CN117587117A - Primer group, kit and application for detecting Leber hereditary optic neuropathy - Google Patents
Primer group, kit and application for detecting Leber hereditary optic neuropathy Download PDFInfo
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Abstract
The invention relates to a primer group for detecting Leber hereditary optic neuropathy, a kit and application thereof, and relates to the field of biotechnology, wherein the primer group for detecting Leber hereditary optic neuropathy comprises 15 groups, and the sequences of the 15 groups of primer groups are shown as SEQ ID No. 1-45. Compared with the existing detection technology, the method can detect the mitochondrial DNA mutation site of the Leber hereditary optic neuropathy by combining a microfluidic chip with a competitive allele specific amplification technology, can detect 15 mitochondrial site mutations related to the Leber hereditary optic neuropathy of the Chinese people comprehensively and accurately, has a small volume of a required PCR reaction system, only needs 5-10 ng of DNA template, can realize high-efficiency, sensitive, stable and multi-site joint detection of the mitochondrial mutation site of the Leber hereditary optic neuropathy, provides important basis for clinical early diagnosis and treatment of the Leber hereditary optic neuropathy, and has wide application prospect and clinical reference value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group for detecting Leber hereditary optic neuropathy, a kit and application thereof.
Background
Leber hereditary optic neuropathy (Leber hereditary optic neuropathy, LHON) is a hereditary eye disease, clinically manifested as acute or subacute central vision drop in both eyes, mostly in young men, one of the main causes of blindness in young people. The disease has been shown to be a maternal genetic disease, the most common "mitochondrial genetic disease", the main cause of which is mutations in certain sites of mitochondrial DNA (mtDNA).
Because LHON has no characteristic clinical manifestation, misdiagnosis is possible only according to clinical manifestation and family history of patients, and the clear gene detection result is helpful for diagnosis, differential diagnosis and prevention of the disease. Since the discovery of the first LHON-related mutation site m.11778G > A by Wallance et al in 1988, more than 70 LHON-related mutation sites were successively reported, wherein m.11778G>A、m.14484T>C、m.3460G>A is the three mutation sites that were originally reported to be associated with LHON, and these three mutation sites are the three primary mutations currently recognized as the most pathogenic, with the remaining mitochondrial DNA mutations being termed secondary mutations. The blindness of the primary mutations is much higher than that of the secondary mutations, which in cooperation with the primary mutations will enhance LHON exogenesis. Mitochondrial genes MT-ND1, MT-ND4 and MT-ND6 are mutant hotspot genes associated with LHON morbidity. In Chinese population, LHON incidence-related secondary mutationsWith m.3394T on MT-ND1 gene>C、m.3635G>A、m.3733G>A、m.3866T>C, performing operation; m.10680G on MT-ND4L Gene>A, A is as follows; m.11696G on MT-ND4 gene>A, A is as follows; m.12338T on MT-ND5 gene>C, performing operation; m.14459G on MT-ND6 gene>A、m.14502T>C；tRNA Met 4435A>G；tRNA Glu 14693A>G and tRNA Thr 15951A>G isosteps.
Currently, in the commercial kit for detecting LHON mitochondrial DNA mutation sites, a single site or 3 common primary mutation sites are mainly detected, and there is no kit for detecting LHON common mtDNA15 site mutation suitable for Chinese people. With the rapid development of molecular biology, currently the most commonly used screening methods for LHON include: allele-specific PCR (AS-PCR), restriction fragment length polymorphism PCR (RFLP-PCR), allele-specific fluorescent probe technique PCR and the like, which have great defects in terms of pollution resistance, sensitivity and clinical use convenience; in addition, the gene sequencing can detect more LHON related mtDNA variation at the same time, but the detection period is long, the cost is high, and the technical requirements for operators and data analysts are high. Therefore, a detection method or detection product which is simple and quick to operate, high in sensitivity and easy to realize multi-site simultaneous detection and automatic interpretation is urgently needed in the market and clinic, and important information is provided for LHON genetic consultation and clinic diagnosis. In view of the above, the invention provides a primer set, a kit and application for detecting Leber hereditary optic neuropathy.
