Gene combination and product for screening and / or auxiliary diagnosis of special diabetes mellitus and application of gene combination and product

Abstract

The invention discloses a gene combination for screening and / or auxiliary diagnosis of special diabetes mellitus, a product and application of the gene combination. The gene combination comprises 54 genes which are screened by the invention and are highly related to the special diabetes mellitus. The invention further provides a kit for screening and / or auxiliary diagnosis of the special diabetes mellitus, and application of the gene combination for screening and / or auxiliary diagnosis of the special diabetes mellitus. The gene combination provided by the invention is used for early screening and early diagnosis of special diabetes mellitus, and is more comprehensive in coverage and high in sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to a gene combination, product and application thereof for screening and / or auxiliary diagnosis of special types of diabetes, belonging to the technical field of biomedicine. Background Art

[0002] At present, diabetes is mainly type I diabetes and type II diabetes. Although patients with special types of diabetes account for the smallest proportion, the patient base is large, and the total number of people in China has reached millions. Studies have shown that about 90% of patients with monogenic diabetes are misdiagnosed as type I or type II diabetes. The clinical prognosis, outcome and treatment options of special types of diabetes are quite different from those of type I and type II diabetes. Currently, nearly 70% of patients do not receive correct and effective treatment options.

[0003] The etiology and pathogenesis of special types of diabetes involve a wide range of fields and progress rapidly. In recent years, the number of types has expanded rapidly. The American Diabetes Association (ADA) revised the diagnostic criteria in 2014 and roughly divided it into eight categories. There are still many unclear types in clinical practice, or overlaps between different types, which bring certain difficulties to clinical diagnosis and treatment. Many subtypes are difficult to lock by clinical diagnosis alone, and are prone to misdiagnosis and mistreatment, which then cause complications and / or co-infection. For example, secondary diabetes caused by endocrine diseases in special types of diabetes can often be cured by the treatment of endocrine diseases. For example, if the diagnosis of diabetes in newborns and infants is caused by mutations in the Kir6.2 and SUR1 genes, sulfonamides are an effective and simple method. In most MODY patients, oral hypoglycemic drugs can control blood sugar for a considerable period of time and avoid the inconvenience caused by insulin injections. Only by understanding special types of diabetes can we broaden and deepen our understanding of normal physiological homeostasis and pathophysiological increases in blood sugar.

[0004] Special types of diabetes have various clinical manifestations and complex diagnosis and treatment. There are also scattered individual case reports in China, but clinical testing generally only targets a single gene and lacks effective and available summary data. More importantly, the molecular genetic tests commonly used in clinical practice are inefficient, have a long cycle, and are out of touch with the academic frontier. Most medical institutions lack knowledge of this special type of diabetes, have difficulty collecting family members, and lack reliable testing methods and conditions. Therefore, there is currently no targeted diagnosis and treatment work in China. Thanks to the rapid development of sequencing technology and equipment, as well as the continuous decline in costs, personal gene sequencing has become a reality, and humans have entered the era of precision medicine. Selecting appropriate detection methods based on the clinical characteristics of patients, and conducting accurate and reasonable data analysis and variant interpretation can not only improve the diagnostic efficiency of genetic diseases. It also helps to deeply understand the relevant molecular mechanisms, so as to formulate personalized plans for subsequent treatment, management, and genetic counseling, thereby meeting the disease diagnosis and treatment needs in the era of precision medicine. The rapid development of genetic testing technology has brought new opportunities for the detection and diagnosis of genetic diseases, as well as challenges in interpretation, analysis, and verification of the pathogenicity of new mutations. The number and structure of chromosomes that cause special types of diabetes are less abnormal, and changes in gene copy number and DNA sequence variation account for the vast majority. With the development of gene sequencing technology, molecular diagnostic kits for genes related to special types of diabetes have become an important means to accurately determine special types of diabetes subtypes. Sanger sequencing (first-generation sequencing) detects DNA sequence changes, including point mutations, small DNA fragment deletions and insertions, etc. It has low throughput and only targets a single gene. The shortcoming of chromosome gene chips is that balanced translocations and inversion structural changes cannot be detected. Second-generation sequencing (NGS) technology generally uses short-read sequencing. Its sequencing length is between 150-400bp. Short-read sequencing has the advantages of high throughput, low average cost per base, and low error rate (about 0.1%).

