A kit for detecting mitochondrial DNA variation based on MLPA-NGS method and application thereof

Abstract

The application discloses a kit for detecting mitochondrial DNA variation based on an MLPA-NGS method and application thereof, and is particularly used for detecting copy number variation (CNV) of each section of human mitochondrial DNA and multiple mutation sites (SNV). The kit comprises a CNV probe composition for human mitochondrial DNA and an SNV probe composition related to diseases, and further comprises a primer composition, which is a universal primer with an index sequence for amplifying the connected probe. The kit prepares each probe into a probe working solution, then takes a DNA sample to be detected, and obtains sequencing data through heating denaturation, incubation with the probe, probe connection, amplification of the universal primer to the connection product, high-throughput sequencing, data analysis by using a self-made program, and sequencing of each disease-related SNV and each section CNV of human mitochondrial DNA. The detection method is simple in operation, low in cost, and high in reliability, and is a good tool for detecting human mitochondrial DNA SNV and CNV.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a kit for detecting mitochondrial DNA variations based on the MLPA-NGS method and its application. Background Technology

[0002] Mitochondria are organelles found in most cells, enclosed by two membranes, and are the primary site of cellular aerobic respiration. Mitochondrial DNA (mtDNA) is the genetic material in mitochondria, and it is a double-stranded circular DNA. A single mitochondrion may contain one or more mtDNA molecules. Normal human mtDNA is 16569 bp in length and contains 37 genes, including 13 genes encoding oxidative phosphorylation respiratory chain complex polypeptides, 2 ribosomal RNAs, and 22 transfer RNAs.

[0003] Due to the lack of multiple damage repair mechanisms found in nuclear genomic DNA, the mutation rate of mtDNA is higher than that of nuclear genomic DNA. During cell division, mutated mtDNA contained in the mitochondria of the mother cell may change in proportion as it randomly separates and enters the daughter cells. mtDNA is maternally inherited; the mtDNA contained in offspring cells generally comes from the mother.

[0004] Mutations in mtDNA can accumulate in somatic cells. Several human genetic diseases have been found to be associated with mtDNA mutations. For example: (1) mutations in the gene MTRNR1, located in the 12S rRNA of mtDNA, such as m.1555A>G and m.1494C>T, can cause deafness after taking aminoglycoside antibiotics; (2) mutations in m.8344A>G and m.3243A>G can cause Leigh syndrome; (3) mtDNA mutations are the molecular basis for the pathogenesis of Leber hereditary optic neuropathy (LHON). Ten primary mutations associated with LHON have been reported, among which m.11778G>A, m.3460G>A, and the gene m.14484T>C account for more than 95% of all mutations.

[0005] The three typical phenotypes caused by mtDNA deletions are Kearns-Sayre syndrome (KSS), Pearson syndrome, and progressive extraocular muscle palsy (PEO), as well as the very rare Leigh syndrome. However, patients with mitochondrial deletion diseases often present with incomplete or overlapping clinical phenotypes, making them unclassifiable as any one of the typical syndromes, which greatly challenges the clinical diagnosis and treatment of these patients. Therefore, developing diagnostic methods for mitochondrial deletions and single-base variations is of significant clinical importance. Summary of the Invention

[0006] To overcome at least one problem in existing technologies, this invention develops a kit for detecting multiple important disease-related mutations and deletions in human mitochondrial DNA (mtDNA) based on the MLPA-NGS principle, providing mitochondrial disease detection services. Specifically, the kit is used for detecting copy number variations (CNVs) and disease-related significant variant sites (SNVs) in human mtDNA. The MLPA-NGS method combines the principles of MLPA and NGS technologies. Its library construction process follows the principles of MLPA (Multiples Ligation-dependent Probe Amplification) technology, and its sequencing process follows the principles of next-generation sequencing technology (NGS). It enables accurate analysis of SNVs and CNVs on genes and is a high-throughput MLPA method. Based on this MLPA-NGS method, multiplex SNV and CNV detection can be performed, thus developing a detection kit suitable for CNV and SNVs in human mtDNA.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention is to provide a composition for detecting mitochondrial DNA variations based on the MLPA-NGS method, comprising a CNV probe composition targeting human mitochondrial DNA and a disease-related SNV probe composition. The CNV probe composition comprises probe pairs respectively designed on multiple regions of mitochondrial DNA, and the SNV probe composition comprises probe sets respectively for detecting variations at multiple sites. The site variations are selected from at least one of m.1555A>G, m.1494C>T, m.8344A>G, m.3243A>G, m.11778G>A, m.3460G>A, and m.14484T>C. Each probe pair and probe set includes a left-side probe and a right-side probe. The composition further comprises a primer composition, which is a pair of universal primers with an index sequence for amplifying the ligated probes. The 5' end of the left-side probe and the 3' end of the right-side probe each contain a universal sequence that serves as the binding sequence during amplification by the universal primers. Specifically, the CNV probe composition comprises probe pairs (each CNV probe pair includes two probes) designed for 27 regions of mitochondrial DNA, and the SNV probe composition comprises seven probe sets (each SNV probe set includes three probes) with site variations of m.1555A>G, m.1494C>T, m.8344A>G, m.3243A>G, m.11778G>A, m.3460G>A, and m.14484T>C. The index sequences in the above universal primers are used to distinguish different samples.

