of the mitochondrial ring gene with a high degree of reliability.

Description of drawings

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with the accompanying drawings, wherein:

Fig. 1 is an agarose gel diagram of mtDNA amplification of primer pair M4 and M5.

Detailed ways

The present invention will be further described in detail below in conjunction with specific examples.

It should be noted that these examples are only used to illustrate the present invention, rather than limit the present invention, the simple improvement of the method under the premise of the present invention, all belong to the protection scope of the present invention.

The design idea of the present invention: first extract the genomic DNA (gDNA) in the whole blood sample, carry out specific mitochondrial genome (mtDNA) long fragment amplification on the extracted gDNA, then perform high-throughput sequencing on the mtDNA, and use biological information analysis software Analyze the sequencing data to obtain information about mtDNA variations (SV, Indel, SNPs).

The experimental samples of this program are tested with samples of known clinical information, as shown in the following table:

serial number Sample serial number Report No report results 1 17C000264 MGNC0007562 chrM:3244 A>G 2 17C003391 MGNC0000595 chrM:11779 G>A 3 19C009909 MGNC0068849 Negative 4 19C172947 MGNO0053873 chrM: 6238-14103 region deletion 5 19C176217 MGNC0090890 chrM:14485 T>C 6 19C177229 MGNC0112608 chrM:8345 A>G 7 20C201995 MGNC0107921 ChrM: 7544-15443 region deletion 8 yf-301 yf-301 Negative

Embodiment one

gDNA extraction from blood samples

The blood sample was extracted using the Magnetic Bead Method Universal Genomic DNA Extraction Kit (DP705-02) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. The following DNA extraction process is the standard process in the kit:

1. Take 250 μl blood sample into a 2ml centrifuge tube, add 20 μl Proteinase K solution and 300 μl lysate GHL, shake and mix, lyse at 75°C for 15 minutes, and invert and mix 3 times during this period, 3-5 times each time. When the number of samples is relatively large, the lysate GHL and Proteinase K can be pre-mixed.

2. Add 300 μl isopropanol, shake and mix for 10 sec.

3. Add 15 μl magnetic bead suspension GH, shake and mix for 1 minute, let stand for 9 minutes, shake and mix for 1 minute every 3 minutes. To ensure the beads are thoroughly resuspended, mix by vortexing before use.

4. Place the centrifuge tube on the magnetic stand and let it stand for 30 sec. After the magnetic beads are completely absorbed, absorb the liquid.

5. Add 900 μl buffer GDZ, shake and mix for 2 minutes.

6. Place the centrifuge tube on the magnetic stand and let it stand for 30 sec. After the magnetic beads are completely absorbed, absorb the liquid.

7. Add 500 μl buffer GDZ, shake and mix for 2 minutes.

8. Place the centrifuge tube on the magnetic stand and let it stand for 30 sec. After the magnetic beads are completely adsorbed, absorb the liquid.

9. Remove the centrifuge tube from the magnetic stand, add 900 μl of washing solution PWD, shake and mix for 2 minutes.

10. Place the centrifuge tube on the magnetic stand and let it stand for 30 sec. After the magnetic beads are completely absorbed, absorb the liquid.

11. Remove the centrifuge tube from the magnetic stand, add 300 μl of washing solution PWD, shake and mix for 2 minutes.

12. Place the centrifuge tube on the magnetic stand and let it stand for 30 sec. After the magnetic beads are completely absorbed, carefully suck off the liquid.

13. Put the centrifuge tube on the magnetic stand and dry it at room temperature for 10-15 minutes.

14. Remove the centrifuge tube from the magnetic stand, add 50 μl of elution buffer TB, shake and mix, place at 56°C, incubate for 10 minutes, and invert and mix 3 times during this period, 3-5 times each time.

15. Transfer the gDNA solution to a new centrifuge tube and measure its concentration with nanodrop. Concentrate or dilute the gDNA concentration to 50ng/μL according to the measured concentration, and then perform subsequent experiments. It is recommended to perform PCR amplification immediately after the extracted gDNA, or store it directly at -20±5°C.

Embodiment two

Amplification of long fragments of mitochondrial DNA

For the mitochondrial circular gene, two pairs of specific primers were designed, and the primer sequences are as follows:

M4-F AAGTGAACTGTATCCGACATCTGGTTCC M4-R TAGCTACCCCCAAGTGTTATGGGC M5-F ATAAAGCCTAAATAGCCCACACGTTCC M5-R GACCCTGAAGTAGGAACCAGATGTCG

Hot Start Version，货号为RR042Q。 1. Reagent preparation: Commercially available PCR kits were used for PCR. The brand of the kit used in this example is TAKARA, and the name is TaKaRa LA

Hot Start Version, the article number is RR042Q.

The test samples need to be amplified by PCR with two pairs of primers M4 and M5 respectively, so two systems need to be prepared. N = number of samples to be tested + 1.