[0005] Currently, there are no related reagents or kits for sequencing screening of pathogenic gene mutations of special types of diabetes, which is in sharp contrast to the necessity and clinical needs of screening for such diseases. Therefore, related reagents or kits for sequencing screening of pathogenic gene mutations of special types of diabetes will provide clinicians with more valuable molecular information and improve the accuracy and sensitivity of diagnosis. It will help to improve the clinical decision-making model of special types of diabetes, improve the level of individualized precision diagnosis and treatment, and fill the gap in the tool of molecular diagnosis of special types of diabetes in China. Summary of the invention

[0006] The purpose of the present invention is: to address the deficiencies of existing diagnostic technologies for special types of diabetes, the present invention provides a gene combination, product and application thereof for screening and / or auxiliary diagnosis of special types of diabetes.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a gene combination for screening and / or auxiliary diagnosis of special types of diabetes, wherein the gene combination includes any one of the following genes or a combination of any multiple genes: ABCC8, AGPAT2, ALMS1, APPL1, BLK, BSCL2, C2ORF37, CAV1, CAVIN1, CEL, CEP19, CFTR, CIDEC, CISD2, DNAJC3, EIF2AK3, FCGR2A, FOXP3, GCK, GLIS3, HNF1A, H NF1B, HNF4A, IER3IP1, IL2RA, IL6, INS, INSR, KCNJ11, KLF11, LEP, LEPR, LMNA, MAFA, NEUROD1, PAX4, PCSK1, PDX1, PLI N1, PNPLA2, PPARG, PPP1R15B, PRKACA, PTF1A, SLC19A2, SPINK1, TCF2, TGFB1, TWNK, USP8, WFS1, WRN, ZFP57, ZMPSTE24.

[0009] In some embodiments, the gene combination includes ABCC8, AGPAT2, ALMS1, APPL1, BLK, BSCL2, C2ORF37, CAV1, CAVIN1, CEL, CEP19, CFTR, CIDEC, CISD2, DNAJC3, EIF2AK3, FCGR2A, FOXP3, GCK, GLIS3, HNF1A, HNF1B, HNF4A, IER3IP1, IL2RA, IL6, INS, INSR, KCNJ11, KLF11, LEP, LEPR, LMNA, MAFA, NEUROD1, PAX4, PCSK1, PDX1, PLIN1, PNPLA2, PPARG, PPP1R15B, PRKACA, PTF1A, SLC19A2, SPINK1, TCF2, TGFB1, TWNK, USP8, WFS1, WRN, ZFP57, and ZMPSTE24.

[0010] In a second aspect, the present invention provides a reagent for screening and / or auxiliary diagnosis of a special type of diabetes, the reagent comprising a reagent for detecting the gene combination described in the first aspect;

[0011] In some embodiments, the reagents include one or a combination of gene mutation detection reagents, sequencing reagents, gene expression detection reagents, RNA detection reagents, antibodies, and affinity proteins.

[0012] In some embodiments, the reagent includes a probe; the sequence of the probe is shown in SEQ ID NO: 1-54.

[0013] Further, the probes include probes for detecting CDS regions, UTR regions and intron regions of genes in the gene combination;

[0014] In a third aspect, the present invention provides the use of the gene combination described in the first aspect or the reagent described in the second aspect in the preparation of a product for screening and / or auxiliary diagnosis of special types of diabetes.

[0015] Furthermore, the application detects and / or analyzes the gene variation in the gene combination by detecting the gene combination.

[0016] Furthermore, the detection and / or analysis is performed by the following methods: first-generation sequencing, second-generation sequencing, third-generation sequencing, single-molecule fluorescence sequencing, nanopore sequencing, gene chip, MLPA, digital PCR, qPCR, enzyme immunoassay, fluorescence immunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, flow cytometry, PLA method or protein blotting.

[0017] In a fourth aspect, the present invention provides a product for screening and / or auxiliary diagnosis of special types of diabetes, the product comprising a reagent for detecting the gene combination described in the first aspect;

[0018] Further, the product includes one or more combinations of in vitro diagnostic products, kits, or detection and / or diagnostic devices;

[0019] Furthermore, the product comprises a kit.

[0020] In a fifth aspect, the present invention provides a method for detecting the gene combination described in the first aspect or the product described in the fourth aspect, characterized in that it comprises the following steps:

[0021] 1) DNA extraction and quality inspection;

[0022] 2) constructing a gene capture library for the gene combination;

[0023] 3) Hybridization capture;

[0024] 4) performing sequencing;

[0025] Furthermore, in step 4), sequencing is performed by bridge PCR.

[0026] In a sixth aspect, the present invention provides a method for constructing a gene capture library for the gene combination described in the first aspect, the construction method comprising the following steps:

[0027] 1) Designing a capture probe for the gene combination;

[0028] 2) Cutting the genomic DNA into fragments of 200-400 bp by enzyme digestion to form fragmented genomic DNA;

[0029] 3) performing end repair on the fragmented genomic DNA and simultaneously performing 3' end A addition;

[0030] 4) adding an index tag to the end-repaired and end-A-added fragmented genomic DNA obtained in step 3) to form a library structure;

[0031] 5) PCR amplification to increase the total amount of the library;

[0032] 6) hybridization capture, using the capture probe described in step 1) to perform capture;

[0033] 7) Clean out the uncaptured library and retain only the target region library;

[0034] 8) Obtaining the target region capture library.

