Human mitochondrial gene mutation detection method, primer set, kit and application thereof

Abstract

The present invention discloses a method for detecting human mitochondrial gene mutation, a primer pair, a kit and its application, and belongs to the field of high-throughput sequencing technology. The present invention provides a set of primers, which can specifically match a specific region of the human mtDNA genome, and the amplicons generated by the primers can cover all sequences including about 400bp near the origin of the mtDNA genome, thereby realizing full gene detection of human mtDNA. In addition, the primer pair can effectively reduce the problem of over-amplification of primers of adjacent amplicons in overlapping regions, avoid the occurrence of primer dimers, realize the completion of mtDNA gene detection of samples in the same tube, reduce costs, and reduce potential risks of operational errors.

Description

Technical Field

[0001] The present invention belongs to the technical field of high-throughput sequencing, and specifically relates to a primer set and a kit for detecting human mitochondrial gene mutations based on second-generation sequencing technology and applications thereof, as well as a method for high-throughput detection of mitochondrial gene mutations. Background Art

[0002] Mitochondria are an important organelle in eukaryotic cells, providing a place for oxidative phosphorylation in cells, and playing a vital role in cell energy metabolism, apoptosis, maintaining intracellular calcium and iron ion balance and other life activities. The human mitochondrial DNA (mtDNA) genome is 16569bp long and has a wide range of applications in maternal kinship identification, forensic testing, and human genetic disease screening.

[0003] Since mtDNA often exists in an environment with high levels of reactive oxygen and lacks histone protection and an effective DNA repair system, its mutation frequency is about 10 times higher than that of the nuclear genome. Currently, clinical testing of mtDNA only focuses on mutation and deletion screening of a few common mitochondrial gene sites, with a low positive detection rate, and it is difficult for most patients to obtain an accurate etiology diagnosis. Therefore, the accurate detection of free mtDNA mutations has unique advantages as a new marker.

[0004] The traditional method for detecting mtDNA genome is the first-generation sequencing, namely the Sanger method, which has the disadvantages of short sequence that can be measured in a single reaction, high sample quality requirements, low throughput, etc., and is time-consuming and labor-intensive. In recent years, the emerging second-generation sequencing technology (NGS) has solved the problem of high-throughput sequencing. Currently, there are two NGS technology routes for detecting mtDNA: (1) using one or a few pairs of primers to amplify the mtDNA sequence by long-fragment amplification, and then mechanically or enzymatically cutting the amplified product and adding sequencing adapters for sequencing; (2) using multiple amplification methods to directly amplify multiple short fragments suitable for NGS sequencing length, and then adding adapters for sequencing. In the two methods described above, the former has a long amplicon and requires the application of a special high-fidelity amplification enzyme. The subsequent interruption and connection also require special enzymes and related reagents, which has a high overall cost and a long operation cycle. The latter has an important problem to be solved, that is, in order to ensure the continuity of the sequencing area, there needs to be a certain overlap between the short amplicons. When the primers of adjacent amplicons are in the same tube, the overlapping area will be over-amplified, resulting in a serious reduction in the target amplification product and the appearance of primer dimers. Normally, to avoid this problem, one sample can only be divided into two or even more tubes, resulting in increased costs and potential risk of operational errors. Summary of the invention

[0005] Based on the above background, the present invention provides a set of primer pairs for multiplex amplification detection of mtDNA, which can effectively reduce the problem of over-amplification of primers of adjacent amplicons in overlapping regions, avoid the occurrence of primer dimers, and achieve mtDNA gene detection of samples in the same tube, thereby reducing costs and reducing potential risks of operational errors.

[0006] One object of the present invention is to provide a primer set, which consists of 131 pairs of primers, and the primer set can specifically match a specific region of the human mtDNA genome. The amplicons generated by the 131 pairs of primers can cover all sequences including about 400bp near the origin of the mtDNA genome, and the 131 pairs of primers can detect the entire human mtDNA genome; the second object of the present invention is to provide a use of the primer set in preparing a product for detecting gene mutations in the human mtDNA genome; the third object of the present invention is to provide a kit, which comprises the primer set; and another object of the present invention is to provide a method for detecting gene mutations in the human mtDNA genome that is not for the purpose of disease diagnosis or treatment.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In the first aspect, the present invention provides a primer set, which is composed of primer pairs No. 1-131 in Table 1, wherein the forward amplification primers in the primer set are a combination of nucleotide sequences shown in SEQ ID NO.1-131, and the reverse amplification primers in the primer set are a combination of nucleotide sequences shown in SEQ ID NO.132-262.

