Gene detection composition for limb dysplasia, its application, kit and detection method

Abstract

The present invention discloses a gene detection composition for limb dysplasia, its application, kit and detection method, belonging to the field of biological detection technology. This detection composition can detect relevant gene mutations of ±20 bp at the boundaries of all exons and introns of 460 limb dysplasia genes at one time, covering 425 limb dysplasia diseases. By using the targeted capture gene panel next-generation sequencing technology, the detection results have the advantages of high throughput, low cost, strong accuracy, high sensitivity, and comprehensive coverage of variations, and have great clinical application value and broader market promotion prospects. In the library construction process, through the principle of targeted hybridization capture, gene mutations that are instructive for limb dysplasia can be detected more efficiently, the cost of gene detection and the difficulty of experimental operation can be reduced, the sequencing depth can be increased, the detection sensitivity can be improved, and it has incomparable accuracy for some complex structural variations and gene complex regions.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a gene detection composition for limb dysplasia, an application, a kit and a detection method. Background Art

[0002] Limb dysplasia is a large category of skeletal system diseases with strong clinical and genetic heterogeneity. The main clinical manifestations include skeletal deformities, short stature, disproportionate growth, and dysplasia of single or a group of bones, etc. According to the latest guidelines of the "International Skeletal Dysplasia Society" in 2019, a total of 461 phenotypes are included in "limb dysplasia", including 437 genes. For example, osteogenesis imperfecta is a limb dysplasia disease with a relatively high incidence, with an incidence of about 1 / 10000 - 1 / 20000, mostly showing autosomal dominant inheritance. The clinical manifestations of this disease mainly include increased bone fragility, easy fracture, progressive joint deformity, blue sclera, dentin hypoplasia, and hearing loss, etc. Hereditary multiple osteochondroma is a relatively common limb dysplasia. This disease is a benign bone tumor, with an incidence of about 1 / 50000, mostly showing autosomal dominant inheritance. The main manifestation is skeletal dysplasia, mainly involving long bones, followed by the pelvis and scapula. Bony prominences of varying sizes often form near the epiphyses of the long bones of the extremities. Generally, the disease starts before the age of 3, and the number and volume of tumors will increase with age. The tumors mainly grow near joints, which can lead to short stature in children and cause deformities such as forearm deformity and genu valgum.

[0003] Therefore, early detection, early diagnosis, and early intervention are important means for preventing and treating such diseases and preventing irreversible serious sequelae in patients. Establishing a reliable and effective molecular diagnosis method to detect pathogenic gene mutations as early as possible is crucial for the correct diagnosis, typing, treatment, prenatal diagnosis, and genetic counseling of limb dysplasia, etc.

[0004] In recent years, it has been gradually emphasized with the development of the whole exome gene detection technology. This method detects all exons of more than 200 million genes. Although the detection range is wide, because there are too many genes included, it is not all applicable to the detection of genes related to limb dysplasia, which greatly increases the detection cost and the difficulty of detection and analysis. Or there are detection panels for a small number of genes, but due to the significant heterogeneity of limb dysplasia, it has obvious characteristics of "different gene mutations for the same disease, different diseases for the same gene mutation". Therefore, it can be seen that detecting a small number of genes is difficult to truly meet the actual clinical needs. Therefore, there is an urgent clinical need to establish a kit for gene detection that is specifically for limb dysplasia, has low cost, high accuracy, and wide coverage. Summary of the Invention

[0005] To solve the above problems existing in the prior art, the object of the present invention is to provide a limb dysplasia gene detection composition and its application, kit and detection method.

[0006] The technical solution adopted by the present invention is: a limb dysplasia gene detection composition, and the composition includes probes shown in SEQ ID NO.1-100.

[0007] The above detection composition provided by the present invention can detect relevant gene mutations of ±20bp at the boundaries of all exons and introns of 460 limb dysplasia genes at one time.

[0008] In the above composition, the probe length is 60-150bp; the step size between probes is 30-50bp.

[0009] An application of a limb dysplasia gene detection composition in preparing a limb dysplasia gene detection kit.

[0010] A limb dysplasia gene detection kit, and the kit includes a limb dysplasia gene detection composition.

[0011] The above kit further includes a library preparation reagent, a purification reagent, an amplification and hybridization capture reagent and a control product; the library preparation reagent includes a fragmentation buffer, a fragmentation reaction solution, a rapid ligation buffer and a rapid ligase; the purification reagent is a purification magnetic bead; during the hybridization capture process, streptavidin magnetic beads are used to capture the hybridization product.

[0012] The kit is a detection product covering 460 limb dysplasia genes.

[0013] A method for detecting limb dysplasia genes, using the high-throughput sequencing method and detecting by using the above-mentioned limb dysplasia gene detection kit.

[0014] The beneficial effects of the present invention are:

[0015] (1) The detection composition provided by the present invention can detect relevant gene mutations of ±20bp at the boundaries of all exons and introns of 460 limb dysplasia genes at one time.

[0016] (2) The kit prepared from the detection composition can cover 425 kinds of limb dysplasia diseases at one time, including the detection of 460 limb dysplasia-related genes. The components of the kit include a library preparation reagent, a magnetic bead purification reagent, a library amplification and hybridization capture reagent, a negative control product, a positive control product, etc.

[0017] (3) This kit uses next-generation sequencing technology, and the test results have the advantages of high throughput, low cost, strong accuracy, high sensitivity, and comprehensive coverage of variations, with great clinical application value and broader market promotion prospects.

[0018] (4) In the library construction process, through the principle of targeted hybridization capture, it can more efficiently detect gene mutations that are instructive for limb dysplasia, reduce the cost and experimental operation difficulty of gene detection, improve the sequencing depth, increase the detection sensitivity, and has incomparably accurate detection results for some complex structural variations and gene complex regions. Description of the Drawings

[0019] Figure 1 For Experiment 3, it is a schematic diagram of the Sanger sequencing peak of the heterozygous variant c.477+5G>T in the subject.

[0020] Figure 2 For Experiment 3, it is a schematic diagram of the Sanger sequencing peak of the wild type in the mother of the subject.