[0035] In a seventh aspect, the present invention provides a system for screening and / or auxiliary diagnosis of special types of diabetes, the system comprising a DNA extraction module, a DNA detection module, and a bioinformatics analysis module; the DNA detection module is used to detect the gene combination described in the first aspect.

[0036] Further, the DNA detection module includes reagents and / or devices for detecting the gene combination;

[0037] Furthermore, the apparatus comprises a processing element, a storage element, and a computer program stored in the storage element and executable on the processing element.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The present invention can quickly and accurately perform early screening and auxiliary diagnosis of special types of diabetes, and provide a scientific basis for diagnosis and treatment for early screening, early diagnosis and early treatment of special types of diabetes; at the same time, the results of gene testing can further provide a theoretical research basis for the pathogenesis, treatment targets, drug screening, and personalized diagnosis and treatment of special types of diabetes;

[0040] 2. Compared with whole genome sequencing and large-scale gene chips, the present invention screens out genes that are highly correlated with special types of diabetes through a large number of clinical sample tests, combined with literature reports and gene database analysis, and develops detection kits based on the screened genes, which can reduce the cost of single testing and screening, and significantly improve the detection rate of special types of diabetes. The accuracy, sensitivity, specificity and stability of the kit are higher. DETAILED DESCRIPTION

[0041] In order to make the present invention more clearly understood, preferred embodiments are described in detail as follows.

[0042] The experimental methods or parameters in the following examples without specifying specific conditions are generally conventional means in the art. The reagents or instruments used without specifying the manufacturer are conventional products that can be purchased commercially and are carried out according to the methods or conditions recommended or disclosed by the reagent or instrument manufacturers.

[0043] The reaction system and reaction conditions in the examples are only examples. The reaction system and reaction conditions in each step of the examples can be adjusted within an acceptable range, and the present invention does not limit this.

[0044] Glossary

[0045] DNA: Deoxyribonucleic Acid (DNA);

[0046] PCR: polymerase chain reaction (PCR);

[0047] Reads: the measured DNA sequence;

[0048] Introns: Introns are intervening sequences in the DNA of eukaryotic cells. These sequences are transcribed in the precursor RNA, removed by splicing, and ultimately do not exist in the mature RNA molecule. The alternating arrangement of introns and exons constitutes a split gene.

[0049] Exon: Exon is a part of the gene of eukaryotic organisms that can be expressed as protein during protein biosynthesis. Exon is a gene sequence that appears in mature RNA, also known as expressed sequence. It is a nucleotide sequence that exists both in the initial transcription product and in the mature RNA molecule.

[0050] CDS region: CDS (coding sequences) is a DNA coding sequence that corresponds one-to-one with the protein sequence, and there is no other sequence unrelated to the protein in the middle of the sequence

[0051] UTR region: UTR region, also known as untranslated region (Untranslated Regions), is the non-coding fragment at both ends of the mRNA molecule.

[0052] Unless otherwise defined, the terms used in the present invention have the meanings commonly understood by those skilled in the art.

[0053] Example 1: Screening of special types of diabetes gene combinations

[0054] 1. Candidate genes

[0055] According to the preliminary in-depth research of the inventor's team, based on the pathogenic genes reported in the prior art that are highly associated with special types of diabetes, and based on literature retrieval and related gene retrieval in disease-related gene databases, a candidate gene library related to special types of diabetes was obtained after screening and analysis.

[0056] 2. Screening of highly correlated genes based on clinical samples:

[0057] Selected samples: 253 clinical peripheral blood samples collected at Shanghai Sixth People's Hospital before January 2024 and stored at -80℃ for testing.

[0058] All clinical research processes passed the hospital's ethical review, and informed consent was signed after the volunteers were informed of the true situation.

[0059] 3. DNA detection and analysis in samples

[0060] According to the instructions, the genomic DNA in the peripheral blood of the subjects was extracted using Kangwei Century's magnetic bead blood DNA extraction kit, and the gene DNA in the sample peripheral blood was detected using an automated extraction workstation. The biotin-labeled probe was hybridized with the sample, and the probe hybridized with the sample target sequence was further captured on the magnetic beads. The captured target sequence was amplified using DNA polymerase, and the sample was purified using Agencourt AMPure XP magnetic beads. The library was quality-checked and quantified, and the sequencing was performed on the machine, as well as data analysis of the sequencing results.

