Mitochondrial DNA highly pathogenic mutation detection kit and detection method based on digital PCR

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a mitochondrial DNA highly pathogenic mutation detection kit based on digital PCR and a detection method. The kit comprises a primer pair for amplifying the high pathogenic mutation of the mitochondrial DNA, a probe and a digital PCR (Polymerase Chain Reaction) premixed solution. The invention provides the primer pair for amplifying the high pathogenic mutation of the mitochondrial DNA and a probe sequence. Detection primers and probes are respectively provided for 14 Chinese common pathogenic mitochondrial DNA variations, about 20 primary mitochondrial DNA diseases can be screened through detection, the detection kit provided by the invention has the characteristics of accuracy, sensitivity and economy, the detection sensitivity is improved to 0.01% of mutation load by optimizing the primer probe design and an amplification system, and the detection kit can be used for detecting the primary mitochondrial DNA diseases of the primary mitochondrial DNA diseases of the primary mitochondrial DNA diseases of the primary mitochondrial DNA diseases of the primary mitochondrial DNA diseases of the primary mitochondrial DNA diseases. The detection can be completed within 6 hours, the clinical decision-making period is greatly shortened, and the kit can be suitable for the fields of conventional screening of mitochondrial DNA diseases, prenatal diagnosis, genetic detection before embryo implantation and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to a kit and a detection method for detecting high-pathogenicity mutations of mitochondrial DNA based on digital PCR. Background Art

[0002] Mitochondrial DNA diseases can affect multiple organs and systems throughout the body, leading to severe maternally inherited syndromes. Mitochondrial DNA (mtDNA) mutations are the core cause of inherited mitochondrial DNA diseases, with highly heterogeneous clinical manifestations, and the mutation load is directly related to the severity of the disease. In recent years, the rapid development of prenatal diagnosis (PND) and preimplantation genetic diagnosis (PGD) technologies has made it possible to intervene early in mitochondrial DNA diseases. However, the limitations of existing detection technologies in clinical applications significantly restrict the diagnostic efficiency and accuracy.

[0003] Currently, the detection of mtDNA mutations mainly relies on technologies such as DNA sequencing, restriction fragment length polymorphism analysis (PCR-RFLP), and high-resolution melting curve (HRM). Although sequencing is regarded as the "gold standard" for gene mutation detection, its detection sensitivity for low-abundance mutations is insufficient, and the operation cycle is long and the cost is high, making it difficult to meet the requirements of prenatal diagnosis for rapid and accurate quantification of the mutation load. Although PCR-RFLP can achieve specific locus screening, it requires electrophoresis verification after enzymatic digestion, with cumbersome steps and incompatible with multiplex detection; although the HRM technology can quickly identify sequence variations, it is easily affected by primer design and PCR conditions, with a high false positive rate, and it is difficult to distinguish homozygous mutations from high-proportion heterozygous mutations. In addition, traditional methods require a large amount of samples and are difficult to adapt to the detection scenarios of single trophoblast cells or trace chorionic villus samples in PGD.

[0004] In recent years, digital PCR (ddPCR) technology, due to its unique droplet partitioning and absolute quantification capabilities, has provided a breakthrough solution for the accurate diagnosis of mtDNA mutations. Compared with fluorescence quantitative PCR (qPCR), ddPCR can achieve absolute copy number analysis without relying on a standard curve, significantly improving the detection sensitivity for low-abundance mutations, especially suitable for the determination of mtDNA mutation load in embryo or prenatal detection samples. However, the number of ddPCR detection kits for specific mtDNA mutations is still small. To meet the growing detection needs of diseases caused by common mitochondrial DNA variations, it is very necessary to provide a detection method based on ddPCR. Summary of the Invention

[0005] To solve the above technical problems, the present invention first provides a kit for detecting high-pathogenicity mutations of mitochondrial DNA based on digital PCR.