[0021] Figure 3 For Experiment 3, it is the Sanger sequencing peak map of the c.937C>T locus of the S13 EXT2 gene in the subject. Detailed Embodiments

[0022] The present invention will be further explained below in conjunction with specific embodiments. Those skilled in the art will understand that the following described embodiments are some embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. All the reagents used are conventional products that can be obtained through commercial purchase.

[0023] Example 1:

[0024] A kit for detecting 425 limb dysplasia diseases at one time based on high-throughput sequencing, including 460 gene variations related to limb dysplasia. The reagents of the kit include library preparation reagents, magnetic bead purification reagents, library amplification and hybridization capture reagents, negative control products, positive control products, etc.

[0025] The hybridization capture probes involved cover all exon regions and introns ±20bp of the 460 genes in Table 1. The length of all probes is 60-150bp. The step size between probes is 30-50bp. The target region is about 1.1M, and the probe coverage is 99.89%.

[0026] Table 1. Table of 460 gene variation types covered by the present invention

[0027]

[0028]

[0029]

[0030] Experimental Example 1:

[0031] The present invention has established a gene detection composition for abnormal limb development, its application, kit and detection method. The components of the kit include library preparation reagents, magnetic bead purification reagents, library amplification and hybridization capture reagents, etc. The kit is stored at -20°C. The kit specification is: 50 person-times per box, and the specific components are shown in Table 2.

[0032] Table 2 Components of the Kit

[0033] Component Volume (uL) Number of tubes Fragmentation buffer 250 1 Fragmentation reaction solution 500 1 Quick ligation buffer 625 2 Quick ligase 250 1 Purification magnetic beads 1000 5 Limb dysplasia probe 460 100 1 Quick hybridization reaction solution 100 1 Hybridization enhancer 750 2 Blocking buffer 250 1 Blocking solution 400 1 High-fidelity reaction solution 625 2 Standard (100 ng / μl) 250 1 Positive control 500 1 Negative control 500 1

[0034] The specific operation of the kit is as follows:

[0035] I. Sample Preparation

[0036] Draw 2 mL of peripheral blood (EDTA anticoagulant) from 30 patients with abnormal limb development, and extract genomic DNA using the QIAamp Whole Blood DNA Extraction Kit (Qiagen, Germany) according to its instructions. Quantify the samples using qubit. Dilute the samples to 10 ng / uL with sterile water, at least 10 uL.

[0037] DNA Fragmentation and End Repair

[0038] 1. Thaw the fragmentation buffer and fragmentation reaction solution, mix well thoroughly, briefly centrifuge to collect at the bottom of the tube, and place on ice for later use. All the following steps are performed on ice.

[0039] Prepare the following reaction in a sterile PCR tube: DNA 10 uL; fragmentation buffer 5 uL; nuclease-free water 25 uL; total: 40 uL.

[0040] 2. Add 10 μL of the fragmentation reaction solution to each sample, mix well by pipetting or shaking, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Immediately place it in a PCR instrument for reaction

[0041] Place the PCR tube in a PCR instrument and run the following program: hot lid 105°C, volume 50 uL; 37°C for 15 min; 65°C for 30 min; 4°C, Hold.

[0042] III. Adapter Ligation

[0043] 1. Take out the quick ligation buffer and quick ligase from -20°C, thaw, mix well thoroughly, briefly centrifuge to collect at the bottom of the tube, and place on ice for later use.

[0044] 2. Prepare the reaction system as follows:

[0045] 50 μL of the product from the previous step; 25 μL of Quick Ligation Buffer; 5 μL of Quick Ligation Enzyme; 15 μL of nuclease-free water; 5 μL of adapter; Total: 100 μL.

[0046] Use a pipette to pipette up and down or vortex to mix well, and briefly centrifuge to collect the reaction solution at the bottom of the tube (it is necessary to pipette up and down thoroughly to mix well).

[0047] 3. Place the PCR tube in the PCR instrument and run the following program: Hot lid at 105 °C, 100 μL; 20 °C for 15 min; 4 °C Hold.

[0048] IV. Product purification:

[0049] Purify the reaction product using purification magnetic beads: Prepare 80% ethanol in advance and equilibrate the magnetic beads at room temperature.

[0050] a. After the magnetic beads are equilibrated to room temperature, vortex to mix the purification magnetic beads well.

[0051] b. Pipette 60 μL of the purification magnetic beads into 100 μL of the Adapter Ligation product, and vortex or pipette up and down 10 times to mix well.

[0052] c. Incubate at room temperature for 5 min.

[0053] d. Briefly centrifuge the centrifuge tube and place it in the magnetic rack to separate the magnetic beads and the liquid. After the solution becomes clear (about 5 min), carefully remove the supernatant.

[0054] e. Keep the centrifuge tube in the magnetic rack all the time, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant.

[0055] f. Repeat step e, for a total of two rinses.

[0056] Keep the centrifuge tube in the magnetic rack all the time, open the lid and dry the magnetic beads in the air for 3 - 5 min until there is no ethanol residue.

[0057] h. Take out the centrifuge tube from the magnetic rack and perform elution:

[0058] Add 22.5 μL of elution buffer (10 mM Tris-HCl, pH 8.0 - 8.5) or sterile ultrapure water for elution, vortex or pipette up and down to mix well, place at room temperature for 2 min, briefly centrifuge the centrifuge tube and place it in the magnetic rack to stand still. After the solution becomes clear (about 5 min), carefully transfer 20 μL of the supernatant to a new EP tube, and do not touch the magnetic beads.

[0059] V. Hybridization capture

[0060] Before the experiment, thaw the following reagents including the limb dysplasia probe 460, rapid hybridization reaction solution, hybridization enhancer, blocking buffer, and blocking solution on ice. Shake for 2 seconds and centrifuge, then place on ice for standby.

[0061] 1. Library hybridization

[0062] a. After thawing the probe on ice, transfer it to a new tube. Add the calculated volume of the library to the old tube, mix well and then transfer to the new tube. Then add the blocking solution and blocking buffer as follows.

[0063] Pre-hybridization mix components: limb dysplasia probe 460 4 μL; blocking solution 8 μL; blocking buffer 5 μL; total: 17 μL.

[0064] Set the following program. The hot lid temperature needs to be set to 85 °C and start running. 95 °C Hold; 95 °C 5 min; 60 °C 15 min - 4 h.