Disclosure of Invention
The invention aims to provide a primer group for detecting Leber hereditary optic neuropathy, a kit and application thereof. The method is used for detecting the mitochondrial DNA mutation site of the Leber hereditary optic neuropathy, and is convenient for genetic consultation and clinical diagnosis.
According to the invention, through consulting and analyzing a large number of Leber hereditary optic neuropathy related documents and clinical practice guidelines of Leber hereditary optic neuropathy, 15 common mutation sites in detected Chinese crowds are determined, wherein the mutation sites comprise m.3394T > C, m.3460G > A, m.3635G > A, m.3733G > A, m.3866T > C, m.4435A > G, m.10680G > A, m.11696G > A, m.11778G > A, m.12338T > C, m.14459G > A, m.14484T > C, m.14502T > C, m.14693A > G and m.15951A > G, and primer design is carried out according to related genetic information, so that the genetic gene information of Leber hereditary optic neuropathy can be detected simultaneously.
The technical scheme for solving the technical problems is as follows:
in a first aspect, a primer set for detecting Leber hereditary optic neuropathy is provided, wherein the primer set comprises 15 groups, and the sequences of the 15 groups of primer sets are shown as SEQ ID No. 1-45.
Further, the 15 primer sets and the corresponding detection sites thereof are respectively:
the first group of primers are shown in a sequence table SEQ ID No.1-3 and are used for detecting m.3394T > C in genetic loci of mitochondrial genes ND 1;
the second group of primers is shown in a sequence table SEQ ID No.4-6 and is used for detecting m.3460G > A in a genetic locus of a mitochondrial gene ND 1;
the third group of primers is shown as a sequence table SEQ ID N o.7-9 and is used for detecting m.3635G > A in genetic loci of mitochondrial genes ND 1;
the fourth group of primers is shown in a sequence table SEQ ID No.10-12 and is used for detecting m.3733G > A in a genetic locus of a mitochondrial gene ND 1;
the fifth group of primers are shown in a sequence table SEQ ID No.13-15 and are used for detecting m.3866T > C in genetic loci of mitochondrial genes ND 1;
the sixth group of primers is shown as SEQ ID No.16-18 of the sequence Listing and is used for detecting mitochondrial gene tRNA Met m.4435A in the genetic locus of (C)>G；
The seventh group of primers are shown in a sequence table SEQ ID No.19-21 and are used for detecting m.10680G > A in a genetic locus of a mitochondrial gene ND 4L;
the eighth group of primers are shown in sequence table SEQ ID No.22-24 and are used for detecting m.11696G > A in genetic loci of mitochondrial gene ND 4;
the ninth group of primers are shown in a sequence table SEQ ID No.25-27 and are used for detecting m.11778G > A in a genetic locus of a mitochondrial gene ND 4;
the tenth group of primers are shown in a sequence table SEQ ID No.28-30 and are used for detecting m.12338T > C in the genetic locus of the mitochondrial gene ND 5;
the eleventh group of primers are shown in sequence table SEQ ID No.31-33 and are used for m.14459G > A in genetic locus of mitochondrial gene ND 6;
the twelfth group of primers are shown in sequence table SEQ ID No.34-36 and are used for detecting m.14484T > C in genetic loci of mitochondrial gene ND 6;
the thirteenth group of primers are shown in a sequence table SEQ ID No.37-39 and are used for detecting m.14502T > C in the genetic locus of mitochondrial gene ND 6;
the fourteenth primer set is shown in SEQ ID No.40-42 of the sequence Listing and is used for detecting mitochondrial gene tRNA Glu m.14693A in the genetic locus of (C)>G；
The fifteenth primer set is shown in a sequence table SEQ ID No.43-45 and is used for detecting mitochondrial gene tRNA Thr m.159551A in the genetic locus of (A)>G。
Further, each of the 15 primer sets comprises two primers designed based on the difference in allele sequence and one common pair primer.