2. Adding samples: Add 1ul DNA to the 19ul amplification reaction mixture.

3. Amplification: The amplification conditions are as follows:

4. Quality inspection of PCR amplification products: use 0.8% agarose gel to perform quality inspection of amplification products, each sample uses primers M4 and M5 for amplification products with at least one comparison marker that can observe a single marker in the target area Specific bands, if not the DNA was re-extracted and PCR amplified. From the amplification products of the samples using primers M4 and M5, a PCR amplification product with good specificity and high yield was selected for subsequent mitochondrial DNA purification. According to Figure 1, the amplification product of M5 was used for subsequent experiments.

Embodiment three

According to the method of the present invention, the library preparation steps are as follows:

The library was prepared using a self-produced kit of our company, the name is Universal Library Preparation Kit (Illumina) (enzyme cutting method) V4.1, and the catalog number is MG0077/96.

1. Nucleic acid fragmentation: prepare the nucleic acid fragmentation reaction system according to the following table

Element volume 10× Fragmentation Buffer V2 2.5μl Fragmentation Mixed Enzyme V2 2.5μl mt DNA amplification product (purified) 100-500ng Enzyme-free water Rehydrate to 25μl

2. Put the above-mentioned PCR tube in the PCR instrument (the hot lid needs to be activated during the reaction), and the reaction procedure is as follows:

step temperature control time 1 4°C 1min 2 32°C 30min 3 65°C 30min 4 4°C HOLD

3. Connection of adapters: Add the following reagents directly to the above reaction solution that has completed DNA end repair:

Element Volume (μl)

5 x T4 Ligase Buffer V2 10μl Adapter (Illumina) 2μl T4 ligase V2 2μl Enzyme-free water 11μl

4. Use the tip of the pipette to gently blow and mix the above solution, and centrifuge briefly to collect all components to the bottom of the tube.

5. Put the above-mentioned PCR tube in the PCR machine. The reaction procedure is as follows:

temperature control time number of cycles 20°C 15min 1 4°C HOLD the

6. Purification of the connection product: The magnetic beads produced by the company are used for purification.

6.1. Place the magnetic beads at room temperature to equilibrate for 20 minutes;

6.2. Add 40 μl of magnetic beads to the system after the reaction, and mix by pumping up and down for at least ten times;

6.3. Put the mixture at room temperature for 5 minutes, so that the DNA is fully adsorbed on the magnetic beads;

6.4. After the adsorption is completed, place it on a magnetic stand at room temperature for 5 minutes;

6.5. After the solution becomes clear, use a pipette to remove the clear liquid;

6.6. Keep the centrifuge tube still on the magnetic stand, add 200μl 80% alcohol, and place it at room temperature for 30s; discard the clarified solution;

6.7. Repeat step 6.6 once, and thoroughly blot the residual liquid at the bottom of the tube;

6.8. Remove the PCR tube from the magnetic stand and place it at room temperature for 5 minutes;

6.9. Add 25 μl of enzyme-free water, mix well by pipetting the tip of the pipette, and place at room temperature for 3 minutes;

6.10. Place the PCR tube on the magnetic stand, and place it at room temperature for 5 minutes or until it is dry;

6.11. Transfer 23 μl of the clarified solution in the reaction tube in step 6.10 for subsequent experiments.

7. PCR enrichment:

7.1 Prepare the reaction system:

Element Volume (μl) DNA purification product 23μl 2xPCR mix 25μl PCR forward primer 1μl Index 1μl

Different Index (biological labels) can be selected; and when performing subsequent capture and enrichment experiments, the biological labels used must match the precise blocking agent. If the UMI connector is used, the biological label needs to be used corresponding to the UMI connector, for example, Index I553 corresponds to A553.

7.2 Set the PCR cycler program:

The number of PCR cycles depends on the amount of DNA loaded and the quality of the DNA. It is recommended to use 6-9 cycles: 6 cycles are recommended for 500ng DNA loading; 9 cycles are recommended for 100ng DNA loading.

8. Purification of PCR products: The magnetic beads produced by our company are used for purification.

8.1. Place the magnetic beads at room temperature to equilibrate for 20 minutes;

8.2. Add 50 μl of magnetic beads to the system after the reaction, and mix by pumping up and down for at least ten times;

8.3. Put the mixture at room temperature for 5 minutes, so that the DNA is fully adsorbed on the magnetic beads;

8.4. After the adsorption is completed, place it on a magnetic stand at room temperature for 5 minutes;

8.5. After the solution becomes clear, use a pipette to remove the clear liquid;

8.6. Keep the centrifuge tube still on the magnetic stand, add 180μl 80% alcohol, leave at room temperature for 30s, then discard the clarified solution;

8.7. Repeat step 5.6 once, and thoroughly blot the residual liquid at the bottom of the tube;

8.8. Remove the PCR tube from the magnetic stand and place it at room temperature for 5 minutes;

8.9. Add 33 μl of TE buffer/deionized water, mix well by pipetting the tip of the pipette, and place at room temperature for 5 minutes;