[0009] In order to solve the problem of over-amplification of primers of adjacent amplicons in the overlapping region, the technical personnel of the present invention unexpectedly found that connecting a specific sequence independently developed by the present invention to the 5' end of the reverse amplification primers shown in SEQ ID NO.132-261 can effectively reduce the problem of over-amplification in the overlapping region and also reduce primer dimers. Specifically, the technical solution is: connecting the nucleotide sequences shown in SEQ ID NO.263-SEQ ID NO.392 to the 5' ends of the reverse amplification primers of the 1st to 130th primer pairs respectively.

[0010] Illustratively, the specific connection relationship between the above-mentioned reverse amplification primer and the specific sequence is as follows: the 5' end of the reverse amplification primer shown in SEQ ID NO.132 is connected to the nucleotide sequence shown in SEQ ID NO.263; the 5' end of the reverse amplification primer shown in SEQ ID NO.133 is connected to the nucleotide sequence shown in SEQ ID NO.264; and so on and so forth until the 5' end of the reverse amplification primer shown in SEQ ID NO.261 is connected to the nucleotide sequence shown in SEQ ID NO.392.

[0011] More preferably, the 5' ends of the forward amplification primer and the reverse amplification primer in the primer set are also connected to a sequencing adapter pairing fragment, and the sequencing adapter pairing fragment comprises a sequence that differs by no more than 3 nucleotides from 15-25 consecutive nucleotides from the 3' end of the sequencing adapter of the illumina, Ion or MGI platform, and the sequencing adapter pairing fragment enables the multiplex PCR amplification products to be added with sequencing adapters of the corresponding sequencing platform through a PCR method.

[0012] In some embodiments of the present invention, the sequencing adapter-paired fragment comprises a sequence that differs by no more than 2 nucleotides from 15-25 consecutive nucleotides from the 3' end of the sequencing adapter of the illumina, Ion or MGI platform.

[0013] In some embodiments of the present invention, the sequencing adapter-paired fragment comprises a sequence that differs by no more than 1 nucleotide from 15-25 consecutive nucleotides from the 3' end of the sequencing adapter of the illumina, Ion or MGI platform.

[0014] In some embodiments of the present invention, the sequencing adapter-paired fragment comprises a sequence identical to 15-25 consecutive nucleotides starting from the 3' end of the sequencing adapter of the illumina, Ion or MGI platform.

[0015] The Illumina, Ion or MGI platform sequencing adapters described in the present invention are existing commercial sequencing adapters known to those skilled in the art. The nucleotide sequence of the sequencing adapter is prior art, and the sequencing adapters can be purchased through conventional commercial channels.

[0016] In a specific embodiment of the present invention, the sequencing adapter pairing fragment suitable for the illumina platform is a combination of the nucleotide sequences shown in (a1) and (a2), the 5' end of the forward amplification primer in the primer set is connected to one of the nucleotide sequences shown in (a1) or (a2), and the 5' end of the reverse amplification primer is connected to the other of the nucleotide sequences shown in (a1) or (a2).

[0017] (a1) the nucleotide sequence shown in SEQ ID NO.393;

[0018] (a2) The nucleotide sequence shown in SEQ ID NO.394.