Wherein SEQ ID No.1, SEQ ID No.4, SEQ ID No.7, SEQ ID No.10, SEQ ID No.13, SEQ ID No.16, SEQ ID No.19, SEQ ID No.22, SEQ ID No.25, SEQ ID No.28, SEQ ID No.31, SEQ ID No.34, SEQ ID No.37, SEQ ID No.40, SEQ ID No.43 are forward primers (F1) in each primer set; SEQ ID No.2, SEQ ID No.5, SEQ ID No.8, SEQ ID No.11, SEQ ID No.14, SEQ ID No.17, SEQ ID No.20, SEQ ID No.23, SEQ ID No.26, SEQ ID No.29, SEQ ID No.32, SEQ ID No.35, SEQ ID No.38, SEQ ID No.41, SEQ ID No.44 are forward primers (F2) in each primer set; the remaining primers of each primer set are common reverse primers (R).
Further, 15 forward primers (F1) were each modified with HEX tag sequence, and 15 forward primers (F2) were each modified with FAM tag sequence. Specifically, the general tag sequences carried by SEQ ID No.1, SEQ ID No.4, SEQ ID No.7, SEQ ID No.10, SEQ ID No.13, SEQ ID No.16, SEQ ID No.19, SEQ ID No.22, SEQ ID No.25, SEQ ID No.28, SEQ ID No.31, SEQ ID No.34, SEQ ID No.37, SEQ ID No.40 and SEQ ID No.43 are shown as SEQ ID No. 46; the general tag sequences carried by SEQ ID No.2, SEQ ID No.5, SEQ ID No.8, SEQ ID No.11, SEQ ID No.14, SEQ ID No.17, SEQ ID No.20, SEQ ID No.23, SEQ ID No.26, SEQ ID No.29, SEQ ID No.32, SEQ ID No.35, SEQ ID No.38, SEQ ID No.41 and SEQ ID No.44 are shown as SEQ ID No. 47.
Wherein the nucleotide sequence of the HEX tag sequence is GAAGGTCGGAGTCAACGGATT (SEQ ID No. 46); the nucleotide sequence of the FAM tag sequence was GAAGGTGACCAAGTTCATGCT (SEQ ID No. 47).
In a second aspect, there is provided a kit for detecting Leber hereditary optic neuropathy, the kit comprising the primer set described above.
Further, the chip in the kit comprises at least 18 chip reaction tanks and at least 2 positioning points, the chip reaction tanks comprise at least 1 internal control quality control, 1 negative quality control and 1 positive quality control, each quality control corresponds to 1 reaction tank, and a group of primer groups are coated in each of the remaining chip reaction tanks.
Further, the sum of the three primer volumes in the primer group in each chip reaction tank is equal.
In a third aspect, a method for preparing a kit for detecting Leber hereditary optic neuropathy is provided, comprising the steps of:
(1) Preparing Kong Weibiao specific primer groups for detecting 15 sites in Leber hereditary optic neuropathy, and respectively coating 15 groups of the primer groups into 15 chip reaction tanks according to Kong Weibiao to obtain a chip semi-finished product;
(2) Preparing a PCR amplification reagent comprising a PCR reaction solution and a PCR buffer solution;
(3) Preparing sealing films and quality control products with corresponding numbers;
(4) The chip, the PCR amplification reagent, the sealing film and the quality control product are assembled into a kit for detecting the Leber hereditary optic neuropathy.
In a fourth aspect, a reagent for detecting Leber hereditary optic neuropathy is provided, comprising the primer set or the kit.
Compared with the prior art, the invention has the beneficial effects that:
(1) The primer group or the kit for detecting the Leber hereditary optic neuropathy genes can detect 15 mutation sites on the mitochondrial DNA of the Leber hereditary optic neuropathy at the same time; the provided 15 groups of primers have similar annealing temperature through optimal design and screening, so that the simultaneous amplification of the 15 groups of primers under the same PCR amplification condition can be realized, the volume of each PCR reaction system is as low as 1.15 mu L, and the DNA sample only needs 5-10 ng, thereby greatly improving the detection efficiency and the reaction sensitivity;
(2) The kit for detecting the Leber hereditary optic neuropathy genes can prevent cross contamination among different reaction tanks, realize uniform distribution of samples among different reaction tanks, and realize multi-site simultaneous detection with high flux, high sensitivity and high accuracy;
(3) The invention is based on KASP technology and micro-fluidic chip technology, can effectively distinguish mutation sites in a wide genomic DNA sample, and is used for assisting genetic consultation and clinical diagnosis of Leber hereditary optic neuropathy.