[0065] b. Place the rapid hybridization reaction solution on a thermostatic mixer and heat at 65 °C for 10 minutes until the precipitate in it dissolves. Shake and mix well, then place at 65 °C for standby.

[0066] c. Add 20 μL of the rapid hybridization reaction solution to the dried pre-hybridization mix to dissolve the dried powder, without pipetting and mixing.

[0067] d. Add 30 μL of hybridization enhancer above the liquid, shake and mix well, then centrifuge.

[0068] e. Place the PCR tube on the PCR instrument that has already run the program and start the hybridization reaction.

[0069] 2. Capture and elution

[0070] 2.1 Shake and mix the streptavidin magnetic beads well, and take 100 μL of the magnetic bead suspension into a 1.5 mL epp.

[0071] 2.2 Add 200 μL of Fast Binding Buffer and pipette and mix well.

[0072] 2.4 Place the epp on the magnetic rack for 1 minute. Aspirate and discard the supernatant, and do not touch the magnetic beads with the pipette tip. Remove the epp.

[0073] 2.5 Repeat steps 2.3 - 2.4 twice, washing the streptavidin magnetic beads a total of 3 times.

[0074] 2.6 After the last wash, add 200 μL of Fast Binding Buffer and shake and mix the magnetic beads well.

[0075] 2.7 Open the lid of the PCR instrument. Without removing the PCR tube, open the tube lid under 60°C incubation. Quickly aspirate all the hybridization solution and add it quickly to 200 μL of streptavidin magnetic beads. Pipette up and down to mix well.

[0076] 2.8 Place the 1.5 mL eppendorf tube on a shaker and rotate at room temperature for 30 minutes.

[0077] 2.9 Remove the eppendorf tube, centrifuge to pellet the liquid to the bottom of the tube, and place the eppendorf tube on a magnetic stand for 1 minute.

[0078] 2.10 Aspirate and discard the supernatant (including the hybridization enhancer), being careful not to touch the magnetic beads with the pipette tip.

[0079] 2.11 Remove the eppendorf tube from the magnetic stand and add 200 μL of Fast Wash Buffer 1 preheated to 70°C (briefly centrifuge 5 minutes before adding to prevent evaporation). Immediately place it on a thermostatic shaker (70°C) and pipette up and down to mix well.

[0080] 2.12 Incubate at 70°C for 5 minutes, place the eppendorf tube on a magnetic stand for 1 minute, and aspirate and discard the supernatant.

[0081] 2.13 Remove the eppendorf tube from the magnetic stand, add 200 μL of Fast Wash Buffer 1 preheated to 70°C, immediately place it on a thermostatic shaker (70°C), pipette up and down to mix well, incubate at 70°C for 5 minutes, and briefly centrifuge to pellet the liquid to the bottom of the tube.

[0082] 2.14 Transfer all the volume to a new 1.5 mL eppendorf tube, place it on a magnetic stand for 1 minute, aspirate and discard the supernatant, being careful not to touch the magnetic beads with the pipette tip.

[0083] 2.15 Remove the eppendorf tube from the magnetic stand, add 200 μL of Wash Buffer 2 preheated to 48°C, pipette up and down to mix well, briefly centrifuge, incubate at 48°C for 5 minutes, and place it on a magnetic stand for 1 minute.

[0084] 2.16 Aspirate and discard the supernatant, and repeat steps 2.21 - 2.24 two more times. Wash a total of three times.

[0085] Remove the eppendorf tube from the magnetic stand, add 45 μL of water, pipette up and down to mix well to obtain magnetic bead slurry. Place it on ice.

[0086] VI. Library Amplification and Purification after Capture

[0087] 1. Thaw the high-fidelity reaction solution and invert to mix well. Briefly centrifuge to collect it at the bottom of the tube. Prepare the following reaction in a sterile PCR tube: 22.5 μL of the above product; 25 μL of high-fidelity reaction solution; 2.5 μL of amplification primer; Total: 45 μL.

[0088] 2. Use a pipette to pipette and mix well or shake and mix well, and briefly centrifuge to collect the reaction solution to the bottom of the tube.

[0089] 3. Place the PCR tube in a PCR instrument and perform the following reaction:

[0090]

[0091] 4. Product purification:

[0092] a. After the magnetic beads are equilibrated to room temperature, vortex and mix well the purified magnetic beads for limb development disorder probe 460.

[0093] b. Pipette 50 μL of the purified magnetic beads for limb development disorder probe 460 and 50 μL of purified water into the above 50 μL of the product, and vortex or pipette 10 times to mix well.

[0094] c. Incubate at room temperature for 5 min.

[0095] d. Briefly centrifuge the centrifuge tube and place it in a magnetic rack to separate the magnetic beads and the liquid. After the solution becomes clear (about 5 min), carefully transfer the supernatant to a new PCR tube and discard the magnetic beads.

[0096] e. Pipette 20 μL of the purified magnetic beads for limb development disorder probe 460 into the supernatant, and pipette 10 times to mix well.

[0097] f. Incubate at room temperature for 5 min.

[0098] g. Briefly centrifuge the centrifuge tube and place it in a magnetic rack to separate the magnetic beads and the liquid. After the solution becomes clear (about 5 min), carefully transfer the supernatant.

[0099] h. Keep the centrifuge tube always placed in the magnetic rack, add 200 μL of freshly prepared 80% ethanol to wash the magnetic beads. Incubate at room temperature for 30 sec, and carefully remove the supernatant.

[0100] i. Repeat step h for a total of two washes.

[0101] j. Keep the centrifuge tube always placed in the magnetic rack, open the lid and dry the magnetic beads in the air for 3 - 5 min until there is no ethanol residue.

[0102] k. Take the centrifuge tube out of the magnetic rack and add 22.5 μL of eluent for elution

[0103] VII. Quality control: Use Qubit to measure the library concentration and use 4200 to check the size distribution of the library.

[0104] Sequencing: The captured regions were sequenced from both ends using the Illumina HiSeq X ten high-throughput sequencing platform, with a length of 150 bp. After sequencing the target regions, adapters and low-quality data in the sequencing data were removed. The data were aligned to the reference genome (hg19 version) using the BWA software (http: / / bio-bwa.sourceforge.net / ), and statistical analyses were performed on data such as sequencing depth, uniformity, and probe specificity.