[0019] Specifically, the connection method of the sequencing adapter paired fragments suitable for the Illumina platform is selected from any one of 1)-4):

[0020] 1) The 5' end of the forward amplification primer in the primer pair 1-131 shown in Table 1 is connected to the nucleotide sequence shown in (a1), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (a2);

[0021] 2) The 5' end of the forward amplification primer in the primer pair 1-131 shown in Table 1 is connected to the nucleotide sequence shown in (a2), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (a1);

[0022] 3) The 5' end of the forward amplification primer in the odd-numbered primer pair shown in Table 1 is connected to the nucleotide sequence shown in (a1), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (a2); the 5' end of the forward amplification primer in the even-numbered primer pair is connected to the nucleotide sequence shown in (a2), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (a1);

[0023] 4) The 5' end of the forward amplification primer in the odd-numbered primer pair shown in Table 1 is connected to the nucleotide sequence shown in (a2), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (a1); the 5' end of the forward amplification primer in the even-numbered primer pair is connected to the nucleotide sequence shown in (a1), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (a2).

[0024] The odd-numbered primer pairs of the present invention refer to the primer pairs shown in Nos. 1, 3, 5, 7 to (2n+1) in Table 1, and the even-numbered primer pairs refer to the primer pairs shown in Nos. 2, 4, 6, 8 to 2n in Table 1, where n=65.

[0025] In another specific embodiment of the present invention, the sequencing adapter pairing fragment suitable for the MGI platform is a combination of the nucleotide sequences shown in (b1) and (b2), the 5' end of the forward amplification primer in the primer set is connected to one of the nucleotide sequences shown in (b1) or (b2), and the 5' end of the reverse amplification primer is connected to the other of the nucleotide sequences shown in (b1) or (b2).

[0026] (b1) the nucleotide sequence shown in SEQ ID NO.395;

[0027] (b2) The nucleotide sequence shown in SEQ ID NO.396.

[0028] Specifically, the connection method of the sequencing adapter paired fragments suitable for the MGI platform is selected from any one of 1) to 4):

[0029] 1) The 5' end of the forward amplification primer in the primer pair 1-131 shown in Table 1 is connected to the nucleotide sequence shown in (b1), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (b2);

[0030] 2) The 5' end of the forward amplification primer in the primer pair 1-131 shown in Table 1 is connected to the nucleotide sequence shown in (b2), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (b1);

[0031] 3) The 5' end of the forward amplification primer in the odd-numbered primer pair shown in Table 1 is connected to the nucleotide sequence shown in (b1), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (b2); the 5' end of the forward amplification primer in the even-numbered primer pair is connected to the nucleotide sequence shown in (b2), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (b1);

[0032] 4) The 5' end of the forward amplification primer in the odd-numbered primer pair shown in Table 1 is connected to the nucleotide sequence shown in (b2), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (b1); the 5' end of the forward amplification primer in the even-numbered primer pair is connected to the nucleotide sequence shown in (b1), and the 5' end of the reverse amplification primer is connected to the nucleotide sequence shown in (b2).

[0033] If not specifically stated, the sequencing adapter pairing fragment of the present invention can be directly connected to the 5' end of the forward amplification primer or the reverse amplification primer or can be connected via a length of 1-10 bases. The 1-10 bases are only for connection and do not affect the amplification effect of the primer set. The 1-10 bases can be obtained by those skilled in the art through conventional technical means, and the present invention does not specifically limit them.

[0034] Those skilled in the art know that connecting the sequencing matching fragment at the 5' end of the primer and adding the sequencing adapter of the corresponding sequencing platform to the amplified product through PCR is not the only method of adding sequencing adapters. Those skilled in the art can also directly add sequencing adapters of different sequencing platforms through TA connection method as needed.

[0035] In a second aspect, the present invention provides a use of the primer set described in the first aspect of the present invention in preparing a product for detecting human mitochondrial gene mutations.

[0036] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0037] In a third aspect, the present invention provides a kit, wherein the kit comprises the primer set described in the first aspect of the present invention, or comprises a buffer containing the primer set described in the first aspect of the present invention.

[0038] Preferably, the kit further comprises a PCR reaction premix, nuclease-free water, and a sequencing adapter suitable for a sequencing platform; the PCR reaction premix comprises a buffer, a DNA polymerase, Mg 2+ , dNTP. In general, the PCR reaction premix can be purchased from commercial sources.