8.10. Place the reaction tube on the magnetic stand and place it at room temperature for 2 minutes;

8.11. Transfer 30 μl of the clarified solution in the reaction tube in step 8.10 to a new 1.5 ml centrifuge tube, and mark the sample information;

8.12. Take 2 μl of library product for quantification using Qubit dsDNA HS Assay Kit, record the concentration of the product, the concentration of Qubit detection library should be >5ng/μL, and the volume should be >10ul;

8.13. Take 1 μl sample and use Agilent 2100Bioanalyzer system (Agilent DNA 1000Kit) to measure the library fragment length, the fragment length is mainly distributed in 200-500bp;

8.14. The quantified products can be directly sequenced using a high-throughput sequencing platform.

Embodiment four

High-throughput sequencing

High-throughput sequencing uses a sequencer and performs sequencing according to the standard operating procedures of the sequencer.

Embodiment five

Sequencing results

Data volume ≥ 0.04G; sequencing depth ≥ 1600×; sequence alignment to the reference genome ratio ≥ 90%.

1. Basic statistics: The mtDNA of 8 samples (mtDNA-1~8) was sequenced for library construction and basic statistics. From the results in the table below, the effective reads in the target region accounted for more than 80%, and the effective data was more than 30MB. The depth is greater than 1600x, and the result meets the detection requirements.

2. SNP, CNV, SV analysis and sample report comparison: The analysis results of 8 samples and their comparison with the capture results are shown in the table below. The detection results of 2 negative samples were still negative for SNPIndel and CNV, and the results were consistent; the detection results of 4 point mutation samples were consistent with the capture method; the results of the two algorithms for CNV analysis of 2 missing samples were inconsistent, and the CNRs were all detected deletions. It shows the deletion, but the specific location of the deletion is different; the SV analysis results of 2 missing samples show a high consistency with the capture results, and the reliability is high.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described with reference to the preferred embodiments of the present invention, those skilled in the art should understand that it can be described in the form Various changes may be made in matter and details thereof without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

A pair of primers for detecting mutations in mitochondrial loop genes, characterized in that the pair of primers is a pair of primers M4 or pair of primers M5; The primer pair M4 is: M4-F: AAGTGAACTGTATCCGACATCTGGTTCC, M4-R: TAGCTACCCCCAAGTGTTATGGGC; The primer pair M5 is: M5-F: ATAAAGCCTAAATAGCCCACACGTTCC, M5-R: GACCCTGAAGTAGGAACCAGATGTCG. A kit for detecting mitochondrial loop gene mutations, characterized in that it comprises the pair of primers according to claim 1. The test kit for detecting mitochondrial ring gene mutation according to claim 2, wherein said kit comprises: 2 parts by volume of PCR amplification solution, 4 parts by volume of 2.5M dNTP, 4 parts by volume of 5M sugar beet The primer pair described in claim 1 of alkali, 0.2 parts by volume of TaKaRa LA Taq HS, 7.8 parts by volume of nuclease-free purified water and 1 part by volume. A detection method for non-disease diagnosis for detection of mitochondrial ring gene mutation, characterized in that it comprises: (1) extract DNA, utilize primer described in claim 1 to carry out long fragment amplification to mtDNA; (2) Fragmentation, end repair, adapter connection, purification and PCR enrichment of the amplified product in the step (1) to prepare a sequencing library, quantification and quality control of the sequencing library, the quantitative sequencing library Using a gene sequencer for high-throughput sequencing to obtain sequencing data; (3) Using biological information analysis software to analyze the sequencing data to obtain information related to mtDNA mutations. According to claim 4, the non-disease diagnosis detection method for detecting mitochondrial ring gene mutations is characterized in that the step (1) is amplified after the step (1) and before the step (2). The product is subjected to quality inspection, and a specificity is better selected from the products amplified by the primer pair M4 and M5 to carry out the step (2). According to claim 5, the non-disease diagnosis detection method for detecting mitochondrial loop gene mutations is characterized in that the quality inspection comprises: using 0.8% agarose gel to carry out the amplified product of the step (1) Quality inspection, so that each sample amplified using the primer pair M4 and M5 has at least one contrast marker that can observe a single specific band in the target region, if not, repeat the step (1). According to claim 4, the detection method for non-disease diagnosis that is used to detect mitochondrial loop gene mutations, is characterized in that, the conditions for carrying out long fragment amplification of mtDNA in the step (1) are:

According to claim 4, the detection method for non-disease diagnosis for detecting mitochondrial ring gene mutations is characterized in that, in the step (2), carrying out quantification and quality control of the sequencing library comprises: the concentration of the sequencing library Greater than 5ng/μL, volume greater than 10ul, the fragment length of the sequencing library is 200-500bp. The use of the primer pair for detecting the mutation of the mitochondrial ring gene according to claim 1 in the preparation of a reagent for detecting the mutation of the mitochondrial ring gene. The application of the kit for detecting mitochondrial ring gene mutations described in claim 2 or 3 in the preparation of reagents for detecting mitochondrial ring gene mutations.

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