55 results about "GC-content" patented technology

The invention discloses a copy number variation detection method, device and equipment based on low-depth whole genome sequencing and a storage medium, and relates to the technical field of medical data processing, the method comprises the following steps: performing sequencing quality control on sample gene data to obtain filtered gene data; performing data preprocessing on the filtered gene data through a dynamic negative reference library and a reference genome to obtain processed gene data and a dynamic Z test result; the dynamic negative reference library is determined according to the GC content of the contrast negative sample set; and performing improved copy number variation detection according to the processed gene data and the dynamic Z test result to obtain a copy number variation detection result. According to the method, sequencing quality control is carried out firstly, and data quality is guaranteed; and then data preprocessing is performed based on a dynamic negative reference library constructed in real time, and finally CNV detection is performed based on optimized processing gene data and a dynamic Z test result, so that systematic deviation is effectively reduced, and the detection reliability is improved.

The present disclosure relates to the technical field of sequencing, and in particular to a method and reagent for preparing a sequencing library. Provided in the present disclosure is a method for preparing a sequencing library. According to the method, by means of additionally adding an additive or an additive combination capable of reducing the DNA secondary structure and / or reducing the degradation of single-stranded DNA in the single-chain cyclization reaction and / or the rolling circle amplification reaction, and additionally adding an additive or an additive combination capable of reducing the DNA secondary structure and reducing the degradation of single-stranded DNA and / or reducing the stability of the double helix structure of double-stranded DNA in the rolling circle amplification reaction of the double-stranded circular library, the ability of the DNA ligase and / or the strand displacement DNA polymerase to bind to a target nucleic acid is enhanced, such that the non-uniform rolling circle amplification efficiency of different circular DNAs caused by the secondary structure is reduced, the probability that the loose single-stranded DNA is easily degraded is reduced, the amplification uniformity of regions with different GC contents and different sequence characteristics in the library is improved, and thus the sequencing quality and the yield of sequencing data are improved.

The application relates to a sarcoma fusion gene nanopore sequencing primer set and application thereof, wherein the primer set comprises five pairs of specific primers (Primer 1-5) with fixed sequences, each primer follows the design principle of 18-25 bp in length, 40%-60% in GC content and strict matching of a target sequence at a 3' end, and the length of an amplification product is 300-800 bp, which is suitable for nanopore sequencing. Through qPCR verification, the primer set has single-peak melting curves, close-to-100% amplification efficiency, strong specificity and excellent amplification performance.

The invention discloses a streptomyces Targetron gene targeting vector as well as a construction method and application of the streptomyces Targetron gene targeting vector. According to the invention, an II-type intron Ll.LtrB from lactococcus lactis is cloned to an escherichia coli-streptomyces shuttle plasmid to construct a streptomyces genetic manipulation tool-vector pSC30 based on targeting of the II-type intron, and gene targeting in streptomyces coelicolor is successfully realized by using the tool. Screening a biosynthetic gene cluster of red pigment in streptomyces roseosporus; and activating a biosynthetic gene cluster of janus doxorubicin in streptomyces spinosus. According to the invention, the conversion rate and the gene targeting efficiency of the Ll.LtrB intron in streptomyces with high GC content are effectively improved, and a new tool is provided for construction of a gene mutation library.

The application discloses a kind of compositions, reagents and its detection method for high-throughput gene sequencing, belong to gene sequencing technical field, the composition includes Tris-HCl buffer 80-120 parts, potassium chloride 40-60 parts, ammonium sulfate 10-20 parts, magnesium chloride 15-25 parts, dNTPs mixed solution 30-50 parts, heat-stable DNA polymerase mutant 8-15 parts, PCR promoter complex 5-12 parts, modified iridium quinoline ketone complex 1-5 parts, glycerol 150-250 parts, Tween-20 1-3 parts, EDTA disodium salt 0.5-2 parts and nuclease-free ultrapure water, by introducing the modified iridium quinoline ketone complex of innovation, effectively solve the technical problems of low amplification efficiency, poor multiplex PCR reaction uniformity and high non-specific amplification background for high GC content, complex secondary structure genomic template, significantly improve the specificity of amplification, uniformity and the quality and reliability of final sequencing data.

A microbial gene editing scheme automatic design method, system and storage medium; the method comprises: selecting a design object for different types of strains, the design object is bacteria or fungi; after determining the target gene, obtaining the target gene information from the NCBI database; the design scheme of bacteria comprises the following steps: determining the gRNA design region; selecting the optimal gRNA sequence; doing sequence complexity and GC content analysis; the design scheme of fungi uses a frameshift scheme: the gRNA of the frameshift scheme is set at any position not overlapping with other genes after the start codon in the gene; select gRNA sequence in gRNA design region, select the optimal gRNA sequence; design two pairs of four primer sequences for homologous arm identification primer; this scheme can replace the complicated and time-consuming manual scheme, save a lot of time, solve the problem of high cost in the design of the best site and the design of sgRNA sequence scheme of artificial gene knockout.