[0061] 4. Screening of genes related to special types of diabetes

[0062] Combined with the test results of clinical samples, mutation analysis was performed, including analysis of the number and rate of mutations in candidate genes; screening of related genes with high numbers and rates of mutations. Further analysis of the differences in gene mutation information in different special types of diabetes subtypes was performed to screen variant genes that were highly correlated with special types of diabetes.

[0063] Further, annotation and database comparison were performed. The databases used for annotation included ClinVar, OMIM, etc., and combined with literature and gene database reports, 54 highly correlated genes were obtained. The gene information is shown in Table 1 below:

[0064] Table 1 Related genes

[0065]

[0066]

[0067]

[0068] Example 2: Probe design

[0069] Probe design is performed for the relevant genes obtained by the present invention.

[0070] Based on further research on the functional regions of each gene, such as exons, introns, and UTR regions, the following highly variant-correlated gene capture regions were analyzed, and probes were further designed based on the gene capture regions.

[0071] The genes captured by the capture probe and the chromosome coordinate information of the genes are shown in Table 2:

[0072] Table 2 Gene regions

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] The locations of the gene regions are all referenced to the gene sequences in the UCSC (hg19) database.

[0088] The probe design adopts a shingled probe design. For the selected gene, the probe is designed based on the latest version of the genome database (Refseq, CCDS, GENECODE, dbSNP) with reference to the hg19 genome on the XCapert online design platform (http: / / nadprobe.njnad.com / login.html). The probe is designed to cover the CDS region, UTR region and intron region in the gene to synthesize the probe package.

[0089] The probe design principles are:

[0090] 1) The probe covers the exon region and the junction of exons and introns of all candidate genes; the exon and intron junction refers to at least 10-100 bp upstream and downstream of the exon; it covers a part of the intron region or UTR region;

[0091] 2) Remove highly repetitive sequences that appear in the human genome;

[0092] 3) The probes are covered by a shingled design, and the probe density can ensure that each DNA template in the area to be detected has a corresponding probe bound to it, and the probe coverage reaches 100%;

[0093] 4) The captured probes are mixed probes with an average probe length of 120 bp.

[0094] According to the homology-based method, two repetitive sequence shielding modes are provided, and the probes designed in both modes will be risk assessed:

[0095] -remove all risk genome region means to shield all risk regions of the genome and only select safe regions for probe design. This method is fast, the probe is safe and reliable, and the on-target rate is high. However, there is also a phenomenon of insufficient coverage of some repetitive regions.

[0096] -remove high risk genome region is designed for the remaining regions after masking the high risk regions of the genome. This method usually provides higher coverage of the target region, but may sacrifice the capture on-target rate to a certain extent.

[0097] For discontinuous SNP sites, one probe is placed at each site by default, and finally the probe package is synthesized and detected and analyzed by high-throughput sequencing technology.

[0098] The designed specific probe is obtained, and the homology between the probe and the target gene sequence it identifies is 80-100%; the TM value is 65 degrees; the average CG content is 45%-48%; there is no special structure such as palindrome sequence. The probe is designed according to the probe capture start and end positions shown in Table 2, and the start and end positions of the gene region captured by the designed probes are respectively;

[0099] In some embodiments, the probe has 100% homology to the target gene sequence it identifies. The sequences of some of the probes are shown in Table 3:

[0100] Table 3 Probe sequences

[0101]

[0102]

[0103]

[0104] Kit composition

[0105] Based on the probe design method, capture probes (biotin-labeled probes) targeting 54 genes were obtained and prepared into a kit.

[0106] The kit further includes a nucleic acid extraction kit, a library construction kit, a hybridization capture kit, a nucleic acid amplification kit, and a sequencing kit.

[0107] The reagents in the kit further include end repair reaction buffer, end repair enzyme, DNA ligation reaction buffer, DNA ligase, PCR amplification primers, PCR premix, hybridization buffer, magnetic beads, magnetic bead washing solution, and hybridization washing solution.

[0108] Example 3: Detection process

[0109] 1. DNA extraction and quality inspection

[0110] The DNA of the sample to be tested is extracted through nucleic acid extraction and nucleic acid quality inspection is performed. After the quality inspection is qualified, the library is constructed.

[0111] The cell structure is destroyed by proteases to release the DNA in the cells, and the positively charged magnetic beads bind to the negatively charged DNA to adsorb and purify the DNA.

[0112] The specific operations are as follows:

[0113] 1.1. Follow the instructions and use Kangwei Century's magnetic bead blood DNA extraction kit to extract genomic DNA from the blood.