[0009] Further, the CNV probe set comprises sequences as shown in SEQ ID NO.1 to SEQ ID NO.54 for binding template DNA; the SNV probe set comprises sequences as shown in SEQ ID NO.55 to SEQ ID NO.75 for binding template DNA, wherein the left probe in the SNV probe set consists of two probes, one targeting wild-type and the other targeting mutant bases. Each SNV site contains two genotypes: wild-type and mutant, and the proportion of mutants in the detected SNVs can be provided after testing.

[0010] Furthermore, in each of the above probe pairs, a phosphate group is added to the 5' end of the right probe for probe attachment.

[0011] Further, the sequences of the universal primers are shown in SEQ ID NO.76 to SEQ ID NO.77, the universal sequence at the 5' end of the left probe is shown in SEQ ID NO.78, and the universal sequence at the 3' end of the right probe is shown in SEQ ID NO.79. Specifically, the overall sequences of the probes participating in the reaction are: left probe: 5'-universal sequence-template DNA binding sequence-3', right probe: 5'-template DNA binding sequence-universal sequence-3'.

[0012] It is understood that in the selection and design of the CNV and SNV probes described above, based on the same MLPA-NGS method, if new probes are designed on mitochondria by changing the CNV or SNV site, it should not be regarded as an essential change to the probes described in this invention.

[0013] A second aspect of the present invention is to provide a kit for detecting mitochondrial DNA variations based on the MLPA-NGS method, comprising a composition for detecting mitochondrial DNA variations as described in any of the first aspects of the present invention.

[0014] Specifically, the kit prepared using the composition can be used to detect at least one of the following diseases: aminoglycoside deafness, Leigh syndrome, Leber hereditary optic neuropathy, Kearns-Sayre syndrome, Pearson syndrome, and progressive extraocular muscle palsy.

[0015] Furthermore, in the above kit, the CNV probe composition and the SNV probe composition are mixed to obtain a probe working solution, wherein the concentration of each probe in the probe working solution is 0.2–20 fmol / μL. Specifically, appropriate amounts of all SNV and CNV probes are mixed to prepare a mixed solution with a probe concentration of 0.2–20 fmol / μL, and the concentration is optimized based on test results to determine the optimal probe working solution. Specifically, the optimal concentration of the probe working solution is 2 fmol / μL.

[0016] Furthermore, in the above-mentioned kit, the concentration of each universal primer in the primer composition is 2 to 200 pMol. Specifically, a concentration of 5 to 40 pMol is preferred, and a concentration of 20 pMol is more preferred. Each primer is prepared separately without mixing. The upstream and downstream primers are combined differently only when amplifying probe ligation products of different samples to obtain products with different indices that can distinguish between samples.

[0017] Furthermore, the kit also includes at least one of MLPA buffer, ligase, ligase buffer, PCR buffer, dNTP, and PCR enzyme.

[0018] Furthermore, the quantities of each component in the kit are as follows: 5 μL of 50-250 ng DNA sample, 1.5 μL of MLPA buffer, 1.5 μL of probe working solution; 6 μL of ligase buffer, 1 μL of ligase; 5 μL of PCR buffer, 4 μL of dNTP, 0.25 μL of PCR enzyme, and 1 μL each of upstream and downstream universal primers.

[0019] It is understood that appropriate changes may be made to the reagents used, their concentrations, and dosages in the above kits, and these changes are not considered essential alterations to the kits themselves.

[0020] Furthermore, in the above-mentioned kit, each CNV probe pair and each SNV probe group has 1 to 4 characteristic sequences for the detection of the SNV or CNV, specifically 2 characteristic sequences; each probe pair or probe group includes a left probe and a right probe, and the characteristic sequences are base sequences of predetermined lengths extracted from the left part of the non-universal primer binding region of the left probe and from the left and right sides of the connection position between the left and right probes; wherein, the sequence length of the characteristic sequence is 10-30 bases, specifically 12-24 bases, and the characteristic sequences are separated by at least one base (e.g., 2 to 20 bases, specifically 3, 4, 5... 19, 20 bases, etc.). In one specific implementation, the specific sequence of each probe pair consists of two segments. The first segment is 12 bases taken from the left side of the region where the left probe binds to the template. The second segment is 6 bases and 12 bases respectively taken from the left and right sides of the connection position of the left and right probes (for CNV, the total length is 18 bases) or 12 bases each (for SNV, the total length is 24 bases).