[0006] The present invention adopts the following technical solutions:

[0007] A mitochondrial DNA highly pathogenic mutation detection kit based on digital PCR, comprising:

[0008] (a) Primer pairs and probes for amplifying the mitochondrial DNA highly pathogenic mutations, where the mitochondrial DNA highly pathogenic mutations are any one or a combination of m.1555A>G, m.3243A>G, m.3271T>C, m.3697G>A, m.8344A>G, m.8993T>G, m.8993T>C, m.9176T>G, m.9176T>C, m.9185T>C, m.11777C>A, m.11778G>A, m.13513G>A, m.14459G>A. The sequences of the forward primer, reverse primer, mutant probe, and normal probe for amplifying each mitochondrial DNA highly pathogenic mutation are shown in SEQ ID No: 1 - SEQ ID No: 56 in sequence;

[0009] (b) Digital PCR premix.

[0010] Preferably, it further includes sterile water and / or oil phase.

[0011] Preferably, the digital PCR premix includes PCR buffer, dNTPs, and DNA polymerase.

[0012] Preferably, the digital PCR premix further includes additives with one or several functions of enhancer, stabilizer, and inhibitor.

[0013] The present invention secondly provides a detection method for the mitochondrial DNA highly pathogenic mutation detection kit based on digital PCR as described above, comprising the following steps:

[0014] S1. Extract mitochondrial DNA from the sample to be tested;

[0015] S2. Use the mitochondrial DNA highly pathogenic mutation detection kit to amplify the extracted mitochondrial DNA;

[0016] S3. Obtain the amplification result, detect whether there is a target mutation in the extracted mitochondrial DNA through fluorescence signal, and calculate the mutation load of the variation to complete the detection.

[0017] Preferably, the specific steps of the amplification are:

[0018] S21. Use the mitochondrial DNA highly pathogenic mutation detection kit to configure a reaction system, put the prepared reaction system into a droplet generation device, and add an oil phase to generate microdroplets for independent PCR reactions;

[0019] S22. Transfer the droplet to a droplet digital PCR instrument and run the PCR program for PCR amplification;

[0020] S23. After the PCR amplification is completed, use a droplet analyzer to detect the fluorescence signal, calculate the mutation load of the mutation according to the number of positive droplets, and complete the detection.

[0021] Preferably, the reaction system for detecting each highly pathogenic mutation of mitochondrial DNA is 25 μl, including 12.5 μL of digital PCR premix, 1 μL of forward primer for amplification, 1 μL of reverse primer, 1 μL of extracted template mitochondrial DNA, 0.5 μL each of mutant probe and normal probe, and 8.5 μL of sterile water.

[0022] Preferably, the PCR program is: pre-denaturation at 95 °C for 5 minutes; denaturation at 95 °C for 30 seconds, then annealing at the annealing temperature set by the primer set for each highly pathogenic mutation of mitochondrial DNA for 60 seconds, with 45 cycles; extension at 98 °C for 10 minutes.

[0023] Preferably, the sample to be tested includes any one of blood, urine, muscle, amniotic fluid, and embryonic cells.

[0024] The beneficial effects of the present invention are as follows:

[0025] This application provides a kit for detecting highly pathogenic mutations of mtDNA based on digital PCR, which is accurate, sensitive, and economical. By optimizing the primer-probe design and amplification system, the detection sensitivity is increased to 0.01% mutation load, and the detection can be completed within 6 hours, greatly shortening the clinical decision-making cycle. It can be applied to the fields of routine screening, prenatal diagnosis, preimplantation genetic testing, etc. of mitochondrial DNA diseases.