Drawings
FIG. 1 is a diagram showing 15 detection information of the position and the corresponding fluorescence expression pattern according to the present invention;
FIG. 2 is a block diagram of a chip substrate of the kit of the present invention;
FIG. 3 is a diagram showing software interpretation of the genotype without site mutation and the fluorescence expression of the corresponding chip:
FIG. 4 is a diagram showing software interpretation of the mutant genotype of m.3394T > C site and the corresponding chip fluorescence expression examples:
FIG. 5 is a diagram showing software interpretation of the mutant genotype of m.11696G > A site and the corresponding chip fluorescence expression examples according to the present invention;
FIG. 6 is a diagram showing software interpretation of the mutant genotype of m.12338T > C locus and the corresponding chip fluorescence expression examples according to the present invention;
FIG. 7 is a diagram showing software interpretation of the mutant genotype of m.14502T > C locus and the corresponding chip fluorescence expression examples according to the present invention;
FIG. 8 is a diagram showing software interpretation of the mutant genotype of m.15951A > G locus and the corresponding chip fluorescence expression examples according to the present invention;
FIG. 9 is a sequencing chart I of the detection result of the gene chip corresponding to the Leber hereditary optic neuropathy of the invention;
FIG. 10 is a sequencing chart II of the detection result of the gene chip of the Leber hereditary optic neuropathy according to the invention.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1: primer design
According to the human mitochondrial DNA genome sequence (sequence number: NC_ 012920.1) disclosed in the national center for Biotechnology information Gene Bank (NCBI), by referring to a large number of Leber hereditary optic neuropathy related documents and the clinical practice guidelines for Leber hereditary optic neuropathy, 15 mutation sites, including m.3394T > C, m.3460G > A, m.3635G > A, m.3733G > A, m.38366T > C, m.4435A > G, m.10680G > A, m.11696G > A, m.11778G > A, m.12338T > C, m.14459G > A, m.14484T > C, m.14502T > C, m.14693A > G, m.3735A > G, and primer design is performed according to the related genetic information.
Based on the competitive allele-specific PCR principle, 15 sets of primers are designed for simultaneously detecting the combination information of the mitochondrial related mutation sites of the Leber hereditary optic neuropathy, each set of primers comprises two primers designed based on the difference of the allele sequence sites and a common matched primer, and the primer sequences corresponding to each set of primers are shown in tables 1-3. The source of each primer of the designed primer set is not particularly limited, and a primer source known in the art may be used. In the embodiment of the invention, the primer group is entrusted to the synthesis of a division of biological engineering (Shanghai).
Table 1 detection sites and sequence numbers of sequence listing corresponding to primer sets of group 15
Table 2 detection sites corresponding to primer sets of 15 and sequence numbers of sequence listing
TABLE 3 primer nucleotide sequences of primer set of 15 sets
Example 2: preparation of the kit
Based on the example 1, the 15 groups of primers are selected simultaneously in the example 2 to prepare a Leber hereditary optic neuropathy gene detection kit, so as to illustrate the kit and the preparation method thereof. The components of the kit mainly comprise a chip, a sealing film, a PCR reaction solution and a PCR buffer solution.
And (3) performing competitive allele-specific PCR amplification, and finally performing fluorescent signal detection on the PCR product, and performing relevant mitochondrial DNA mutation site detection on the sample according to fluorescent signal grouping. The primer groups can be used for realizing multi-site simultaneous detection, and can achieve the purposes of detecting the Leber hereditary optic neuropathy genes with high accuracy and high flux (see the attached figure 1 of the specification for details).
The kit for SNP typing based on the principle of competitive allele-specific PCR, wherein the PCR reaction is completed on a microfluidic chip substrate (Beijing Boao classical biotechnology Co., ltd., product No. G020010, description figure 2), the preparation method steps are as follows:
(1) The 15 site specific primer groups are formulated Kong Weibiao, the specific primer groups are respectively coated into a chip reaction tank according to Kong Weibiao, a chip semi-finished product is obtained, the chip specification is 4 persons/sheet, each chip is provided with a positioning hole, an internal reference quality control, a negative quality control and a positive quality control, and the corresponding detection indexes of the chip reaction tank are shown in Table 4 in detail.