[0021] Specifically, after performing MLPA-NGS testing on subjects using the aforementioned kit, in order to analyze the number of reads for each SNV or CNV allele in the fastQ file, it is necessary to first design the characteristic sequences of the corresponding probes as described above. In one specific implementation, each SNV probe set contains two sets of characteristic sequences, each set targeting the two alleles of the SNV. Each set of characteristic sequences contains two segments with lengths of 12 and 24 bases, respectively; the two sets of probe characteristic sequences for the two alleles differ only by one base in the second specific sequence. The above constitutes the characteristic sequences for all detection sites. If the probe for one SNV containing degenerate bases is considered as two sets of probes, then any set of characteristic sequences corresponds one-to-one with the corresponding probe. Each CNV probe pair contains only one set of characteristic sequences consisting of two segments with lengths of 12 and 18 bases, respectively.

[0022] It is understood that when designing characteristic sequences, the number, length, position, etc. of the sequences can be appropriately adjusted. Provided that the analysis of the amplification products is guaranteed, such adjustments should not be considered as essential changes to the characteristic sequences described in this invention.

[0023] Based on the probe and primer sequences derived from Illumina next-generation sequencing adapter sequences, amplification itself is also a library preparation process before Illumina sequencing. Understandably, adjustments can be made to use other suitable high-throughput sequencing platforms, such as Roche / 454 sequencing, ABI SOLiD sequencing, Ion Torrent sequencing, and CG sequencing.

[0024] The sequence information involved in the above kit is shown in the table below:

[0025] Table 1 – Information on Probes, Primers, and Characteristic Sequences

[0026]

[0027]

[0028]

[0029]

[0030] A third aspect of the present invention is to provide a method of using a kit as described in any of the second aspects of the present invention, comprising the steps of: denaturing a DNA sample; hybridizing an SNV probe composition and a CNV probe composition with the DNA sample; ligating the hybridization probes using a ligase and a ligase buffer; performing PCR amplification of the probe ligation product with a primer composition; and sequencing the PCR amplification product to obtain sequencing results.

[0031] Furthermore, the specific steps for using the above-mentioned reagent kit include:

[0032] Step S1, DNA denaturation and probe hybridization: Denature the DNA sample; mix MLPA buffer and probe working solution thoroughly and perform hybridization reaction. The reaction program is as follows: 95℃ for 2 min, 65℃ to 55℃, incubate for 1 hour for each degree drop, and then maintain at 54℃ for 3-10 hours to obtain the hybridization product.

[0033] Step S2: Prepare a ligase main solution containing ligase buffer, add the ligase to the ligase main solution and mix well, heat at 54°C for 1 minute, add to the hybridization product at a constant temperature of 54°C and mix well, continue incubation for 25 minutes, heat at 98°C for 5 minutes, and cool to 20°C and pause to achieve the ligation of the hybridization probe and obtain the ligation product.

[0034] Step S3: Perform PCR amplification reaction with the ligation product and PCR reaction solution. The PCR reaction solution includes PCR reaction buffer, dNTPs, universal upstream and downstream primers with indexes, and Taq enzyme. The reaction conditions are: 95℃ for 30s, 60℃ for 30s, 72℃ for 60s, 35 cycles; incubate at 72℃ for 20min, and finally incubate at 15℃ to obtain the PCR amplification product.

[0035] Step S4: Take an appropriate amount of sample from each of the PCR amplification products, mix them evenly, and send them to an NGS sequencer for sequencing to obtain sequencing results (specifically, a fastQ file).

[0036] In one specific implementation scheme, the above kit can be used as follows: (1) On the first day, DNA denaturation and probe hybridization are performed by adding 5 μL of DNA sample (50-250 ng) to a PCR tube, denaturing at 98°C for 5 minutes, and cooling to 25°C. Mix 1.5 μL of MLPA buffer (from MRC-Holland) with 1.5 μL of probe working solution, add to the sample tube, and mix thoroughly. (2) Continue the thermal cycling program: 95°C for 2 minutes, 65°C to 55°C, incubating for 1 hour for each degree Celsius drop, and then maintaining at 54°C for 3-10 hours. (3) On the second day, prepare the ligase-65 main solution: each reaction contains 25 μL. dH2O + 3μL ligase buffer B + 3μL ligase buffer A, then add 1μL ligase-65 enzyme, and gently pipette to mix evenly; buffers A, B and ligase-65 are all from MRC-Holland; place the mixture in a PCR instrument (54℃) and heat for 1 minute, then add to the PCR tube that is incubating at 54℃, mix well, and continue incubation for 25 minutes; (4) heat the above reaction at 98℃ for 5 minutes, and cool to 20℃ to pause, and remove the PCR tube; (5) the PCR enzyme used in PCR amplification is HS Taq enzyme from Takara; 50μL PCR reaction solution includes the following components: 5μL reaction buffer, 4μL 1 μL each of dNTPs, universal upstream and downstream primers with index (20 pMol), 10 μL of ligation product, 0.25 μL of enzyme, and water to make up to 50 μL; the reaction conditions are: 95℃ for 30s, 60℃ for 30s, 72℃ for 60s, 35 cycles; incubate at 72℃ for 20min, and finally incubate at 15℃; (6) Take an appropriate amount of sample from each PCR amplification product, mix them evenly, freeze and store them, and send them to the NGS sequencer produced by Illumina for sequencing to obtain the sequencing fastQ file.