[0105] Experimental Example 2: The enterprise reference products of this kit were detected according to the operation steps of Experimental Example 1, and the results should all meet the detection performance requirements.

[0106] Table 3. Enterprise reference products

[0107]

[0108]

[0109] (1) Accuracy: Ten enterprise reference products for accuracy were detected using the kit described in Example 1, and all the results were positive, with a positive coincidence rate of 100%.

[0110] (2) Specificity: Three enterprise reference products for specificity were detected using the kit described in Example 1, and all the detection results should be negative.

[0111] (3) Minimum detection limit: Ten reference products for the minimum detection limit with a concentration not higher than 5 ng / μL were detected using the kit described in Example 1, and all the results were positive, with a coincidence rate of 100%.

[0112] Experiment 3: The kit and the whole exome sequencing method were used to detect gene mutations in 30 patients with limb dysplasia respectively.

[0113]

[0114]

[0115] It is not difficult to see from the above results that the library construction and sequencing cost of this kit is reduced by more than half compared with the whole exome library construction and sequencing scheme, but the average sequencing depth is more than twice that of the whole exome sequencing. Finally, it is not difficult to see from the detection results that among the 30 patients, 23 cases had relevant mutations detected by WES (76.67%), and 25 cases had relevant mutations identified by the present invention (83.33%), including all 23 cases detected by WES.

[0116] Such as Figure 1As shown in Table 4 data, further analysis of the original sequencing data for the discrepant results found that among the 2 cases identified more by the present invention, 1 case was the intron +5 region (Table 4), which might be the region not covered by WES, while the present invention covered the relevant region. After Sanger verification, the deletion at this locus had the above-mentioned mutation ( Figure 1 ).

[0117] Such as Figure 1 As shown in Table 5 data, for the last discrepant result sample, after Sanger verification, it was also consistent with the detection result of the kit of the present invention ( Figure 1 ). This variation belonged to the exon region, but due to the high GC content in this region, the WES sequencing quality was not high, and the actual sequencing depth was only 30×, while the present invention had a high sequencing depth, and the actual sequencing depth was about 80×, so it could accurately detect this locus.

[0118] Table 4: Detection information of the c.477+5G>T locus mutation of the RPL13 gene in the S7 family of the tested subjects by the present kit.

[0119]

[0120] Tested subject: c.477+5G>T heterozygous variation, peak map as Figure 1 shown; Mother of the tested subject: wild type, peak map as Figure 2 shown. Such as Figure 1 and 2 shown, are the Sanger sequencing peak maps of the c.477+5G>T locus of the RPL13 gene in the S7 family of the tested subjects.

[0121] Table 5. Detection information of the c.937C>T mutation of the EXT2 gene in the S13 tested subject by the present kit

[0122]

[0123] Tested subject: c.937C>T heterozygous variation, peak map as Figure 3 shown. Such as Figure 3 shown, is the Sanger sequencing peak map of the c.937C>T locus of the EXT2 gene in the S13 tested subject.