TABLE 4 chip reaction cell correspondence detection index
(2) The PCR amplification reaction reagent is prepared mainly comprising probes, enzymes and dNTPs, and is provided with sealing films with corresponding numbers and PCR buffer solution (1 XTE buffer solution: prepared from Tris and EDTA), and the kit is assembled according to specifications.
(3) The semi-finished chip, the sealing film, the PCR reaction liquid, the PCR buffer solution and the quality control (human genome DNA) prepared by the steps are assembled into the Leber hereditary optic neuropathy gene detection kit.
Example 3: detection of mitochondrial DNA mutation site related to Leber hereditary optic neuropathy
A method for detecting Leber hereditary optic neuropathy mitochondrial DNA mutation based on the kit prepared in example 2, comprising the steps of:
1. blood of 100 blood donors without blood relationship is collected, and genomic DNA is extracted (the sample applicable to the kit is human genomic DNA extracted from whole blood). The extracted genome DNA needs to be subjected to concentration measurement, and the detected genome DNA needs to meet the concentration of 10 ng/mu L-50 ng/mu L;
2. 100 persons of 15 pieces of SNP site information were detected by using the kit prepared in example 2, and the following operations were performed according to the procedure for use:
a) Preparation of amplification System
And (5) liquid component split charging is carried out between reagent split charging. And taking out the PCR reaction solution and the PCR buffer solution, standing at room temperature until the PCR reaction solution and the PCR buffer solution are completely melted, mixing the mixture by vortex oscillation, and instantly centrifuging the mixture to the bottom of the tube. In a clean bench, a corresponding number of autoclaved 200. Mu. LEP tubes (centrifuge tubes) were prepared, and each tube was filled with PCR reaction solution and PCR buffer, and labeled.
Transferring the separated reagent to a sample preparation area. Sample DNA was removed from-20deg.C, left at room temperature until completely melted, vortexed, mixed well, and centrifuged transiently to the bottom of the tube. In a clean workbench, the sample DNA is taken out and added into a corresponding marked process EP tube, vortex oscillation is carried out for uniform mixing, and instantaneous centrifugation is carried out until the tube bottom is ready for use. The total volume of each human PCR reaction was 40. Mu.L, and the composition of the PCR reaction system for each human sample is shown in Table 5.
TABLE 5 reaction system
| Sequence number | Reactant composition | Volume (mu L) |
| 1 | PCR reaction solution | 20 |
| 2 | PCR buffer | 10 |
| 3 | DNA template | 10 |
| 4 | Total volume (mu L) | 40 |
b) Chip sample adding
In the sample preparation room, taking out the chip in the kit from the refrigerator with the temperature of 2-8 ℃ to restore the chip to room temperature, opening the package in the ultra-clean workbench, horizontally placing the chip, sucking 38 mu L of the PCR amplification system prepared in the step a) by using a manual pipette, vertically pumping liquid into the chip from the sample hole on the right side of the chip until the liquid reaches the sample hole on the left side of the chip through the sample inlet channel, immediately stopping sample feeding, wiping the residual liquid in the sample inlet hole and the sample outlet hole by using dust-free paper, and finally sealing the sample inlet hole and the sample outlet hole by using a sealing film (see the figure 2 of the specification).
c) Chip centrifugation
And (5) turning on a power supply of the centrifugal heat-sealing integrated machine. After the centrifugal cabin door is opened, the chip with the sample hole sealed after the sample is added is placed in a centrifugal rotor in the centrifugal heat-sealing integrated machine. When the centrifugal heat sealing integrated machine is placed, the unfilled corner of the chip is positioned at the upper right corner, and the centrifugal heat sealing integrated machine is balanced. After placing the chip, covering a centrifugal cabin door, centrifuging by using a point centrifugal key, and opening the cabin door to take out the chip after centrifuging.