[0037] It is understood that appropriate adjustments can be made to the reagents, temperature, incubation time, sequencing instruments, etc., in the above-described method of use. These adjustments should not be considered as essential changes to the method of use of the above-described kit, provided that they do not affect the detection and analysis.

[0038] Furthermore, in the above-described method of use, each probe pair and each probe group has a characteristic sequence for mitochondrial detection, the design of which and its sequence information are detailed in the first aspect of the present invention described above.

[0039] Furthermore, the above-described method of use also includes a step of analyzing the results based on the sequencing results, performing at least one of the following analyses: analyzing the number of probe reads, determining the SNV ratio, and determining the CNV status of mitochondrial DNA.

[0040] Furthermore, in the above method of use, when used to analyze the number of probe reads and determine the SNV ratio, it includes the following steps: taking the combination of 1 to 4 (specifically 2) characteristic sequences of each pair of probes as the text to be searched, and counting the number of reads containing each text to be searched (taking each read in the fastQ file as the search object, and using the findall function in Python regular expressions as the search function); wherein, for each SNV, if the sum of the number of reads of the two alleles is less than 100, it is considered unqualified and not analyzed; through the quality control SNV, for each sample, the mutant reads detected at the site are divided by the total number of reads of the site SNV to obtain the genotyping value of the sample SNV, and the value range of the genotyping value is [0,1].

[0041] Furthermore, when used to determine the SNV proportion, the SNV is calculated as follows: the genotyping value of a sample's SNV minus the mean genotyping value of the negative samples. If the result is positive and the ratio to the standard deviation is greater than 2, then within a 95.45% confidence interval, the SNV of that sample exhibits variation, and the proportion of variation is the genotyping value of the SNV minus the mean genotyping value of the negative samples. If the result is positive and the ratio to the standard deviation is greater than 3, then within a 99.73% confidence interval, the SNV of that sample exhibits variation, and the proportion of variation is the genotyping value of the SNV minus the mean genotyping value of the negative samples.

[0042] Furthermore, in the above method of use, when used to determine whether CNVs exist in mitochondrial DNA, the reads of each CNV in the sample to be tested are compared with the reads of the corresponding CNV in the known negative sample. The ratio is calculated to determine whether there is a change greater than a certain range, and whether the changes have a common relationship between adjacent CNV sites, so as to determine whether CNVs exist on mitochondrial DNA.

[0043] Furthermore, when determining the presence of CNVs in mitochondrial DNA, the reads of each CNV in the test sample are compared with the corresponding CNV reads in a known negative sample to obtain the ratio of the test sample. A t-test (two-tailed, heteroscedastic) is then performed between any two or more geographically adjacent ratios and all ratios of the sample. The significance level is α = 0.01; a value less than this is considered significant, indicating the presence of a CNV. Additionally, to determine the presence of a CNV at a single locus, the absolute value of the difference between the CNV ratio at that location and the mean is compared with the standard deviation. If the difference is greater than three standard deviations, a CNV is considered present. However, since the probe is significantly affected by SNVs, interference from SNVs should be excluded when determining the presence of CNVs at a single locus.

[0044] It is understood that this invention is not limited to the CNVs and SNVs currently detected in mitochondria based on the MLPA-NGS principle. Other CNVs and SNVs on mitochondria designed based on the MLPA-NGS principle, if following the same method, are not considered to be a substantial change to the above-described analytical methods.

[0045] A fourth aspect of the present invention provides an application of a composition as described in any of the first aspects of the present invention, or a kit as described in any of the second aspects of the present invention, wherein the kit is used to test a mitochondrial DNA sample of a subject for MLPA-NGS detection.

[0046] Furthermore, in the above applications, at least one of the following results is obtained by MLPA-NGS detection: probe read count, SNV classification, and CNV status.

[0047] Furthermore, in the above applications, DNA sample preparation includes: collecting peripheral blood and preparing a DNA sample using a blood DNA extraction kit. It is understood that other forms of samples may also be used. Specifically, genomic DNA (gDNA) is extracted from whole blood samples or other materials.