[0026] This application provides detection primers and probes for 14 common pathogenic mitochondrial DNA variations in China respectively. Through detection, about 20 primary mitochondrial DNA diseases can be screened. Among them, m.1555A>G can screen for DEAF and MASID; m.3243A>G can screen for MELAS, Leigh Syndrome, MIDD, SNHL, CPEO, MM, FSGS, and ASD; m.3271T>C can screen for MELAS, DM, and MERRF; m.3697G>A can screen for MELAS, Leigh Syndrome, LDYT, and BSN; m.8344A>G can screen for MERRF and leukoencephalopathy; m.8993T>G and m.8993T>C can screen for NARP and Leigh Syndrome; m.9176T>G can screen for Leigh syndrome, spastic paraplegia, and spinocerebellar ataxia; m.9176T>C can screen for FBSN, Leigh Syndrome, and spinocerebellar ataxia; m.9185T>C can screen for Leigh Syndrome, ataxia, NARP, and CMT; m.11777C>A can screen for Leigh Syndrome; m.11778G>A can screen for Leigh Syndrome and progressive dystonia; m.13513G>A can screen for Leigh Syndrome, MELAS, and LHON; m.14459G>A can screen for LDYT, Leigh Disease, and dystonia.

[0027] The kit of this application adopts standardized premixed components, simplifies the operation process, reduces the detection cost by more than 70% compared with NGS, and supports the detection of multiple types of samples such as blood, urine, muscle, amniotic fluid, and embryonic cells, which is suitable for large-scale screening and promotion in primary medical institutions.

[0028] This application can provide an efficient, accurate and economical solution for the prevention, diagnosis and reproductive intervention of mitochondrial DNA diseases, has significant clinical application value and market competitiveness, and has great market prospects and social value. Detailed implementation manners

[0029] The technical solutions of the present invention will be described in more detail below in combination with experiments.

[0030] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this article can be purchased through the market or prepared by existing methods.

[0031] The corresponding relationship between diseases and abbreviations in this article is as follows:

[0032] DEAF: Mitochondrial inherited deafness, MASID: Mitochondria-related autism spectrum disorder with intellectual disability, MELAS: Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes syndrome, Leigh Syndrome: Subacute necrotizing encephalomyelopathy, MIDD: Maternal inherited diabetes and deafness, SNHL: Mitochondria-related sensorineural deafness, CPEO: Chronic progressive external ophthalmoplegia, MM: Mitochondrial myopathy, FSGS: Focal segmental glomerulosclerosis, ASD: Autism spectrum disorder, DM: Mitochondrial inherited diabetes, MERRF: Myoclonic epilepsy with ragged red fibers, LDYT: Leber hereditary optic neuropathy with dystonia, NARP: Ataxia and retinitis pigmentosa syndrome, FBSN: Familial bilateral striatal necrosis, CMT: Hereditary neural muscular atrophy, LHON: Leber hereditary optic neuropathy.

[0033] 1. Identification of mtDNA high-pathogenic mutation detection sites

[0034] The mtDNA high-pathogenic mutations referred to in this application specifically refer to mtDNA mutations that are clearly recognized as pathogenic and can cause primary mitochondrial DNA diseases. The specific evaluation method is as follows: Based on the mitochondrial DNA variant interpretation criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG / AMP), mtDNA variants evaluated as "pathogenic" and "likely pathogenic" and mtDNA variants with "confirmed" pathogenicity annotation in the Mitomap database.

[0035] This invention included a total of 14 common high-pathogenic mtDNA variant sites in China, which can detect approximately 20 primary mitochondrial DNA diseases, namely:

[0036] m.1555A>G: Screening for DEAF, MASID;

[0037] m.3243A>G: Screening for MELAS, Leigh Syndrome, MIDD, SNHL, CPEO, MM, FSGS, ASD;

[0038] m.3271T>C: Screening for MELAS, DM, MERRF;

[0039] m.3697G>A: Screening for MELAS, Leigh Syndrome, LDYT;

[0040] m.8344A>G: Screening for MERRF, leukoencephalopathy;

[0041] m.8993T>G: Screening for NARP, Leigh Syndrome;

[0042] m.8993T>C: Screen for NARP, Leigh Syndrome;

[0043] m.9176T>G: Screen for Leigh syndrome, spastic paraplegia, spinocerebellar ataxia;

[0044] m.9176T>C: Screen for FBSN, Leigh Syndrome, spinocerebellar ataxia;

[0045] m.9185T>C: Screen for Leigh Syndrome, ataxia, NARP, CMT;

[0046] m.11777C>A: Screen for Leigh Syndrome;

[0047] m.11778G>A: Screen for Leigh Syndrome, progressive dystonia;

[0048] m.13513G>A: Screen for Leigh Syndrome, MELAS, LHON;

[0049] m.14459G>A: Screen for LDYT, Leigh Disease, dystonia.