[0124] The present invention is not limited to the above optional embodiments. Anyone can obtain other various forms of products under the inspiration of the present invention, and all belong to the protection scope of the present invention. The above specific embodiments should not be construed as limiting the protection scope of the present invention. Those of ordinary skill in the art should understand that without departing from the scope of the present invention, the technical solutions recorded in the foregoing embodiments can be modified, or some or all of the technical features can be equivalently replaced. At the same time, these modifications or replacements will 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. Sequence Listing <110> Beijing Jishuitan Hospital; Beijing Huaxin Aomei Gene Biotechnology Co., Ltd. <120> Gene Detection Composition for Limb Developmental Abnormalities, and Applications, Kits and Detection Methods Thereof <160> 50 <170> SIPOSequenceListing 1.0 <210> 1 <211> 100 <212> DNA <213> Artificial Sequence <400> 1 gaccatgaca actcgctgag tgtcagcatc ccccaaccgt ccccgctgag ggtcctcctg 60 gggacctccc tcaccatccc ctgctatttc atcgacccca 100 <210> 2 <211> 100 <212> DNA <213> Artificial Sequence <400> 2 tgcaccctgt gaccaccgcc ccttctaccg ccccactggc cccaagaatc aagtggagcc 60 gtgtgtccaa ggagaaggag gtagtgctgc tggtggccac 100 <210> 3 <211> 100 <212> DNA <213> Artificial Sequence <400> 3 tgaagggcgc gtgcgggtca acagtgccta tcaggacaag gtctcactgc ccaactaccc 60 ggccatcccc agtgacgcca ccttggaagt ccagagcctg 100 <210> 4 <211> 100 <212> DNA <213> Artificial Sequence <400> 4 ggcatcgtgt tccattacag agccatctct acacgctaca ccctcgactt tgacagggcg 60 cagcgggcct gcctgcagaa cagtgccatc attgccacgc 100 <210> 5 <211> 100 <212> DNA <213> Artificial Sequence <400> 5 gataccccat ccacactccc cgggaaggct gctatggaga caaggatgag tttcctggtg 60 tgaggacgta tggcatccga gacaccaacg agacctatga 100 <210> 6 <211> 100 <212> DNA <213> Artificial Sequence <400> 6 ggtgaggtct tttatgcaac atctccagag aagttcacct tccaggaagc agccaatgag 60 tgccggcggc tgggtgcccg gctggccacc acgggccagc 100 <210> 7 <211> 100 <212> DNA <213> Artificial Sequence <400> 7 tctacctggc ctggcaggct ggcatggaca tgtgcagcgc cggctggctg gccgaccgca 60 gcgtgcgcta ccccatctcc aaggcccggc ccaactgcgg 100 <210> 8 <211> 100 <212> DNA <213> Artificial Sequence <400> 8 ggtgaagact ttgtggacat cccagaaaac ttctttggag tggggggtga ggaggacatc 60 accgtccaga cagtgacctg gcctgacatg gagctgccac 100 <210> 9 <211> 100 <212> DNA <213> Artificial Sequence <400> 9 tgcctcgaaa catcactgag ggtgaagccc gaggcagcgt gatccttacc gtaaagccca 60 tcttcgaggt ctcccccagt cccctggaac ccgaggagcc 100 <210> 10 <211> 100 <212> DNA <213> Artificial Sequence <400> 10 cttcacgttt gcccctgaaa taggggccac tgccttcgct gaggttgaga atgagactgg 60 agaggccacc aggccctggg gctttcccac acctggcctg 100 <210> 11 <211> 100 <212> DNA <213> Artificial Sequence <400> 11 ggggtcgtct tccactaccg cccgggaccc acccgctact cgctgacctt tgaggaggca 60 cagcaggcct gcctgcgcac gggggcggtc attgcctcgc 100 <210> 12 <211> 100 <212> DNA <213> Artificial Sequence <400> 12 gataccccat tgtgagcccc cggaccccat gcgtgggtga caaggacagc agcccagggg 60 tcaggaccta tggcgtgcgc ccatcaacag agacctacga 100 <210> 13 <211> 100 <212> DNA <213> Artificial Sequence <400> 13 ggggaggtgt tcttcgccac acgccttgag cagttcacct tccaggaagc actggagttc 60 tgtgaatctc acaatgctac gctggccacc acgggccagc 100 <210> 14 <211> 100 <212> DNA <213> Artificial Sequence <400> 14 tctacgccgc ctggagccgc ggcctggaca agtgctatgc cggctggctg gccgacggca 60 gcctccgcta ccccatcgtc accccaaggc ctgcctgcgg 100 <210> 15 <211> 100 <212> DNA <213> Artificial Sequence <400> 15 ggcatttcag cggttccttc tccaggagaa gaagagggtg gcacacccac atcaccctct 60 ggtgtggagg agtggatcgt gacccaagtg gttcctggtg 100 <210> 16 <211> 100 <212> DNA <213> Artificial Sequence <400> 16 tggctgctgt ccccgtagaa gaggagacaa ctgctgtacc ctcaggggag actactgcca 60 tcctagagtt caccaccgag ccagaaaacc agacagaatg 100 <210> 17 <211> 100 <212> DNA <213> Artificial Sequence <400> 17 gggatccttc ctacttggcc tcccactggc gcagcaacag aggaaagtac agaaggccct 60 tctgcaactg aagtgccctc tgcctcagag gaaccatccc 100 <210> 18 <211> 100 <212> DNA <213> Artificial Sequence <400> 18 cctcagaggt gccattcccc tcagaggagc catccccctc agaggaacca ttcccctcag 60 tgaggccatt cccctcagtg gagctgttcc cctcagagga 100 <210> 19 <211> 100 <212> DNA <213> Artificial Sequence <400> 19 gccattcccc tccaaggagc catccccctc agaggaacca tcagcctcgg aagagccgta 60 tacaccttca ccccccgtgc ccagctggac tgagctgccc 100 <210> 20 <211> 100 <212> DNA <213> Artificial Sequence <400> 20 agctctgggg aggaatctgg ggcccctgat gtcagtggtg acttcacagg cagtggagat 60 gtttcaggac accttgactt cagtgggcag ctgtcagggg 100 <210> 21 <211> 100 <212> DNA <213> Artificial Sequence <400> 21 acagggcaag tggactgccc tctggagacc tggactccag tggtcttact tccacagtgg 60 gctcaggcct gcctgtggaa agtggactac cctcagggga 100 <210> 22 <211> 100 <212> DNA <213> Artificial Sequence <400> 22 tgaagagaga attgagtggc ccagcactcc tacggttggt gaactgccct ctggagctga 60 gatcctagag