[0048] Understandably, the analysis of the above results may be for non-diagnostic purposes, used to obtain intermediate results of the correlation analysis.

[0049] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects:

[0050] This invention provides a kit and analytical method based on the MLPA-NGS principle for CNV detection and disease-related SNV detection in mitochondrial DNA. The kit consists of probe working solution, MLPA buffer, heat-resistant ligation solution, universal primers, and PCR reaction solution, and its operation is similar to MLPA technology. The fastQ file obtained after sequencing can be automatically analyzed. The analysis includes SNV genotyping and CNV detection. This kit uses next-generation sequencing, providing a large amount of information, high sensitivity for SNV and CNV detection, low reagent cost, simple operation, and low skill requirements for operators, facilitating large-scale deployment. Attached Figure Description

[0051] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings:

[0052] Figure 1 This is a schematic diagram of the probe characteristics of the MLPA-NGS method for SNV detection in one embodiment of the present invention;

[0053] Figure 2 This is a schematic diagram of the CNV analysis results performed in one embodiment of the present invention, using sample 10 as a control and sample 9 as an example. Detailed Implementation

[0054] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0055] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0056] In the following embodiments, the design concept for detecting copy number variation (CNV) and disease-associated significant variant sites (SNV) of human mitochondrial DNA based on the MLPA-NGS method is briefly described as follows: First, genomic DNA (gDNA) is extracted from whole blood samples or other materials. Since mtDNA is not fundamentally different from genomic DNA, it will be extracted together. The extracted gDNA is subjected to MLPA-NGS operation, including unwinding, template and probe incubation, probe ligation, and probe amplification. Then, the amplification products are subjected to high-throughput sequencing. A self-written program is used to analyze the sequencing results for mutation sites and CNVs to provide the variation status of the detection sites.

[0057] When designing an SNV probe, its SNV detection mode is as follows: Figure 1 As shown, the design principle of SNV is: for a certain SNV with wild-type and mutant alleles A and G respectively, when the degenerate bases designed on the probe hybridize with the corresponding T and C on the template, they can be successfully ligated; if they do not match on the template, they cannot be successfully ligated or the ligation rate is low. SNVs are detected based on this principle.

[0058] Example 1 - Design and Use of the Reagent Kit

[0059] This embodiment designs a kit for detecting mitochondrial DNA variations based on the MLPA-NGS method and describes the operation method of the kit. The design specifically includes:

[0060] (1) Extraction of gDNA from blood samples;

[0061] Blood samples were extracted using a blood genomic DNA extraction kit (centrifuge column type, catalog number DP318) manufactured by Tiangen Biotech (Beijing) Co., Ltd. The standard procedure is as follows: before use, add anhydrous ethanol to buffer GD and wash buffer PW according to the instructions, and affix a label indicating that ethanol has been added to the bottle.

[0062] 1) Add 1.5 ml of peripheral blood to 5 ml of erythrocyte lysis buffer and incubate at 4°C for 10 minutes. Invert the container several times during this period to mix. Centrifuge at 1200 RPM for 10 minutes and discard the supernatant. Carefully observe the number of white blood cells at the bottom of the tube.

[0063] 2) Add 20 μL of proteinase K solution and mix well.

[0064] 3) Add 200 μL of buffer solution GB, mix thoroughly by inverting, and incubate at 56°C for 10 minutes, inverting and mixing several times during the incubation period. The solution should become clear (if the solution is not completely clear, please extend the lysis time until the solution becomes clear).

[0065] 4) Add 200 μL of anhydrous ethanol and mix thoroughly by inverting the container. At this point, flocculent precipitate may appear.

[0066] 5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column CB3 in the collection tube), centrifuge at 12000 RPM (13,400 x g) for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column CB3 into the collection tube.

[0067] 6) Add 500 μL of buffer GD to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 RPM (13,400 x g) for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column CB3 into the collection tube.

[0068] 7) Add 700 μL of washing buffer PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 RPM (13,400 x g) for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column CB3 into the collection tube.

[0069] 8) Add 500 μL of wash buffer PW to the adsorption column CB3, centrifuge at 12000 RPM (13, 400 x g) for 30 seconds, and discard the waste liquid in the collection tube.

[0070] 9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 RPM (13,400 x g) for 2 minutes, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0071] 10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 100 μL of elution buffer TB to the center of the adsorption membrane, incubate at room temperature for 2–5 minutes, centrifuge at 12000 RPM (13, 400 x g) for 2 minutes, and collect the solution into the centrifuge tube.

[0072] 11) Discard the adsorption column. Clearly label the centrifuge tubes.

[0073] Measure DNA concentration and maintain DNA samples at -80 degrees Celsius.