[0039] In a fourth aspect, the present invention provides a method for detecting human mitochondrial gene mutations for purposes other than disease diagnosis and treatment, the method comprising the following steps:

[0040] (1) DNA extraction from samples;

[0041] (2) First round of PCR amplification

[0042] Using the DNA obtained in step (1) as a template, preparing a PCR reaction solution containing the primer set described in the first aspect of the present invention, performing PCR amplification, and collecting the amplified product;

[0043] (3) Purification of PCR amplification products;

[0044] (4) Second round of PCR amplification

[0045] Using the purified product obtained in step (3) as a template, adding a sequencing adapter to perform PCR amplification to obtain a library;

[0046] (5) Purify the library;

[0047] (6) Library quality control;

[0048] (7) Sequencing on a machine;

[0049] (8) Bioinformatics analysis

[0050] Preferably, the sample in step (1) includes but is not limited to fresh or frozen tissue, moderately or severely degraded tissue, paraffin-embedded tissue, whole blood, and saliva.

[0051] In a specific embodiment of the present invention, the PCR reaction solution in step (2) is: 12.5 μl of PCR reaction premix, 1.5-6 μl of the primer set described in the first aspect of the present invention, 0-0.5 μl of DMSO, 2 μl of DNA template, and the system is supplemented to 25 μl with nuclease-free water; the final concentration of the primer set in the PCR reaction solution is 25-100 nM, specifically 25 nM, 50 nM, 100 nM; the volume concentration of DMSO in the PCR reaction solution is 0-2%.

[0052] Preferably, the final concentration of the primer set in the PCR reaction solution is 50 nM, and the DMSO content is 0.

[0053] In a specific embodiment of the present invention, the PCR reaction conditions in step (2) are: pre-denaturation at 95°C for 15 min; 98°C for 1 min, 58°C for 2 min, 60°C for 4 min, 64°C for 1 min, 72°C for 1 min, for a total of 5 cycles; then 95°C for 30 sec, 62-72°C for 3 min, for a total of 18 cycles; and temporary storage at 8°C.

[0054] Preferably, the annealing temperature during the PCR reaction is 95° C. for 30 seconds, 66° C. for 3 minutes, for a total of 18 cycles.

[0055] In the detection method described in the present invention, sample DNA can be extracted using a commercially available kit. The sequencing adapters used in the second round of PCR that are suitable for each sequencing platform are prior art and can be obtained from a commercially available kit. The amplification products and library purification and library quality control methods are all conventional technical means in the art and will not be elaborated herein.

[0056] In a fifth aspect, the present invention provides a method for constructing a gene library for detecting human mitochondrial gene mutations based on NGS, the method comprising the following steps:

[0057] (1) DNA extraction from samples;

[0058] (2) First round of PCR amplification

[0059] Using the DNA obtained in step (1) as a template, preparing a PCR reaction solution containing the primer set described in the first aspect of the present invention, performing PCR amplification, and collecting the amplified product;

[0060] (3) Purification of PCR amplification products;

[0061] (4) Second round of PCR amplification

[0062] Using the purified product obtained in step (3) as a template, adding a sequencing adapter to perform PCR amplification to obtain a library;

[0063] (5) Purify the library;

[0064] (6) Library quality control.

[0065] The present invention defines each step in the gene library construction method as described in the fourth aspect of the present invention.

[0066] The technical solution provided by the present invention has the following beneficial technical effects:

[0067] (1) The present invention designs 131 pairs of primers for human mitochondrial DNA (mtDNA). The amplicons generated by the 131 pairs of primers are distributed in a shingled manner and can cover all sequences outside about 400 bp near the origin of the mtDNA genome. Therefore, the 131 pairs of primers provided by the present invention can detect the entire human mtDNA genome.

[0068] (2) Under normal circumstances, when the primer pairs obtained according to the above method are in the same tube, the primers of adjacent amplicons will have the problem of over-amplification of overlapping regions, resulting in a serious reduction in the target amplification product. To avoid this problem, the prior art is to divide the sample into 2 tubes or multiple tubes for operation, which not only increases the cost but also increases the potential risk of operational errors. The present invention creatively adds a specific sequence to the outside of the above reverse primer, and the specific sequence enables the 131 pairs of primers provided by the present invention to react in one tube without causing excessive amplification of overlapping regions, and can inhibit primer dimers.