[0114] 1.2. Use enzyme digestion to break the genomic DNA into small fragments of about 200-400 bp.

[0115] a) In this example, 3 μg of DNA was fragmented using the enzyme digestion method according to standard procedures.

[0116] 1.3. Purify the sample using Agencourt AMPure XP magnetic beads, with a volume ratio of beads to sample of 1.8:1, and elute with 50 μl of nuclease-free water. The specific steps are as follows:

[0117] a) Place AMPure XP beads at room temperature for at least 30 minutes.

[0118] b) Vortex the reagent to mix until the color is uniform.

[0119] c) Add 1.5 times the amount of AMPure XP beads to a 1.5 ml low-absorption centrifuge tube, take 50 μl of DNA fragment product, pipette and mix 10 times, and leave at room temperature for 5 minutes.

[0120] d) Place the tube on the magnetic stand until the solution becomes clear (approximately 3 to 5 minutes).

[0121] e) Keep the centrifuge tube on the magnetic stand. Be careful not to touch the magnetic beads when discarding the supernatant in the centrifuge tube.

[0122] f) While keeping the centrifuge tubes on the magnetic rack, add 500 μl of 70% ethanol to each centrifuge tube.

[0123] g) Leave the tube for 1 minute to allow all the magnetic beads to settle and discard the ethanol.

[0124] h) Repeat steps f and g once.

[0125] i) Dry the samples in a 37°C heat block for 5 min or until any residual ethanol disappears.

[0126] j) Add 50 μl of nuclease-free water, mix well on a vortex mixer, and incubate at room temperature for 2 min.

[0127] k) Keep the centrifuge tube on the magnetic stand for 2 to 3 minutes until the solution is clear.

[0128] l) Transfer about 50 μl of the supernatant to a new 1.5 ml centrifuge tube and discard the beads.

[0129] (II) Library construction

[0130] The library was constructed based on the screened related genes.

[0131] The genomic DNA is cut into 200-400bp fragments by enzyme digestion, the fragmented genomic DNA is end-repaired and 3' end is added with A, and then an index marker is added to form a library structure. After PCR amplification, the total amount of the library is increased to facilitate subsequent probe capture.

[0132] The specific operations are as follows:

[0133] 2.1 DNA fragmentation, end repair and A addition

[0134] 2.1.1 According to the library construction information table, take 500 ng of DNA sample and prepare the reaction system as follows:

[0135] Element Amount required for 1 library Input DNA 500ng FEA Buffer V2 5μl Nuclease-free water Make up to a total volume of 50 μl Total master mix volume 40μl FEA Enzyme Mix V2 10.0μl Total reaction volume 50.0μl

[0136] 2.1.2 Vortex to mix, centrifuge and perform the following reaction on a PCR instrument:

[0137] Reaction temperature Reaction time 37℃ 6min 65℃ 30min 4℃ ∞

[0138] 2.2 Adapter Ligation

[0139] 2.2.1 Take out Rapid Ligation Buffer V2 and Rapid DNA Ligase V2 from -20℃, thaw and mix thoroughly, centrifuge briefly to collect to the bottom of the tube, and place on ice for later use

[0140] 2.2.2 Vortex and centrifuge 50 μl of the reaction product from the previous step and prepare the reaction system as follows:

[0141] Element Amount required for 1 library Rapid Ligation Buffer V2 25μl Rapid DNALigase V2 5μl Nuclease-free water 15μl DNA adapter-S for Illumina 5μl Total master mix volume 100μl

[0142] 2.2.3 Vortex to mix, centrifuge and perform the following reactions:

[0143]

[0144]

[0145] 2.3. Use Agencourt AMPure XP magnetic beads to purify samples and sort fragments, and elute with 32 μl. The specific steps are as follows:

[0146] a) Place AMPure XP beads at room temperature for at least 30 minutes.

[0147] b) Vortex the reagent to mix until the color is uniform.

[0148] c) Add 16.5 μl of water to the product and transfer the entire volume to a new centrifuge tube.

[0149] d) Add 60 μl of AMPure XP beads to a 1.5 ml low-absorption centrifuge tube, mix by pipetting 10 times, and leave at room temperature for 5 min.

[0150] e) Place the tube on the magnetic rack until the solution is clear (approximately 3 to 5 minutes).

[0151] f) Keep the centrifuge tube on the magnetic stand and transfer the supernatant to a new 1.5 ml centrifuge tube.

[0152] g) Add 20 μl of magnetic beads to the centrifuge tube and let stand at room temperature for 5 min.

[0153] h) Place the centrifuge tube on a magnetic rack until the solution becomes clear (approximately 3 to 5 minutes) and discard the supernatant.