[0074] (2) Preparation of working solution for mtDNA mutation detection probe

[0075] Following the principles of MLPA-NGS technology, a pair or set of probes is designed for each analyte site and region. Each pair or set of probes includes a left probe (indicated by L) and a right probe (indicated by R), which bind seamlessly to the template. After probe design is complete, a phosphate group is added to the 5' end of the right probe during synthesis.

[0076] Due to the large amount of variation in the D-loop region, CNV probes were not designed in this region. The probe pair sequences that bind to the template in the designed CNV probe composition are shown in SEQ ID NO.1 to SEQ ID NO.54. In the probe sequences, the 5' end of the left probe (L probe) has a universal sequence: 5'-CCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO.78), and the 3' end of the right probe (R probe) has a universal sequence: 5'-TCCAACCCTTAGGGAACCCCGATC-3' (SEQ ID NO.79), to facilitate amplification.

[0077] The probe sequences that bind to the template in the designed SNV probe composition are shown in SEQ ID NO. 55 to SEQ ID NO. 75. Each SNV probe set includes three probes, with the left probe consisting of two probes targeting wild-type and mutant bases, respectively. In the probe sequences, the left probe (L probe) has a universal sequence at its 5' end: 5'-CCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO. 78), and the right probe (R probe) has a universal sequence at its 3' end: 5'-TCCAACCCTTAGGGAACCCCGATC-3' (SEQ ID NO. 79), to facilitate amplification.

[0078] After each CNV probe and SNV probe is synthesized, it is dissolved and mixed with distilled water to prepare a mixed solution with a concentration of 0.2-20 fmol / uL for each probe. The concentration is optimized according to the test results (the optimal concentration is 2 fmol / uL) and used as the probe working solution.

[0079] (3) Design the analytical sequence (characteristic sequence) of the probe.

[0080] After adjacent probes complete ligation, amplification, and sequencing, sequencing results are obtained. These results are stored in a fastQ file, containing the sequence and related information for each probe fragment; these sequences are called reads. To analyze the number of reads amplified by the designed probes, the characteristic sequences of the probes are searched in the fastQ file. Reads containing these specific sequences are categorized as belonging to that probe and counted. Each probe pair's specific sequence consists of two segments: the first segment is 12 bases taken from the left side of the left probe's binding region to the template; the second segment is 6 bases to the left and 12 bases to the right of the left probe's ligation site (for CNVs, this is a total length of 18 bases) or 12 bases to the left and right of the right probe's ligation site (for SNVs, this is a total length of 24 bases). The two sequences are separated by at least one base. Relevant characteristic sequence information is shown in the characteristic sequence information section of Table 1.

[0081] (4) Experimental operation procedures for the reagent kit;

[0082] The library preparation steps are as follows:

[0083] On day one, DNA denaturation and probe hybridization were performed as follows: 5 μL of DNA sample (50-250 ng) was added to a PCR tube, denatured at 98°C for 5 minutes, and then cooled to 25°C. 1.5 μL of MLPA buffer (from MRC-Holland) was mixed with 1.5 μL of the prepared probe working solution and added to the sample tube, mixing thoroughly. The thermal cycling program was continued: 95°C for 2 minutes, then 65°C to 55°C, incubating for 1 hour for each degree Celsius decrease, and finally maintaining at 54°C for 3-10 hours. On day two, the ligase-65 main solution was prepared: each reaction contained 25 μL dH₂O + 3 μL ligase buffer B + 3 μL ligase buffer A, followed by 1 μL of ligase-65 enzyme. The mixture was gently pipetted to mix thoroughly. Buffers A and B, and ligase-65 were all from MRC-Holland. Heat the mixture in a PCR instrument (54℃) for 1 minute, then add it to the PCR tube that is incubating at a constant temperature of 54℃, mix well, and continue incubation for 25 minutes. Heat the above reaction at 98℃ for 5 minutes, then cool to 20℃ and pause. Remove the PCR tube.

[0084] For PCR amplification, the PCR enzyme used was HS Taq enzyme from Takara (catalog number: R007Q). A 50 μL PCR reaction solution contained the following components: 5 μL reaction buffer, 4 μL dNTPs, 1 μL each of forward and reverse primers (20 pMol), 10 μL ligation product, 0.25 μL enzyme, and water to a final volume of 50 μL. The sequences of the forward and reverse primers are shown in SEQ ID NO.76 and SEQ ID NO.77, respectively. The poly(N) sequences in both primers are index sequences used to distinguish samples. The reaction conditions were: 95℃ for 30 s, 60℃ for 30 s, 72℃ for 60 s, for 35 cycles; incubation at 72℃ for 20 min, and a final incubation at 15℃. Take an appropriate amount of sample from each PCR amplification product, mix them thoroughly, freeze and store them, and send them to Nanjing Novogene Biotechnology Co., Ltd. for sequencing. Use Qubit 2.0 to perform preliminary quantification of library concentration, use Agilent 2100 to detect the integrity of library DNA fragments and the size of inserted fragments, and use an Illumina high-throughput sequencer (such as HiSeq2500 / HiSeq4000 / HiSeqX / MiSeq) to perform paired-end 150bp sequencing to obtain the sequencing fastQ file.