[0069] (3) Secondly, the primer pairs provided by the present invention intentionally avoid the main polymorphic sites on the mtDNA genome during design, adapt to the variable sample sequences, and improve the amplification efficiency.

[0070] (4) The average length of the amplicon generated by the primer pair provided by the present invention is 216 bp, which is suitable for detecting low-quality samples, such as moderately to severely degraded samples or samples from paraffin-embedded tissues.

[0071] (5) Furthermore, the outside of the primer pair can be connected to a characteristic sequence adapted to different sequencing platforms, and the amplified product can be added to the sequencing adapter of the corresponding sequencing platform by PCR, so as to facilitate high-throughput detection using mainstream platforms such as Illumina, MGI, Ion, etc. Those skilled in the art can also choose to directly add sequencing adapters of different sequencing platforms by TA connection.

[0072] (6) The kit provided by the present invention is low-cost and easy to operate. NGS library construction and sequencing can be completed within one day. Together with the automated bioinformatics pipeline, the test can be completed and a test report can be issued within three days. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Flowchart of human mtDNA gene mutation detection.

[0074] Figure 2 Electrophoresis of the first-round PCR products in optimization experiment 3.

[0075] Figure 3 Electrophoresis of the final product of the Chinese library in optimization experiment 3.

[0076] Figure 4Relative coverage of each amplicon in PB samples in the primer pool D2.3 detection system.

[0077] Figure 5 Relative coverage of each amplicon of NA12878 sample in the primer pool D2.3 detection system.

[0078] Figure 6 The relative coverage of each amplicon in the PB sample in the control primer pool detection system.

[0079] Figure 7 The relative coverage of each amplicon of NA12878 sample in the control primer pool detection system. DETAILED DESCRIPTION

[0080] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0081] Primer pairs

[0082] The present invention designs 131 pairs of primers for human mtDNA, and the nucleotide sequences of the primer pairs are shown in the following table. The amplicons generated by the 131 pairs of primers are distributed in a shingled manner, and can cover all sequences outside about 400 bp near the origin of the mtDNA genome.

[0083] Table 1 Primer pairs nucleotide sequences

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] In order to solve the problem of over-amplification of overlapping regions of primers of adjacent amplicons, the present invention connects a specific sequence to the 5' end of the reverse amplification primer (SEQ ID NO.132-SEQ ID NO.261) of primer pair 1-130, and the specific sequence is shown in SEQ ID NO.263-SEQ ID NO.392. Exemplarily, the 5' end of the reverse amplification primer shown in SEQ ID NO.132 is connected to the specific sequence shown in SEQ ID NO.263; the 5' end of the reverse amplification primer shown in SEQ ID NO.133 is connected to the nucleotide sequence shown in SEQ ID NO.264; sequentially, until the 5' end of the reverse amplification primer shown in SEQ ID NO.261 is connected to the nucleotide sequence shown in SEQ ID NO.392; those skilled in the art can connect the specific sequence to the 5' end of the reverse amplification primer by conventional technical means.

[0094] Table 2 Specific sequences

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] Example 1 Human mtDNA gene mutation detection method

[0101] 1. Sample Preparation

[0102] Human genomic DNA NA12878, purchased from Coriell;

[0103] Human peripheral blood leukocyte DNA (abbreviated as PB in the present invention) was obtained from a medical laboratory.

[0104] 2. Primer Pool Preparation

[0105] 2.1 The primer pool consists of 131 pairs of primers, wherein the forward amplification primers are a combination of nucleotide sequences shown in SEQ ID NO.1-131, and the reverse amplification primers in the 1st to 130th pairs of primers are the connection products obtained by connecting the nucleotide sequences shown in SEQ ID NO.263-SEQ ID NO.392 shown in Table 2 to the 5' ends of the reverse amplification primers of SEQ ID NO.132-SEQ ID NO.261 shown in Table 1, respectively, and the reverse amplification primer of the 131st pair of primers is shown in SEQ ID NO.262. The nucleic acid connection method described in the present invention is a conventional technical means well known to those skilled in the art.