[0050] 2. Digital PCR-based Kit for Detecting High-pathogenicity Mutations in Mitochondrial DNA

[0051] 2.1 Composition

[0052] The detection kit includes primer pairs and probes for amplifying high-pathogenicity mutations in mitochondrial DNA, ddPCR premix, and also includes sterile water and oil phase.

[0053] In this application, the ddPCR premix includes PCR buffer, dNTPs, and DNA polymerase. According to requirements, it can also include additives with one or several functions of enhancer, stabilizer, and inhibitor. The ddPCR premix in this application can use commercially available premixes, such as the ddPCR premix from Bio-Rad.

[0054] 2.2 Design Principles of Primers and Detection Probes

[0055] Use Beacon Designer 8 to design primer sequences. The design of primers and probes should meet the following indicators:

[0056] The primer length is (18 - 25 bp), the Tm value is (60 ± 5 °C), the GC content is (40 - 60%), the difference in Tm values between the forward and reverse primers does not exceed 5 °C, consecutive repeating bases at the 3' end are avoided (such as GGG / CCC), and the product length is 80 - 200 bp. The optimal indicators for probe design should be: the probe length is (20 - 27 bp), and the Tm value is 5 - 10 °C higher than that of the primer.

[0057] Finally, the sequences of the forward primer, reverse primer, mutant probe, and normal probe for amplifying each of the highly pathogenic mutations of the mitochondrial DNA are shown in SEQ ID No: 1 - SEQ ID No: 56 in sequence;

[0058]

[0059]

[0060]

[0061] 2.3 Detection method

[0062] Detection is carried out using the detection kit provided by this application, and the steps include:

[0063] S1. Extract the mitochondrial DNA from the sample to be tested.

[0064] S2. Use the highly pathogenic mutation detection kit for mitochondrial DNA to amplify the extracted mitochondrial DNA. Specifically:

[0065] S21. Configure the reaction system using the highly pathogenic mutation detection kit for mitochondrial DNA. The reaction system for detecting each highly pathogenic mutation of the mitochondrial DNA is 25 μl, including 12.5 μL of ddPCR premix, 1 μL of the forward primer for amplification, 1 μL of the reverse primer, 1 μL of the extracted template mitochondrial DNA, 0.5 μL each of the probes for detecting the mutant site and the normal site, and 8.5 μL of sterile water; put the prepared reaction system into the droplet generation device and add the oil phase to generate microdroplets for independent PCR reactions;

[0066] S22. Transfer the droplets to a microdroplet ddPCR instrument and run the PCR program for PCR amplification. The PCR program is: pre-denaturation at 95 °C for 5 minutes; denaturation at 95 °C for 30 seconds, then anneal for 60 seconds according to the annealing temperature set for each primer set of the highly pathogenic mutations of the mitochondrial DNA (the annealing temperature is set as the Tm value of the primer with the lower Tm value minus 5 °C), cycle 45 times; extension at 98 °C for 10 minutes.

[0067] After the PCR amplification in S3 is completed, a droplet analyzer is used to detect the fluorescence signal. The droplets of each sample sequentially pass through a dual-color detector to determine whether there is a target mutation in the mitochondrial DNA extracted by the fluorescence signal detection. The droplets with fluorescence signals are determined to be positive, and the droplets without fluorescence signals are negative. The QuantaSoft software is used to record the number of positive droplets in each sample and calculate the mutation load of the variation, thus completing the detection.

[0068] Example 1

[0069] The m.3243A>G variation is the most common pathogenic mtDNA variation in China. The blood samples of a family with 1 m.3243A>G carrier were detected using the above kit and method. Samples 1-8 are respectively 8 members of the family. The detection results are shown in Table 1.