[0154] i) While keeping the centrifuge tubes on the magnetic rack, add 500 μl of 70% ethanol to each centrifuge tube.

[0155] j) Leave the tube for 1 minute to allow all the magnetic beads to settle and discard the ethanol.

[0156] k) Repeat steps i and j once.

[0157] l) Dry the samples in a 37°C heating block for 5 min or until any residual ethanol disappears.

[0158] m) Add 25 μl of nuclease-free water, mix well on a vortex mixer, and incubate at room temperature for 2 min.

[0159] n) Keep the centrifuge tube on the magnetic rack for 2 to 3 minutes until the solution is clear.

[0160] o) Transfer about 25 μl of the supernatant to a new 1.5 ml centrifuge tube and discard the beads.

[0161] 4. Amplify the ligation product. The PCR reaction conditions are: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 65℃ annealing for 30s, 72℃ extension for 30s, 6 cycles in total; and finally 72℃ extension for 5min. Thus, the PCR product is obtained. The reaction system is as follows:

[0162] Element Amount required for 1 library Purified or sorted Adapter Ligation products 20.0μl VAHTS HiFi Amplification Mix 25.0μl VAHTS UDIPrimer for Illumina 5.0μl Total reaction volume 50.0μl

[0163] Among them: PCR primer sequences are as follows:

[0164] Universal primer:

[0165] 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG CTCTTCCGATCT-3' (SEQ ID NO: 55);

[0166] Index primer:

[0167] 5'-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAG ACGTGTGCTCTTCCGATCT-3' (SEQ ID NO: 56).

[0168] 5. Purify the sample using Agencourt AMPureXP magnetic beads, with a volume ratio of beads to sample of 1.5:1, and elute with 30 μl. The specific steps are as follows:

[0169] a) Place AMPureXP beads at room temperature for at least 30 minutes.

[0170] b) Vortex the reagent to mix until the color is uniform.

[0171] c) Add 1.5 times the amount of AMPureXP beads to a 1.5 ml low-absorption centrifuge tube, take 50 μl of PCR product, pipette and mix 10 times, and leave at room temperature for 5 minutes.

[0172] d) Place the tube on the magnetic stand until the solution becomes clear (approximately 3 to 5 minutes).

[0173] e) Keep the centrifuge tube on the magnetic stand. Be careful not to touch the magnetic beads when discarding the supernatant in the centrifuge tube.

[0174] f) While keeping the centrifuge tubes on the magnetic rack, add 500 μl of 70% ethanol to each centrifuge tube.

[0175] g) Leave the tube for 1 minute to allow all the magnetic beads to settle and discard the ethanol.

[0176] h) Repeat steps f and g once.

[0177] i) Dry the samples in a 37°C heat block for 5 min or until any residual ethanol disappears.

[0178] j) Add 15 μl of nuclease-free water, mix well on a vortex mixer, and incubate at room temperature for 2 min.

[0179] k) Keep the centrifuge tube on the magnetic stand for 2 to 3 minutes until the solution is clear.

[0180] l) Transfer about 15 μl of the supernatant to a new 1.5 ml centrifuge tube and discard the beads.

[0181] (III) Hybridization capture

[0182] The target region library in the whole genome library is captured using the gene probe set. Before capturing, the mixed pre-library is vacuumed and concentrated into dry powder, and then a subsequent hybridization capture experiment is performed. The specific operation is as follows:

[0183] 3.1 Use biotin-labeled probe to hybridize with the sample. The reaction conditions are: 95℃, 5min denaturation, then keep at 65℃ for 16-24h. The reaction system is as follows:

[0184] Volume(μL) 2×Hybridization Buffer 8.5 Hybridization Buffer Enhancer 2.7 xGen Lockdown Probe pools 4 Nuclease-free water 1.8 Total master mix volume 17

[0185] 3.2 Use streptavidin-labeled magnetic beads to capture the probe hybridized with the sample target sequence onto the magnetic beads through the binding of biotin and streptavidin. The steps are as follows:

[0186] a) Incubate the mixture at room temperature for 30 min on a thermostatic oscillator to ensure that the sample is completely mixed. Centrifuge briefly.

[0187] b) Separate the beads and buffer on a magnetic stand and remove the supernatant.

[0188] c) Resuspend the beads in 500 μl Wash Buffer 1 and vortex for 5 seconds.

[0189] d) Incubate the sample at room temperature for 15 minutes. Vortex and mix occasionally. Centrifuge briefly.

[0190] e) Separate the beads on a magnetic rack and remove the supernatant.

[0191] f) Wash the beads 3 times with 500 μl Wash Buffer 2: i) Incubate the sample at 65°C for 10 min in a circulating water bath. Vortex intermittently. ii) Invert to mix. iii) Centrifuge clockwise.