[0106] 2.2 Further, based on the 131 pairs of primers obtained according to the above method, sequencing adapter paired fragments suitable for the illumina sequencing platform were connected. Specifically, the 5' end of each forward amplification primer was connected to the nucleotide sequence shown in SEQ ID NO.393, and the 5' end of each reverse amplification primer was connected to the nucleotide sequence shown in SEQ ID NO.394.

[0107] 2.3 Mix equal volumes of the primer pairs obtained in step 2.2 (each primer pair has the same molar concentration) to prepare a primer pool for PCR amplification reaction.

[0108] 3. Library Preparation Process

[0109] 3.1 First round of PCR

[0110] 3.1.1 Prepare PCR reaction solution as shown in the table below and add corresponding DNA sample as template to each tube;

[0111]

[0112] 3.1.2 Cover the tube, shake evenly and then centrifuge quickly;

[0113] 3.1.3 Carry out PCR amplification according to the procedure shown in the table below;

[0114]

[0115] 3.2 Purification of the first round of PCR products

[0116] 3.2.1 After taking each tube out of the PCR instrument, centrifuge quickly to throw the liquid to the bottom of the tube;

[0117] 3.2.2 Add 1.2 times the volume of purified magnetic beads (AMPure, Beckman, the same below) to each tube, shake to mix, centrifuge quickly, and let stand at room temperature for 5 minutes;

[0118] 3.2.3 Transfer each tube to the magnetic rack and place at room temperature for 5 minutes or until the liquid is clear. Carefully aspirate the supernatant, taking care to avoid touching the magnetic beads.

[0119] 3.2.4 Add 150 μL 70% ethanol to each tube for washing. After incubation for 30 seconds, remove the ethanol and avoid touching the magnetic beads. Repeat the washing process.

[0120] 3.2.5 Cover the tubes, centrifuge them quickly, place them on a magnetic rack, and carefully remove the remaining ethanol from the tubes;

[0121] 3.2.6 Place at room temperature for 2-3 minutes until the beads are dry, add 64 μL of nuclease-free water to each tube, cover the tube, shake to mix and centrifuge quickly;

[0122] 3.2.7 Transfer each tube to a magnetic rack, place at room temperature for 5 minutes, and transfer the supernatant to a new tube.

[0123] 3.3 Second round of PCR

[0124] 3.3.1 Prepare the second round of PCR reaction solution as shown in the table below;

[0125]

[0126]

[0127] 3.3.2 Cover the tube, shake evenly and then centrifuge quickly;

[0128] 3.3.3 Perform PCR amplification according to the procedure shown in the table below.

[0129]

[0130] 3.4 Library purification

[0131] 3.4.1 After taking each tube out of the PCR instrument, quickly centrifuge it to throw the liquid to the bottom of the tube;

[0132] 3.4.2 Add 1 volume of purified magnetic beads to each tube, shake to mix, centrifuge quickly, and let stand at room temperature for 5 minutes;

[0133] 3.4.3 Transfer each tube to the magnetic rack and place at room temperature for 5 minutes or until the liquid is clear. Carefully aspirate the supernatant, taking care to avoid touching the magnetic beads.

[0134] 3.4.4 Add 150 μL 70% ethanol to each tube for washing. After incubation for 30 seconds, remove the ethanol and avoid touching the magnetic beads. Repeat the washing process.

[0135] 3.4.5 Cover the tubes, centrifuge them quickly, place them on a magnetic rack, and carefully remove the remaining ethanol from the tubes;

[0136] 3.4.6 Place at room temperature for 2-3 minutes until the beads are dry. Add 32 μL of nuclease-free water to each tube, cover the tube, shake to mix and centrifuge quickly;

[0137] 3.4.7 Transfer each tube to a magnetic rack, place at room temperature for 5 minutes, and transfer the supernatant to a new tube.

[0138] 3.5 Library quality control

[0139] The library was quantified using Qubit, and the library fragment size and purity were analyzed using Qseq100. The mass concentration was converted to molar concentration based on the library fragment size.