[0085] Example 2 – Analysis of Sequencing Results from the Reagent Kit

[0086] This embodiment analyzes the results from the FastQ file obtained in Example 1. Sequencing results sent by the sequencing company are typically compressed files; after decompression, the forward sequencing file is >0.1G. The analysis uses the decompressed forward sequencing file as the analysis file, and the specific analysis steps include:

[0087] The combination of two characteristic sequences of each pair of probes is used as the text to be searched. Each read in the fastQ file is used as the search object. The findall function in Python regular expressions is used as the search function to count the number of reads containing each text to be searched.

[0088] For each SNV, if the sum of read counts for the two alleles is less than 100, it is considered substandard and not analyzed. For quality control SNVs, the genotyping value of each sample is obtained by dividing the number of mutant reads by the total number of reads for that sample's SNVs. The genotyping value ranges from [0,1]. For negative samples, the mean plus or minus the standard deviation of each SNV is calculated, which represents the normal range for a negative SNV. The data shows that in negative samples, the mean of this genotyping value is generally less than 0.05.

[0089] The SNV is calculated as follows: the SNV genotype value of a sample minus the mean genotype value of the negative samples. If the result is positive and the ratio to the standard deviation is greater than 2, then within a 95.45% confidence interval, the SNV of that sample exhibits variation, and the proportion of variation is the SNV genotype value minus the mean genotype value of the negative samples. If the result is positive and the ratio to the standard deviation is greater than 3, then within a 99.73% confidence interval, the SNV of that sample exhibits variation, and the proportion of variation is the SNV genotype value minus the mean genotype value of the negative samples.

[0090] When determining the presence of CNVs in mitochondria, to ascertain the existence of a CNV at a single site or between consecutive sites, the ratios of the CNVs at corresponding sites in the test sample and the negative sample are calculated. The analysis examines whether there are significant variations in these ratios, and whether these variations share commonalities among consecutive sites. Specifically, the reads of each CNV in the test sample are compared with the reads of the corresponding CNVs in a known negative sample to obtain the ratio for the test sample. A t-test (two-tailed, heteroscedastic) is then performed between any two or more adjacent ratios and all ratios in the sample. The significance level is α = 0.01; values ​​less than this are considered significant, indicating the presence of a CNV. To determine the presence of a CNV at a single site, the absolute value of the difference between the CNV ratio at that site and the mean is compared with the standard deviation. If the difference exceeds three standard deviations, a CNV is considered present. However, because the probe is significantly affected by SNVs (Specially Occurring Viruses), extreme caution should be exercised when determining the presence of CNVs at a single site, and SNV interference should be excluded.

[0091] Example 3 – Validation of the analytical method for the kit

[0092] This embodiment uses certain samples to validate the analysis method in Example 2. Specifically, samples with known clinical information obtained through capture sequencing are used for testing, as follows: Sample 1 has m.1555A>G; Sample 2 has m.1494C>T; Sample 3 has m.8344A>G; Sample 4 has m.3243A>G; Sample 5 has m.11778G>A; Sample 6 has m.3460G>A; Sample 7 has m.14484T>C; Sample 8 has a deletion in the m:7503-15621 region; Sample 9 is negative; Sample 10 is negative. All samples are from the hospital's sample bank and were obtained with informed consent from patients who came to the hospital for treatment. The informed consent forms were approved by the hospital's ethics committee in advance.

[0093] The analysis results of the above samples are as follows:

[0094] (1) Basic statistics: The mtDNA of 10 samples (mtDNA-1 to 10) were constructed and sequenced. Basic statistics were performed. The results showed that the effective reads in the target region accounted for more than 80%, the effective data was more than 80MB, and the average depth was more than 5000x. The results met the detection requirements.

[0095] Sequencing all reads Analyzable reads Analyzable proportions Sample 1 511037 413940 81％ Sample 2 470899 390846 83％ Sample 3 449700 368754 82％ Sample 4 436772 375624 86％ Sample 5 596945 501434 84％ Sample 6 525360 462317 88％ Sample 7 565072 485962 86％ Sample 8 469486 389674 83％ Sample 9 536725 456217 85％ Sample 10 543920 446015 82％

[0096] Analysis revealed that the CNV sequencing depth of each sample was >5000×; and the proportion of indexes that could be assigned to specific detection sites was >80%.