[0192] g) Separate the beads and buffer on a magnetic rack and remove the supernatant.

[0193] h) Mix the beads with 30 μl of nuclease-free water and vortex for 5 seconds to resuspend the beads.

[0194] 3.3 Use DNA polymerase to amplify the captured target sequence. The PCR reaction conditions are: 98℃ pre-denaturation for 2min; 98℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 1min, 12 cycles in total; final extension at 72℃ for 10min. Thus, the PCR product is obtained, and the reaction system is as follows:

[0195]

[0196] Among them: PCR primer sequences are as follows:

[0197] Illumina P5 primer: 5'-AATGATACGGCGACCACCGAGATCTACAC-3' (SEQ ID NO: 57);

[0198] Illumina P7 primer: 5'-CAAGCAGAAGACGGCATACGAGAT-3' (SEQ ID NO: 58).

[0199] 3.4 Use Agencourt AMPureXP magnetic beads to purify the sample, the volume ratio of magnetic beads to sample is 1.8:1, and elute with 30μl nuclease-free water. The specific steps are as follows:

[0200] a) Place AMPureXP beads at room temperature for at least 30 minutes.

[0201] b) Vortex the reagent to mix until the color is uniform.

[0202] c) Add 1.8 times the amount of AMPureXP beads to a 1.5 ml low-absorption centrifuge tube, take 50 μl of PCR product, pipette and mix 10 times, and place at room temperature for 5 minutes.

[0203] d) Place the tube on the magnetic stand until the solution becomes clear (approximately 3 to 5 minutes).

[0204] e) Keep the centrifuge tube on the magnetic stand and be careful not to touch the magnetic beads when discarding the supernatant in the centrifuge tube.

[0205] f) While keeping the centrifuge tubes on the magnetic rack, add 500 μl of 70% ethanol to each centrifuge tube.

[0206] g) Leave the tube for 1 minute to allow all the magnetic beads to settle and discard the ethanol.

[0207] h) Repeat steps f and g once.

[0208] i) Dry the sample in a 37°C heating block for 5 min or until the residual ethanol disappears completely. Add 30 μl of nuclease-free water, mix well on a vortex mixer, and incubate at room temperature for 2 min. Keep the centrifuge tube on the magnetic stand for 2 to 3 min until the solution is clear.

[0209] j) Transfer about 30 μl of the supernatant to a new 1.5 ml centrifuge tube and discard the beads.

[0210] (IV) Library quality inspection and quantification

[0211] The library obtained in the previous step was quantified using Qubit2.0 (Invitrogen) and quality checked using Agilent 2100 Bioanalyzer.

[0212] (V) Sequencing

[0213] The samples were subjected to double-end sequencing using the Illumina novaseq6000 program to obtain sequencing results. For detailed operation procedures, please refer to the Novasek6000 operating instructions.

[0214] (VI) Bioinformatics Analysis

[0215] The sequencing data was compared with the human genome, and the unmatched sequences and repeated sequences were removed for variation analysis. The sequencing results produced by Novaseq are DNA sequences in the form of fastq. The original data generated by illumina Novaseq were quality controlled to obtain high-quality DNA sequence data, reads were located on the human genome, and the original variation information was obtained based on the reads location information. The original variation information was quality controlled to obtain high-quality variation sites, and the generated high-quality variation sites were annotated using multiple databases. The annotated variation site information was updated to ADMD (Amplicongene Diabetes Mellitus Database), and ADMD was used to further annotate the variation sites. Suspicious sites were screened in conjunction with clinical manifestations. For samples where no suspicious sites were found, CNV (copy number variation) screening was performed, and the diagnosis report was compiled. The pathogenic gene was identified through Sanger sequencing verification.

[0216] Example 4: Verification of detection effect in clinical samples

[0217] 1. Source of samples in the verification sample set:

[0218] Selected samples: 54 clinical samples diagnosed with special types of diabetes with genetic characteristics of the Chinese population and 199 control samples collected at Shanghai Sixth People's Hospital between March 2019 and January 2024. The samples are collected peripheral blood samples.

[0219] All clinical research processes passed the hospital's ethical review, and informed consent was signed after the volunteers were informed of the true situation.

[0220] 2. Detection and analysis using the detection kit of the present invention

[0221] For the kit, please refer to Example 2; for the detection process, please refer to the detection process in Example 3.

[0222] 3. Experimental results

[0223] (1) Kit and mutant gene detection results

[0224] Based on the results of special type diabetes panel kit detection and Sanger sequencing of 253 samples, the detection kit of the present invention was used to perform DNA extraction, library construction, hybridization capture, sequencing and analysis on the samples, and compared with the gold standard Sanger sequencing results. The results are shown in the following table:

[0225]

[0226] The test data was analyzed and the performance of the kit is as follows:

[0227] Accuracy = (true positive + true negative) / total number of samples from three sites = (41 + 212) / (253) = 100%.