[0070] Table 1 Detection Results

[0071]

[0072] It can be seen that the ddPCR kit of the present application demonstrates high sensitivity and the ability to detect low-abundance mutations. The mutant (G) ratios of samples 7 and 8 are as low as 0.35% and 0.36% (corresponding to G copy numbers of only 0.46 and 0.15), indicating that the kit of the present application can accurately detect extremely low-abundance mutations of <1%. At the same time, it can distinguish subtle differences in mutation load (such as 13.75% of sample 5 compared with 12.04% of sample 6), and can detect samples with low, medium, and high mutation loads. According to this kit, it can effectively guide the clinical genetic counseling work for mtDNA disease families.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. Digital PCR-based kit for detecting highly pathogenic mutations of mitochondrial DNA, characterized in that, Comprising: (a) Primer pairs and probes for amplifying highly pathogenic mutations of the mitochondrial DNA, wherein the highly pathogenic mutations of the mitochondrial DNA are any one or a combination of more than one of m.1555A>G, m.3243A>G, m.3271T>C, m.3697G>A, m.8344A>G, m.8993T>G, m.8993T>C, m.9176T>G, m.9176T>C, m.9185T>C, m.11777C>A, m.11778G>A, m.13513G>A, m.14459G>A; the sequences of the forward primer, reverse primer, mutant probe, and normal probe for amplifying each highly pathogenic mutation of the mitochondrial DNA are shown in SEQ ID No:1 - SEQ ID No:56 in sequence; (b) Digital PCR premix.

2. The mitochondrial DNA high-pathogenic mutation detection kit based on digital PCR according to claim 1, characterized in that, Also comprising sterile water and / or oil phase.

3. The mitochondrial DNA high-pathogenicity mutation detection kit based on digital PCR according to claim 1, wherein The digital PCR premix comprises PCR buffer, dNTPs, and DNA polymerase.

4. The mitochondrial DNA high-pathogenic mutation detection kit based on digital PCR according to claim 3, wherein The digital PCR premix further comprises additives with one or several functions of enhancer, stabilizer, and inhibitor.

5. A detection method for a mitochondrial DNA high-pathogenicity mutation detection kit based on digital PCR as described in any one of claims 1-4, characterized in that, Comprising the following steps: S1. Extract mitochondrial DNA from the sample to be tested; S2. Amplify the extracted mitochondrial DNA using the highly pathogenic mutation detection kit for mitochondrial DNA; S3. Obtain the amplification result, detect whether there is a target mutation in the extracted mitochondrial DNA through fluorescence signal, and calculate the mutation load of the variation to complete the detection.

6. The detection method of the mitochondrial DNA high-pathogenic mutation detection kit based on digital PCR according to claim 5, characterized in that, The specific steps of the amplification are: S21. Configure the reaction system using the highly pathogenic mutation detection kit for mitochondrial DNA, put the prepared reaction system into the droplet generation device, and add the oil phase to generate microdroplets for independent PCR reactions; S22. Transfer the droplets to a microdroplet digital PCR instrument and run the PCR program for PCR amplification.

7. The detection method of the mitochondrial DNA high-pathogenic mutation detection kit based on digital PCR according to claim 5, characterized in that The reaction system for detecting each highly pathogenic mutation of the mitochondrial DNA is 25 μl, including 12.5 μL of digital PCR premix, 1 μL of forward primer for amplification, 1 μL of reverse primer, 1 μL of extracted template mitochondrial DNA, 0.5 μL each of mutant probe and normal probe, and 8.5 μL of sterile water.

8. The detection method of the mitochondrial DNA high-pathogenic mutation detection kit based on digital PCR according to claim 5, characterized in that, The PCR program is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, then anneal at the annealing temperature set according to the primer set of each highly pathogenic mutation of the mitochondrial DNA for 60 seconds, cycle 45 times; extension at 98°C for 10 minutes.

9. The detection method of the mitochondrial DNA high-pathogenic mutation detection kit based on digital PCR according to claim 5, wherein, The sample to be tested includes any one of blood, urine, muscle, amniotic fluid, and embryonic cells.

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