ggctctgcct ctggagttgg ggatctcagt 100 <210> 23 <211> 100 <212> DNA <213> Artificial Sequence <400> 23 ggacttcctt ctggagaagt tctagagacc tctgcctctg gagtaggaga cctcagtggg 60 cttccttctg gagaagttct agagaccact gcccctggag 100 <210> 24 <211> 100 <212> DNA <213> Artificial Sequence <400> 24 tagaggacat cagcgggctt ccttctggag aagttctaga gaccactgcc cctggagtag 60 aggacatcag cgggcttcct tctggagaag ttctagagac 100 <210> 25 <211> 100 <212> DNA <213> Artificial Sequence <400> 25 cactgcccct ggagtagagg acatcagcgg gcttccttct ggagaagttc tagagaccac 60 tgcccctgga gtagaggaca tcagcgggct tccttctgga 100 <210> 26 <211> 100 <212> DNA <213> Artificial Sequence <400> 26 gaagttctag agaccactgc ccctggagta gaggacatca gcgggcttcc ttctggagaa 60 gttctagaga ccactgcccc tggagtagag gacatcagcg 100 <210> 27 <211> 100 <212> DNA <213> Artificial Sequence <400> 27 ggcttccttc tggagaagtt ctagagaccg ctgcccctgg agtagaggac atcagcgggc 60 ttccttctgg agaagttcta gagaccgctg cccctggagt 100 <210> 28 <211> 100 <212> DNA <213> Artificial Sequence <400> 28 agaggacatc agcgggcttc cttctggaga agttctagag accgctgccc ctggagtaga 60 ggacatcagc gggcttcctt ctggagaagt tctagagacc 100 <210> 29 <211> 100 <212> DNA <213> Artificial Sequence <400> 29 gctgcccctg gagtagagga catcagcggg cttccttctg gagaagttct agagaccgct 60 gcccctggag tagaggacat cagcgggctt ccttctggag 100 <210> 30 <211> 100 <212> DNA <213> Artificial Sequence <400> 30 aagttctaga gaccgctgcc cctggagtag aggacatcag cgggcttcct tctggagaag 60 ttctagagac cgctgcccct ggagtagagg acatcagcgg 100 <210> 31 <211> 100 <212> DNA <213> Artificial Sequence <400> 31 gcttccttct ggagaagttc tagagactgc tgcccctgga gtagaggaca tcagcgggct 60 tccttctgga gaagttctag agactgctgc ccctggagta 100 <210> 32 <211> 100 <212> DNA <213> Artificial Sequence <400> 32 gaggacatca gcgggcttcc ttctggagaa gttctagaga ctgctgcccc tggagtagag 60 gacatcagcg ggcttccttc tggagaagtt ctagagactg 100 <210> 33 <211> 100 <212> DNA <213> Artificial Sequence <400> 33 ctgcccctgg agtagaggac atcagcgggc ttccttctgg agaagttcta gagactgctg 60 cccctggagt agaggacatc agcgggcttc cttctggaga 100 <210> 34 <211> 100 <212> DNA <213> Artificial Sequence <400> 34 agttctagag actgctgccc ctggagtaga ggacatcagc gggcttcctt ctggagaagt 60 tctagagact gctgcccctg gagtagagga catcagcggg 100 <210> 35 <211> 100 <212> DNA <213> Artificial Sequence <400> 35 cttccttctg gagaagttct agagactgct gcccctggag tagaggacat cagcgggctt 60 ccttctggag aagttctaga gactgctgcc cctggagtag 100 <210> 36 <211> 100 <212> DNA <213> Artificial Sequence <400> 36 aggacatcag cgggcttcct tctggagaag ttctagagac tactgcccct ggagtagagg 60 agatcagcgg gcttccttct ggagaagttc tagagactac 100 <210> 37 <211> 100 <212> DNA <213> Artificial Sequence <400> 37 tgcccctgga gtagatgaga tcagtgggct tccttctgga gaagttctag agactactgc 60 ccctggagta gaggagatca gcgggcttcc ttctggagaa 100 <210> 38 <211> 100 <212> DNA <213> Artificial Sequence <400> 38 gttctagaga cttctacctc tgcggtaggg gacctcagtg gacttccttc tggaggagaa 60 gttctagaga tttctgtctc tggagtagag gacatcagtg 100 <210> 39 <211> 100 <212> DNA <213> Artificial Sequence <400> 39 ggcttccttc tggagaggtt gtagagactt ctgcctctgg aatagaggat gtcagtgaac 60 ttccttcagg agaaggtcta gagacctctg cttctggagt 100 <210> 40 <211> 100 <212> DNA <213> Artificial Sequence <400> 40 agaggacctc agcaggctcc cttctggaga agaagttcta gagatttctg cctctggatt 60 tggggacctc agtggacttc cttctggagg agaaggtcta 100 <210> 41 <211> 100 <212> DNA <213> Artificial Sequence <400> 41 gagacctctg cttctgaagt agggactgac ctcagtgggc ttccttctgg aagggagggt 60 ctagagactt cagcttctgg agctgaggac ctcagtgggt 100 <210> 42 <211> 100 <212> DNA <213> Artificial Sequence <400> 42 tgccttctgg aaaagaagac ttggtggggt cagcttctgg agacttggac ttgggcaaac 60 tgccttctgg aactctagga agtgggcaag ctccagaaac 100 <210> 43 <211> 100 <212> DNA <213> Artificial Sequence <400> 43 aagtggtctt ccctctggat ttagtggtga gtattctggg gtggaccttg gaagtggccc 60 accctctggc ctgcctgact ttagtggact tccatctgga 100 <210> 44 <211> 100 <212> DNA <213> Artificial Sequence <400> 44 ttcccaactg tttccctagt ggattctaca ttggtggaag tggtcacagc ctccactgca 60 agtgaactgg aagggagggg aaccattggc atcagtggtg 100 <210> 45 <211> 100 <212> DNA <213> Artificial Sequence <400> 45 caggagaaat atctggactg ccctccagtg agctggacat tagtgggaga gctagtggac 60 tcccttcagg aactgaactc agtggccaag catctgggtc 100 <210> 46 <211> 100 <212> DNA <213> Artificial Sequence <400> 46 tcctgatgtc agtggggaaa tacctggact ctttggtgtc agtggacagc catcagggtt 60 tcctgacact agtggggaaa catctggagt gactgagctt 100 <210> 47 <211> 100 <212> DNA <213> Artificial Sequence <400> 47 agcgggctgt cctctggaca accaggtatt agtggagaag catctggagt tctttatggc 60 actagtcaac cctttggcat aactgatctg agtggagaaa 100 <210> 48 <211> 100 <212> DNA <213> Artificial Sequence <400> 48 catctggggt ccctgatctc agtgggcagc cttcagggtt accagggttc agtggggcaa 60 catcaggagt ccctgacctg gtttctggta ccacgagtgg 100 <210> 49 <211> 100 <212> DNA <213> Artificial Sequence <400> 49 cagcggtgaa tcttctggga ttacatttgt ggacaccagt ttggttgaag tggcccctac 60 tacatttaaa gaagaagaag gcttagggtc tgtggaactc 100 <210> 