[0140] 4. Sequencing

[0141] 4.1 Dilute each library to 5 nM according to the concentration, take a small amount of equal volume and mix;

[0142] 4.2 Add an equal volume of 0.2N NaOH for denaturation, and then dilute to 15pM with HT1 solution;

[0143] 4.3 Take 594 μL of 15 pM mixed library and add 6 μL of 12.5 pM PhiX library control;

[0144] 4.4 Add the above 600 μL to the sequencing kit and load it into the Illumina Miseq sequencer for sequencing. The sequencing read length is PE150, and at least 1M reads are allocated to each sample.

[0145] 5. Data Analysis

[0146] The data were split according to different sample Index barcodes to obtain the original data FASTQ files. The human genome Hg19 standard sequence was used for sequence alignment and splicing, the coverage depth of each amplicon was identified, and subsequent parameter calculations were performed.

[0147] Optimization experiment 1 Optimization of primer set and DMSO concentration

[0148] The samples and detection methods are the same as those in Example 1, except for the primers and DMSO concentrations in the first round of PCR reaction solution in step 3.1.1, as shown in the following table:

[0149]

[0150] According to the method provided in Example 1, the grouping and test results are as follows:

[0151] Table 3 Test results

[0152]

[0153]

[0154] According to the test results shown in the above table, it can be seen that when the primer concentration is 50nM and the DMSO content is 0, the overall performance parameters of the test are higher than those of other groups. Therefore, the final concentration of the primer group in the first round of PCR reaction solution of the present invention is preferably 50nM, and the preferred DMSO content is 0.

[0155] Optimization test 2: annealing temperature optimization

[0156] The samples and detection methods are the same as those in Example 1, with the only difference being the annealing temperature of the first round of PCR reaction in step 3.1.3. The annealing temperatures for the two samples are set to 62°C, 64°C, 66°C, 68°C, 70°C, and 72°C, respectively. Based on the comparison of the electrophoresis results, alignment rate, and on-target rate of the first round PCR products and the final products of the library, the most preferred annealing temperature of the present invention is 66°C.

[0157] Optimization experiment 3: Optimization of primer pair concentration and ratio

[0158] The samples and detection methods are the same as those in Example 1, except for the ratio of each primer pair in the preparation process of the primer pool in step 2 (specifically step 2.3). Specifically, equal volumes of primer pairs 1-131 are mixed to obtain primer pool D1 (each primer pair has the same molar concentration). As shown in the table below, the concentrations of the corresponding primer pairs are reduced or increased to obtain primer pools D2.1, D2.2 and D2.3. The four primer pools with different ratios are specifically shown in the table below:

[0159]

[0160]

[0161] The test results were as follows:

[0162] 6.1 Electropherogram

[0163] The electrophoresis of the first round PCR products is shown in Figure 2 As shown, the electrophoresis diagram of the final product of the library is as follows Figure 3 The meanings of the lanes in the figure are as follows: M: 50 bp ladder; 1: NA12878, D2.1; 2: NA12878, D2.2; 3: PB, D2.1; 4: PB, D2.2; 5: NA12878, D2.3; 6: PB, D2.3.

[0164] 6.2 Sequencing results

[0165]

[0166] According to the electrophoresis and sequencing results, it can be seen that the present invention prefers the ratio of the D2.3 primer pool, that is, the optimal molar concentration ratio of the 131 pairs of primers provided by the present invention is (the 4th, 8th, 11th, 13th, 28th, 31st, 38th, 56th, 72nd, 79th, 82nd, 90th, 96th, 113th, 122nd, 128th, 131st pairs of primers): (the 33rd, 37th, 55th, 69th, 121st, 129th pairs of primers): (the 3rd, 15th, 26th, 29th, 35th, 39th, 42nd, 53rd, 59th, 64th, 65th, 67th, 71st, 73rd, 76th, 83rd, 88th, 93rd, 99th, 104th, 125th, 126th pairs of primers): (all other primer pairs) = 0.625:2:1.5:1.

[0167] Verification experiment on specific sequences in primers

[0168] Preparation of control primer pool D0

[0169] The preparation method of the 131 pairs of primers in the control primer pool is the same as step 2 of Example 1, except that the specific sequence connected to the 5' end of the reverse amplification primer shown in Table 2 is different from that in Example 1. Specifically, in the control primer pool, the first 4 bases of SEQ ID NO. 263-392 are replaced with random base sequences respectively.