[0228] Sensitivity = (true positive / (true positive + false negative)) = (41) / (41+0) = 100%.

[0229] Specificity = (true negative / (true negative + false positive)) = (212) / (212+0) = 100%.

[0230] 4. Precision evaluation

[0231] Three clinical samples were selected, including two positive samples and one negative sample. The experiments were repeated three times within and between batches respectively. The experiments were performed on the same person at the same time within the batch, and on the same person at different times between batches. In the two positive samples, the positive sites detected before could be stably detected, with a repeatability of 100% and a CV% of 0%, which was less than 15%, showing very good stability.

[0232] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A gene combination for screening and / or auxiliary diagnosis of special types of diabetes, characterized in that: The gene combination includes any one of the following genes or a combination of any multiple genes: ABCC8, AGPAT2, ALMS1, APPL1, BLK, BSCL2, C2ORF37, CAV1, CAVIN1, CEL, CEP19, CFTR, CIDEC, CISD2, DNAJC3, EIF2AK3, FCGR2A, FOXP3, GCK, GLIS3, HNF1A, HNF1B, HNF4A, IER3IP1 , IL2RA, IL6, INS, INSR, KCNJ11, KLF11, LEP, LEPR, LMNA, MAFA, NEUROD1, PAX4, PCSK1, PDX1, PLIN1, PNPLA2 , PPARG, PPP1R15B, PRKACA, PTF1A, SLC19A2, SPINK1, TCF2, TGFB1, TWNK, USP8, WFS1, WRN, ZFP57, ZMPSTE24.

2. The gene combination according to claim 1, characterized in that: The gene combination includes ABCC8, AGPAT2, ALMS1, APPL1, BLK, BSCL2, C2ORF37, CAV1, CAVIN1, CEL, CEP19, CFTR, CIDEC, CISD2, DNAJC3, EIF2AK3, FCGR2A, FOXP3, GCK, GLIS3, HNF1A, HNF1B, HNF4A, IER3IP1, IL2RA, IL6, INS, INSR, KCNJ11, KLF11, LEP, LEPR, LMNA, MAFA, NEUROD1, PAX4, PCSK1, PDX1, PLIN1, PNPLA2, PPARG, PPP1R15B, PRKACA, PTF1A, SLC19A2, SPINK1, TCF2, TGFB1, TWNK, USP8, WFS1, WRN, ZFP57 and ZMPSTE24.

3. A reagent for screening and / or auxiliary diagnosis of special types of diabetes, characterized in that: The reagents include reagents for detecting the gene combination according to any one of claims 1-2.

4. The reagent according to claim 3, characterized in that The reagent includes a probe, and the sequence of the probe is shown in SEQ ID NOs: 1-54.

5. Use of the gene combination described in any one of claims 1-2 or the reagent described in any one of claims 3-4 in the preparation of products for screening and / or auxiliary diagnosis of special types of diabetes.

6. The use according to claim 5, characterized in that The application detects and / or analyzes the gene variation in the gene combination by detecting the gene combination.

7. A product for screening and / or auxiliary diagnosis of special types of diabetes, characterized in that: The product comprises a reagent for detecting the gene combination according to any one of claims 1-2.

8. A method for detecting the gene combination according to any one of claims 1 to 2, or the product according to claim 7, characterized in that: The following steps are involved: 1) DNA extraction and quality inspection; 2) constructing a gene capture library for the gene combination; 3) Hybridization capture; 4) Perform sequencing.

9. A method for constructing a gene capture library for the gene combination according to any one of claims 1 to 2, characterized in that: The construction method comprises the following steps: 1) Designing capture probes for the gene combination; 2) Cutting the genomic DNA into fragments of 200-400 bp by enzyme digestion to form fragmented genomic DNA; 3) performing end repair on the fragmented genomic DNA and simultaneously performing 3' end A addition; 4) adding an index tag to the end-repaired and end-A-added fragmented genomic DNA obtained in step 3) to form a library structure; 5) PCR amplification to increase the total amount of the library; 6) hybridization capture, using the capture probe described in step 1) for capture; 7) Clean out the uncaptured library and retain only the target region library; 8) Obtaining the target region capture library.

10. A system for screening and / or auxiliary diagnosis of special types of diabetes, characterized in that: The system includes a DNA extraction module, a DNA detection module, and a bioinformatics analysis module; the DNA detection module is used to detect the gene combination described in any one of claims 1-2.

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