50 <211> 100 <212> DNA <213> Artificial Sequence <400> 50 agtggcctcc cttccggaga ggcagatctg tcaggcaaat ctgggatggt ggatgtcagt 60 ggacagtttt ctggaacagt cgattccagt gggtttacat 100 <210> 51 <211> 100 <212> DNA <213> Artificial Sequence <400> 51 cccagactcc ggaattcagt ggcctaccaa gtggcatagc tgaggtcagt ggagaatcct 60 ccagagctga gattgggagc agcctgccct cgggagcata 100 <210> 52 <211> 100 <212> DNA <213> Artificial Sequence <400> 52 ttatggcagt ggaactccat ctagtttccc cactgtctct cttgtagaca gaactttggt 60 ggaatctgta acccaggctc caacagccca agaggcagga 100 <210> 53 <211> 100 <212> DNA <213> Artificial Sequence <400> 53 gaagggcctt ctggcatttt agaactcagt ggtgctcatt ctggagcacc agacatgtct 60 ggggagcatt ctggatttct ggacctaagt gggctgcagt 100 <210> 54 <211> 100 <212> DNA <213> Artificial Sequence <400> 54 ccgggctgat agagcccagc ggagagccac caggtactcc atattttagt ggggattttg 60 ccagcaccac caatgtaagt ggagaatcct ctgtagccat 100 <210> 55 <211> 100 <212> DNA <213> Artificial Sequence <400> 55 gggcaccagt ggagaggcct caggacttcc agaagttact ttaatcactt ctgagttcgt 60 ggagggtgtt actgaaccaa ctatttctca ggaactaggc 100 <210> 56 <211> 100 <212> DNA <213> Artificial Sequence <400> 56 caaaggcccc ctgtgacaca cacaccccag ctttttgagt ccagtggaaa agtctccaca 60 gctggggaca ttagtggagc taccccagtg ctccctgggt 100 <210> 57 <211> 100 <212> DNA <213> Artificial Sequence <400> 57 ctggagtaga agtatcatca gtcccagaat ctagcagtga gacgtccgcc tatcctgaag 60 ctgggttcgg ggcatctgcc gcccctgagg ccagcagaga 100 <210> 58 <211> 100 <212> DNA <213> Artificial Sequence <400> 58 agattctggg tcccctgatc tgagtgaaac cacctctgca ttccacgaag ctaaccttga 60 gagatcctct ggcctaggag tgagcggcag cactttgaca 100 <210> 59 <211> 100 <212> DNA <213> Artificial Sequence <400> 59 tttcaagaag gcgaggcgtc cgctgcccca gaagtgagtg gagaatccac caccaccagt 60 gatgtgggga cagaggcacc aggcttgcct tcagccactc 100 <210> 60 <211> 100 <212> DNA <213> Artificial Sequence <400> 60 ccacggcttc tggagacagg actgaaatca gcggagacct gtctggtcac acctcgcagc 60 tgggcgttgt catcagcacc agcatcccag agtctgagtg 100 <210> 61 <211> 100 <212> DNA <213> Artificial Sequence <400> 61 gacccagcag acccagcgcc ctgcagagac gcatctagaa attgagtcct caagcctcct 60 gtactcagga gaagagactc acacagtcga aacagccacc 100 <210> 62 <211> 100 <212> DNA <213> Artificial Sequence <400> 62 gcccccgcca ggtcctgtgc agaggagccc tgtggagctg ggacctgcaa ggagacagag 60 ggacacgtca tatgcctgtg cccccctggc tacactggcg 100 <210> 63 <211> 100 <212> DNA <213> Artificial Sequence <400> 63 gaccaggagg tatgtgagga gggctggaac aagtaccagg gccactgtta ccgccacttc 60 ccggaccgcg agacctgggt ggatgctgag cgccggtgtc 100 <210> 64 <211> 100 <212> DNA <213> Artificial Sequence <400> 64 gcaatttgag aactggcgcc ccaaccagcc tgacaacttt tttgccgctg gagaggactg 60 tgtggtgatg atctggcacg agaagggcga gtggaatgat 100 <210> 65 <211> 100 <212> DNA <213> Artificial Sequence <400> 65 gtggcctgcg gagagccccc tgtggtggag catgccagga ccttcgggca gaagaaggac 60 cggtatgaga tcaattccct ggtgcggtac cagtgcacag 100 <210> 66 <211> 100 <212> DNA <213> Artificial Sequence <400> 66 tgtcaacagt cagttttcaa tttgtcgagg gaattatcaa ttttggtcaa agtctttttg 60 atacgcagtg ctgtgagtct tgcggatgga ttttgatacc 100 <210> 67 <211> 100 <212> DNA <213> Artificial Sequence <400> 67 accgggtctg agaaccatct gttgggtatg tttggccttt gttgatccac acagactacc 60 ttcctcatat cttcaaaact tgggtcattg ggaaccacat 100 <210> 68 <211> 100 <212> DNA <213> Artificial Sequence <400> 68 accattgctc accatccgcc tggccacttc ccacaaaaca agtccaaagg cccaaatatc 60 gaccctttta taagaatcga aacaatccac ctggatggtt 100 <210> 69 <211> 100 <212> DNA <213> Artificial Sequence <400> 69 tcatctagaa cttcgggggc catgtagcgc ttggtgccca cacggggatt gttccccaca 60 tcaagctgat tggtgctctg ggaatgcatg actgccaggc 100 <210> 70 <211> 100 <212> DNA <213> Artificial Sequence <400> 70 acccaaatct gctatgcaac actgtccatt cttcttaacc agaatatttt tgctctttaa 60 atctcgatgg gcaatggctg gtttcccttg ggtcccaaat 100 <210> 71 <211> 100 <212> DNA <213> Artificial Sequence <400> 71 atctctatgt gcaaatgtgc aagaccacta gctatggaca gcactattcg aaggcagcta 60 actgtatcca gagtagtaag ctgaagatag tcgtacaacg 100 <210> 72 <211> 100 <212> DNA <213> Artificial Sequence <400> 72 acctaagata ttttcatgcc tcagcatcac agtgttgtac aattccgttt ccctgaacca 60 tgacttctca tcacgggagg agaagatctt cacggcaaca 100 <210> 73 <211> 100 <212> DNA <213> Artificial Sequence <400> 73 accgacacac tccaacagtg taatctggcg agccactgtt ctttgtacca gaaaaggaag 60 accagagcca cttcctgatg tacacgaatg atccaataaa 100 <210> 74 <211> 100 <212> DNA <213> Artificial Sequence <400> 74 actgctaaag tgctgtctcc aacattggtg gtgatgagcc cttcgatagt gccatactcc 60 acgtctcggg gattgaggcg ttcttggttg cgccttttaa 100 <210> 75 <211> 100 <212> DNA <213> Artificial Sequence <400> 75 attttcggag agcaactccc agcaggcagg ctaaaagaca tactgcgaac actacagaga 60 gaataatgag gccaacctcc aagtggaaat tctgtgttcc 100 <210> 76 <211> 100 <212> DNA <213> Artificial Sequence <400> 76 acctttagtg ggcagctggg ccgtgatgtt cctgttacac cagtcccctt ggcagcactc 60 cacggcttgg ccaggggacg gcggggtctt acaggtcatc 100 <210> 77 <211> 100 <212> DNA <213> Artificial