[0097] (2) Comparison of SNV and CNV analysis with sample reports;

[0098] The results of MLPA-NGS analysis of 10 samples were compared with those of the capture + NGS analysis method in the existing technology (the results were sent to Shanghai KingMed Diagnostics Laboratory). The table below shows the results of the MLPA-NGS analysis of the samples and their comparison with the capture results.

[0099]

[0100] As shown in the table above, the SNV and CNV test results for the two negative samples remained negative, indicating consistent results. The test results for the seven point mutation samples were consistent with the capture method; although the mutation ratio varied, the error was within acceptable limits. The CNV analysis results for the one missing sample showed inconsistencies between the two algorithms: the capture + NGS result indicated a missing region in the 7503-15621 area, while the MLPA-NGS result showed a missing region in the 7617-15071 area. This is because the CNV probes designed for MLPA-NGS are discontinuous, only showing variations at the probe's location. Therefore, the results of the two methods are considered largely consistent and highly reliable.

[0101] Using sample 10 as a control, mitochondrial DNA analysis was performed on all samples. Taking sample 9 as an example, the CNV analysis results are as follows: Figure 2 As shown, the CNV variation range at each site is within 30%, which meets the quality control requirements.

[0102] As can be seen from the above embodiments, the kit developed in this invention for detecting copy number variations (CNVs) and multiple mutation sites (SNVs) in various regions of human mitochondrial DNA (mtDNA) can be analyzed using a self-developed program to obtain the sequencing information of disease-related SNVs and CNVs in various regions of human mtDNA. Compared with existing methods for mitochondrial mutation and copy number detection (e.g., Xing Jinliang, Mo Qinqin, Guo Shanshan: A method for simultaneous detection of mtDNA copy number and mutation based on NGS (application number: 201910576636.3)), the method involved in this invention can detect deletions or additions of segments in mitochondrial DNA, not just changes in the copy number of mitochondrial DNA relative to genomic DNA. Capture or amplification methods can lead to significant variations in the copy number of target fragments relative to the template, resulting in distortion of CNV detection. MLPA is the recognized gold standard for CNV detection. MLPA-NGS technology, developed based on MLPA, inherits MLPA's CNV detection capabilities and has higher accuracy than other existing NGS library preparation methods. In mutation detection, compared with existing capture methods or PCR amplification methods (e.g., Pan Shi, Xuan Wei, Li Lulu, Wang Qicheng, Lin Bin, Qian Haifeng, Sun Zhibin, Song Jinyu: Primers, methods and kits for human mitochondrial whole genome detection (application number 201911286581.9)), the method described in this invention has a clear detection target and can obtain more accurate data under the same sequencing volume conditions. Furthermore, by detecting only the identified sites and not sites outside the target, it avoids being troubled by the pathogenicity of accidental mutations, which is beneficial for the application for medical device registration certificates. In summary, its detection method is simple to operate, low in cost, and highly reliable, making it an excellent tool for detecting human mtDNA SNVs and CNVs.

[0103] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for detecting mitochondrial DNA variations based on the MLPA-NGS method, characterized in that, The composition comprises a CNV probe composition targeting human mitochondrial DNA and a disease-related SNV probe composition. The CNV probe composition comprises probe pairs designed for multiple regions of mitochondrial DNA, and the SNV probe composition comprises probe sets for detecting multiple site variations. The site variations are selected from at least one of m.1555A>G, m.1494 C>T, m.8344 A>G, m.3243A>G, m.11778 G>A, m.3460 G>A, and m.14484 T>C. Each probe pair and probe set includes a left-side probe and a right-side probe. The composition also includes a primer composition, which is a pair of universal primers with an index sequence for amplifying the ligated probes. The 5' end of the left-side probe and the 3' end of the right-side probe each contain a universal sequence that serves as the binding sequence during amplification by the universal primers. The CNV probe composition comprises sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 54 for binding template DNA; the SNV probe composition comprises sequences as shown in SEQ ID NO. 55 to SEQ ID NO. 75 for binding template DNA, wherein the left probe in each SNV probe set consists of two probes targeting wild-type and mutant bases respectively; the sequences of the universal primers are shown in SEQ ID NO. 76 to SEQ ID NO. 77, the universal sequence at the 5' end of the left probe is shown in SEQ ID NO. 78, and the universal sequence at the 3' end of the right probe is shown in SEQ ID NO.

79.

2. A kit for detecting mitochondrial DNA variations based on the MLPA-NGS method, characterized in that, The kit contains the composition for detecting mitochondrial DNA variations as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The CNV probe composition and the SNV probe composition are mixed to obtain a probe working solution, wherein the concentration of each probe in the probe working solution is 0.2~20 fmol / μL; and / or, in the primer composition, the concentration of each universal primer is 2~200 pMol; and / or, the kit further includes at least one of MLPA buffer, ligase, ligase buffer, PCR buffer, dNTP, and PCR enzyme.

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