[0170] According to the mtDNA detection method disclosed in Example 1, primer pool 2.3 and control primer pool were used to detect PB samples and human genomic DNA NA12878 samples respectively. In the primer pool 2.3 detection system, the relative coverage of each amplicon is as follows: Figure 4 and Figure 5 As shown; in the control primer pool detection system, the relative coverage of each amplicon is as follows Figure 6 and Figure 7 shown.

[0171] according to Figure 4-7 From the results of the relative coverage of the amplicons, it can be seen that when the specific sequence described in the present invention is replaced in the primers, the primers of adjacent amplicons will have problems of over-amplification of overlapping regions and / or the appearance of primer dimers, which will reduce the target amplification product. Compared with the primer pool D2.3, the relative coverage of each amplicon fluctuates significantly, and the coverage of some amplicons is reduced to 0 or close to 0.

[0172] The specific sequence provided by the present invention is the best sequence obtained by the inventor through a large number of optimization tests. For example, when the first four bases in the specific sequence shown in SEQ ID NO.263-392 are randomly replaced, it can be seen from the results shown in the figure that the relative coverage of each amplicon fluctuates toward an extreme value compared with the primer pool D2.3. Based on the above experimental results, it can be obtained that connecting the specific sequence provided by the present invention to the outside of the reverse amplification primer can effectively solve the problem of excessive amplification of the overlapping region of the primers of adjacent amplicons, inhibit primer dimers, and enable the detection to be performed in the same tube, thereby reducing costs and reducing the potential risk of operational errors.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set, characterized in that: The primer set consists of primer pairs No. 1-131 in Table 1, wherein the forward amplification primers in the primer set are a combination of nucleotide sequences shown in SEQ ID NO.1-131, and the reverse amplification primers in the primer set are a combination of nucleotide sequences shown in SEQ ID NO.132-262.

2. The primer set according to claim 1, characterized in that The 5' ends of the reverse amplification primers shown in SEQ ID NO.132-261 are connected to the nucleotide sequences shown in SEQ ID NO.263-SEQ ID NO.392 respectively.

3. The primer set according to any one of claims 1-2, characterized in that: The 5' ends of the forward amplification primer and the reverse amplification primer in the primer set are also connected to a sequencing adapter pairing fragment, which contains a sequence that differs by no more than 3 nucleotides from 15-25 consecutive nucleotides from the 3' end of the sequencing adapter of the illumina, Ion or MGI platform.

4. The primer set according to claim 3, characterized in that: The sequencing adapter paired fragment is directly connected to the 5' end of the forward amplification primer or the reverse amplification primer or is connected through a length of 1-10 bases.

5. Use of the primer set according to any one of claims 1 to 4 in preparing a product for detecting human mitochondrial gene mutations.

6. A kit comprising the primer set according to any one of claims 1 to 4, or a buffer containing the primer set according to any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that The kit also includes a PCR reaction premix, nuclease-free water, and a sequencing adapter suitable for a sequencing platform.

8. A method for detecting human mitochondrial gene mutation for purposes other than disease diagnosis and treatment, the method comprising the following steps: (1) DNA extraction from samples; (2) First round of PCR amplification Using the DNA obtained in step (1) as a template, preparing a PCR reaction solution containing the primer set described in any one of claims 1 to 4, performing PCR amplification, and collecting the amplified product; (3) Purification of PCR amplification products; (4) Second round of PCR amplification Using the purified product obtained in step (3) as a template, adding a sequencing adapter to perform PCR amplification to obtain a library; (5) Purify the library; (6) Library quality control; (7) Sequencing on a machine; (8) Bioinformatics analysis 9. The method according to claim 8, characterized in that The samples described in step (1) include fresh or frozen tissue, moderately or severely degraded tissue, paraffin-embedded tissue, whole blood, and saliva.

10. The method according to claim 9, characterized in that In the PCR reaction solution described in step (2), the final concentration of the primer set is 25-100 nM, and the volume concentration of DMSO is 0-2%.

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