d) Chip heat seal
The chips were inserted into the heat-seal trays (up to 4 chips each time), with the chip unfilled corner at the upper left corner. The device screen interface corresponding to the chip position is lighted orange after the chip is inserted, the chip is inserted without problems, after the temperature is stable, the heat sealing key is lighted, the heat sealing key is clicked to carry out heat sealing, when the interface corresponding to the chip position is lighted green, the heat sealing is finished, and the chip can be taken out after the tray is taken out of the bin.
e) Chip amplification
The above chip was placed in a chip amplification apparatus with the chip film side facing down and the unfilled corner facing up and left for amplification, and PCR amplification was performed according to the amplification reaction procedure shown in Table 6.
TABLE 6 nucleic acid amplification reaction procedure
3. Chip scanning
In the amplification product analysis area, a microarray scanner Luxscan10K/D and a Leber hereditary optic neuropathy gene detection analysis system are used for signal reading and result interpretation (see figure 1 of the specification for details), and specific judging principles are shown in Table 7.
TABLE 7 Leber hereditary optic neuropathy mitochondrial DNA site mutation interpretation principle corresponding to fluorescent signals
4. Detection results and genotyping results
The Leber hereditary optic neuropathy gene detection analysis system is used for scanning, signal reading and result interpretation, and 100 samples are used for detecting 5 parts of m.3394 homogenizing mutation (shown in figure 4 of the specification), 1 part of m.11696 homogenizing mutation (shown in figure 5 of the specification), 4 parts of m.12338 homogenizing mutation (shown in figure 6 of the specification), 1 part of m.14502 homogenizing mutation (shown in figure 7 of the specification) and 2 parts of m.15951 homogenizing mutation (shown in figure 8 of the specification).
In addition, the 100 samples are subjected to gene sequencing detection, and the sequencing result (see the specification of figures 9-10) is 100% consistent with the chip detection result, so that the stability of the technology is proved.
In summary, the invention adopts the microfluidic chip combined with competitive allele specific amplification technology to detect the mitochondrial DNA mutation site of the Leber hereditary optic neuropathy, compared with the existing detection technology, the invention can simultaneously detect and accurately type 15 site mutations of mitochondria related to the Leber hereditary optic neuropathy of a Chinese person, and the required PCR reaction system has small volume, the DNA template only needs 5-10 ng, and the invention can realize the efficient, sensitive, stable and multi-site combined detection of the mitochondrial mutation site of the Leber hereditary optic neuropathy, thereby providing important basis for the clinical early diagnosis and treatment of the Leber hereditary optic neuropathy and having wide application prospect and clinical reference value.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (9)
1. The primer group for detecting the Leber hereditary optic neuropathy is characterized by comprising 15 groups, wherein the sequences of the 15 groups of primer groups are shown as SEQ ID No. 1-45.
2. The primer set for detecting Leber hereditary optic neuropathy according to claim 1, wherein 15 primer sets and corresponding detection sites thereof are respectively:
the first group of primers are shown in a sequence table SEQ ID No.1-3 and are used for detecting m.3394T > C in genetic loci of mitochondrial genes ND 1;
the second group of primers is shown in a sequence table SEQ ID No.4-6 and is used for detecting m.3460G > A in a genetic locus of a mitochondrial gene ND 1;
the third group of primers are shown in a sequence table SEQ ID No.7-9 and are used for detecting m.3635G > A in genetic loci of mitochondrial genes ND 1;
the fourth group of primers is shown in a sequence table SEQ ID No.10-12 and is used for detecting m.3733G > A in a genetic locus of a mitochondrial gene ND 1;
the fifth group of primers are shown in a sequence table SEQ ID No.13-15 and are used for detecting m.3866T > C in genetic loci of mitochondrial genes ND 1;