Sequence <400> 77 tttccctgct cataaacctg gaagcagcct ttctggtaga cgtggaagcc atcgttgatg 60 ctcagtgagg aaaagcactg ctggccttca cagtggtcct 100 <210> 78 <211> 100 <212> DNA <213> Artificial Sequence <400> 78 cctagtggcc atggcaggtt ttgcagcaca tctggcggaa gtaggctcgg ctgcagaact 60 gaaatttgag caccaggggg cagtaggcga ccttgttcac 100 <210> 79 <211> 100 <212> DNA <213> Artificial Sequence <400> 79 ccttcagggc cgtccccggg ggttgggctg tcgcacttgg cctcacactg ctgcgtggtg 60 ggcggccgca gggcctccgt gcactcgtgc gacgcctggc 100 <210> 80 <211> 100 <212> DNA <213> Artificial Sequence <400> 80 cctcacccca ctcgccagcc acccagcggg ccggggggca gcggcgcaag ttgcagcgca 60 tggtggccgg tggcttggcg gcgggtgagc agtgcgccgg 100 <210> 81 <211> 100 <212> DNA <213> Artificial Sequence <400> 81 cctcagacca gtcgagggcc gcccactccg gagggcaagt ggggccgtgg caggcctcca 60 gtacaggtgg gcgcggctgc gggcatgcgc tgtcgtccag 100 <210> 82 <211> 100 <212> DNA <213> Artificial Sequence <400> 82 cgccttctcc tccgcggcag agacgcggcg ctggcacacg accgagcggc tgcgcacgcc 60 tgcatcgcag ctgcggctgc agagcgacca gttccctaca 100 <210> 83 <211> 100 <212> DNA <213> Artificial Sequence <400> 83 ctctggaggg caaggctccg tgttgcaggc gcgctgcctt ttgggcagct tgctgtgggc 60 actgcagtag tggggggcga ccgcggagct gtccagctgg 100 <210> 84 <211> 100 <212> DNA <213> Artificial Sequence <400> 84 ccgcctgcac actgggccga gcacttggtc cagggcgcat agtgccagga gtaggggggc 60 agcgagtcac gggcgatggg ggcattgaag cggtagcgga 100 <210> 85 <211> 100 <212> DNA <213> Artificial Sequence <400> 85 ccatgacgat gagagatgca ttaatcggtc ccagggcttc gaggctctgg acctggtctg 60 gcccctgtcg cagttgaaag gtggtcccag ctagaggcag 100 <210> 86 <211> 100 <212> DNA <213> Artificial Sequence <400> 86 ccggccccag gtgaggctgg gctgaagacg ccctcgatgg tctcgcaggc actgccgtca 60 ccgccacaca ctcggcactt gtcctcccgc aggtcggagc 100 <210> 87 <211> 100 <212> DNA <213> Artificial Sequence <400> 87 ccttgcattc gccactgacg caaatgtcca ccgtgtctgg acggcagggt gtcccgtcca 60 ccacggctgc cgccctctcc gtgtagaagt tgaagccttc 100 <210> 88 <211> 100 <212> DNA <213> Artificial Sequence <400> 88 cctccccggt acgttttcca cttgtagaat ttcccacgga aagggatgct gtcaaattca 60 gaacactgca cttctctgaa gtcctgggag ccagggggac 100 <210> 89 <211> 100 <212> DNA <213> Artificial Sequence <400> 89 cctggggctg tcgcagtgac ggctagaaga ggacacgccg ccgccacagg tccggctgca 60 gtcgccccat ggagtccacg gcccccaggc tccgtccaca 100 <210> 90 <211> 100 <212> DNA <213> Artificial Sequence <400> 90 ccctctgggc gcgacccaaa ggggacacag acccgtttgt agcaccactg ggtgggggga 60 gacaggaagg agtgagtcca gcccggagga cactcgtcgg 100 <210> 91 <211> 100 <212> DNA <213> Artificial Sequence <400> 91 cccccttgtc gatggtgtgc gtctggcaca gcgtgccctc ggcggccggg atgctgttgg 60 tgatgcaccg gttgctcttg ctcagacacc acagctcgct 100 <210> 92 <211> 100 <212> DNA <213> Artificial Sequence <400> 92 ccccgtattt acactgacgc gatttgactc catgctgaaa gcggcattgc tcatctgcat 60 cgtaggcttg gcccggtgcc actgtcgggt acacaaagtc 100 <210> 93 <211> 100 <212> DNA <213> Artificial Sequence <400> 93 ctctagaaag ctggtgatgt agtcacggct gcaggatgac cacacgaatg ggttggtctt 60 catggtaatg tgggcagcca tgagcttggc tgggtcctga 100 <210> 94 <211> 100 <212> DNA <213> Artificial Sequence <400> 94 cgtgtgcccg atctcgtggg caatggtgaa cgctgtggcc aggccaatgt cctcattgac 60 gctgcagctt ctctcgcgct cacacattcc gcccaccggg 100 <210> 95 <211> 100 <212> DNA <213> Artificial Sequence <400> 95 ccgtgtgatg agcactgctg tgtcatggtt agccacaccg ttctctggaa tggcattgcc 60 atggccgctg tggttcacga tggatttctg ccacttacag 100 <210> 96 <211> 100 <212> DNA <213> Artificial Sequence <400> 96 caatgttcat gatggccagg acatactgct ccacatcccg gcgcccgtga taggccacca 60 tcatcttgtc agccaccacc agggtctcca cgtagcgctc 100 <210> 97 <211> 100 <212> DNA <213> Artificial Sequence <400> 97 tcggctgacc gatcgcttca ggcctggctg gccacgctct gtttcattcc ccaggggcct 60 ggcaggcggt ggcttcaagg tccgcagcca ccatggccgc 100 <210> 98 <211> 100 <212> DNA <213> Artificial Sequence <400> 98 cctctcactc cacaggctgt gtccaggtgg gggtgacgca gagaggaacg cttgtacacc 60 acatgtggtc cactttcctc cgggctccga gaacccttgg 100 <210> 99 <211> 100 <212> DNA <213> Artificial Sequence <400> 99 ccaggcctcc acaggtgctg atggccacat gggaggtgct ggcctggccc tgcaggtgac 60 cagcgtagag gcagtggggc cgggccgccc tctgccaggc 100 <210> 100 <211> 100 <212> DNA <213> Artificial Sequence <400> 100 caggccctcc cgtgtccagt actccacgga gacgtgccct gccagtagac gggagctgcg 60 ggtcaggttc agcaggaagt gggtgctggg cgaggccact 100

Claims

1. A gene detection composition for limb dysplasia, characterized in that, The composition comprises probes as shown in SEQ ID NO. 1-100.

2. Use of the gene detection composition for limb dysplasia as described in claim 1 in the preparation of a kit for gene detection of limb dysplasia.

3. A kit for detecting genes with abnormal limb development, characterized in that, The kit comprises the gene detection composition for limb dysplasia as described in claim 1.

4. The limb dysplasia gene detection kit according to claim 3, wherein The kit is a detection product covering 460 genes related to limb dysplasia.

Images