the sixth group of primers is shown as SEQ ID No.16-18 of the sequence Listing and is used for detecting mitochondrial gene tRNA Met m.4435A in the genetic locus of (C)>G；
The seventh group of primers are shown in a sequence table SEQ ID No.19-21 and are used for detecting m.10680G > A in a genetic locus of a mitochondrial gene ND 4L;
the eighth group of primers are shown in sequence table SEQ ID No.22-24 and are used for detecting m.11696G > A in genetic loci of mitochondrial gene ND 4;
the ninth group of primers are shown in a sequence table SEQ ID No.25-27 and are used for detecting m.11778G > A in a genetic locus of a mitochondrial gene ND 4;
the tenth group of primers are shown in a sequence table SEQ ID No.28-30 and are used for detecting m.12338T > C in the genetic locus of the mitochondrial gene ND 5;
the eleventh group of primers are shown in sequence table SEQ ID No.31-33 and are used for m.14459G > A in genetic locus of mitochondrial gene ND 6;
the twelfth group of primers are shown in sequence table SEQ ID No.34-36 and are used for detecting m.14484T > C in genetic loci of mitochondrial gene ND 6;
the thirteenth group of primers are shown in a sequence table SEQ ID No.37-39 and are used for detecting m.14502T > C in the genetic locus of mitochondrial gene ND 6;
the fourteenth primer set is shown in SEQ ID No.40-42 of the sequence Listing and is used for detecting mitochondrial gene tRNA Glu m.14693A in the genetic locus of (C)>G；
The fifteenth primer set is shown in a sequence table SEQ ID No.43-45 and is used for detecting mitochondrial gene tRNA Thr m.159551A in the genetic locus of (A)>G。
3. A primer set for detecting Leber hereditary optic neuropathy according to claim 1 or 2, wherein each of 15 primer sets comprises two primers designed based on allele sequence differences and a common pair primer.
4. The primer group for detecting Leber hereditary optic neuropathy according to claim 1, wherein the sequence of the universal tag is shown as SEQ ID No.46 in SEQ ID No.1, SEQ ID No.4, SEQ ID No.7, SEQ ID No.10, SEQ ID No.13, SEQ ID No.16, SEQ ID No.19, SEQ ID No.22, SEQ ID No.25, SEQ ID No.28, SEQ ID No.31, SEQ ID No.34, SEQ ID No.37, SEQ ID No.40 and SEQ ID No. 43; the general tag sequences carried by SEQ ID No.2, SEQ ID No.5, SEQ ID No.8, SEQ ID No.11, SEQ ID No.14, SEQ ID No.17, SEQ ID No.20, SEQ ID No.23, SEQ ID No.26, SEQ ID No.29, SEQ ID No.32, SEQ ID No.35, SEQ ID No.38, SEQ ID No.41 and SEQ ID No.44 are shown as SEQ ID No. 47.
5. A kit for detecting Leber hereditary optic neuropathy, comprising any one of the 15 sets of primers or at least 2 sets of primers according to any one of claims 1 to 4.
6. The kit for detecting Leber hereditary optic neuropathy according to claim 5, wherein the chip in the kit comprises at least 18 chip reaction tanks, at least 2 positioning points, the chip reaction tanks comprise at least 1 internal control quality control, 1 negative quality control and 1 positive quality control, each quality control corresponds to 1 reaction tank, 1 group of primer groups based on any one of claims 1-5 are respectively coated in each of the remaining chip reaction tanks, and one group of primer groups is coated in each of the chip reaction tanks.
7. The kit for detecting Leber hereditary optic neuropathy according to claim 6, wherein the sum of three primer products in the primer set in each of the chip reaction tanks is equal.
8. A method for preparing a kit for detecting Leber hereditary optic neuropathy according to any one of claims 5 to 7, comprising the steps of:
(1) Preparing Kong Weibiao specific primer groups for detecting 15 sites in Leber hereditary optic neuropathy, and respectively coating 15 groups of the primer groups into 15 chip reaction tanks according to Kong Weibiao to obtain a chip semi-finished product;
(2) Preparing a PCR amplification reagent comprising a PCR reaction solution and a PCR buffer solution;
(3) Preparing sealing films and quality control products with corresponding numbers;
(4) The chip, the PCR amplification reagent, the sealing film and the quality control product are assembled into a kit for detecting the Leber hereditary optic neuropathy.
9. A reagent for detecting Leber hereditary optic neuropathy, comprising the primer set of any one of claims 1 to 4 or the kit of any one of claims 5 to 8.
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