The application belongs to the technical field of gene detection, and discloses a method and system for judging the influence of homologous regions in NGS gene variation detection. The method comprises the following steps: obtaining a first type of gene and a plurality of second type of genes according to a public database; obtaining corresponding original sequence files of the first type of gene and the second type of gene based on a local gene database, obtaining corresponding sequence alignment files based on a local BAM database, and combining them to obtain known NGS gene data; calculating the GC content, the proportion of repeat units, and the similarity ratio in the original sequence file, and calculating the alignment quality in the corresponding sequence alignment file to construct a training sample; inputting the training sample into a classification model to obtain an optimized classification model; extracting the GC content, the proportion of repeat units, the similarity ratio, and the alignment quality from the NGS gene data to be detected and inputting them into the optimized classification model to judge whether the variation detection is affected. The application can judge the influence of homologous regions in variation detection without the aid of prior information of homologous regions.

The present invention relates to a Klotho messenger-RNA (mRNA), wherein the mRNA has a 5' CAP region, a 5' un-translated region (5'-UTR), a coding region encoding a Klotho polypeptide, a 3' untranslated region (3'-UTR) and a poly-adenosine Tail (poly-A tail), wherein the Klotho polypeptide comprises the KL1 domain of human Klotho, preferably wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80 % sequence identity to SEQ ID NO: 1, and wherein the coding region encoding the Klotho polypeptide preferably has a GC content of at least 54 %.

This invention discloses a method for constructing a multi-target amplicon abundance standard for microorganisms and its application, belonging to the fields of molecular biology detection and microbiome analysis. This standard material screens sequences of common human gut microbiota strains, designs and adds universal primers, and obtains 14 DNA fragments of different lengths through gene synthesis, cloning, and purification, which are then mixed according to a preset abundance. It retains natural characteristics such as primer binding sites and GC content, and can systematically correct technical deviations in amplicon sequencing, solving the problem of species abundance distortion. This standard material functions as both a non-homologous internal reference and a homologous external reference, and can be used for laboratory proficiency testing, reagent kit performance evaluation, and cross-platform data calibration, promoting the leap from relative qualitative to absolute quantitative research in microbiome studies and providing metrological support for the standardization and precision of detection results.

The invention belongs to the technical field of gene engineering, and particularly relates to an arthrobacter carbodii gene knockout vector and method. The vector comprises a target gene upstream and downstream homologous arm amplification sequence and a target gene double PAM integration fragment; wherein the target gene is a gene segment to be knocked out; the lengths of upstream and downstream arms of the target gene are respectively 1000bp, and the lengths of amplified homologous arm genes are respectively 2000bp; a basic group at the tail of the sequence of the double PAM integrated fragment is adenine or guanine, and the GC content is 40-60%. According to the knockout vector and the corresponding knockout method, the stable knockout of the gene in the Arthrobacter carbodii can be realized, the knockout efficiency is high, and the knockout vector and the corresponding knockout method have important significance for researching the gene function of the Arthrobacter carbodii and also have a certain reference value for other strains of the Arthrobacter.

The invention discloses a siRNA sequence design method based on deep learning and an activity prediction device, and relates to the technical field of artificial intelligence assisted biology. The method comprises the following steps: carrying out global scanning on target mRNA, intercepting the target mRNA into RNA sequence fragments according to a preset step length and a preset window width, and enumerating all available candidate siRNA sequence fragments; filtering out sequence fragments of which the GC content is out of a preset range in the candidate siRNA sequence fragments, and excluding 15-polymer sequences of which the occurrence frequency is greater than a preset threshold value in the transcriptome; and inputting the siRNA sequence data, the mRNA sequence and structure data and experimental information into a deep learning model, outputting an activity predicted value, analyzing the siRNA sequence with a high activity sequence, and outputting a potential siRNA sequence. According to the method, the activity of the siRNA in a real experiment scene can be more accurately predicted, and efficient design of the siRNA sequence is carried out.

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant for improving amplification efficiency of a high GC sample and application of the DNA polymerase mutant. According to the invention, DNA polymerase mutants KODG and KODC are obtained through site-specific mutagenesis, and on the basis of retaining the capability of amplifying a DNA template with conventional GC content, the capacity of amplifying a DNA template with high GC content (gt; compared with the prior art, the method has the advantages that the amplification efficiency of a DNA template (3kb, 60%) is improved, effective amplification of difficult-to-amplify fragments with 3kb, 95% GC content and the like is successfully realized, and the technical bottleneck of amplification failure caused by high thermal stability and difficulty in complete melting of the existing polymerase in a high-GC region is overcome, so that the application range of the DNA polymerase in complex template amplification is expanded, and the application prospect is broad. And a more reliable enzymology tool is provided for related detection and identification of high-GC-content samples.

The application discloses a wheat salt-tolerant related gene DNA sequence capture sequencing method and belongs to the technical field of molecular biology and genomics. The method comprises the following steps: synthesizing a group of biotin-labeled DNA capture probes according to the salt-tolerant related gene candidate region coordinate interval, the probes are unevenly spaced in the target region, the interval distance is dynamically adjusted by the GC content fluctuation curve in the region, and the probe density is increased when the gradient change rate of the GC content is greater than a threshold value; taking wheat genome DNA as a template, the probes are competitively hybridized with the denatured genome DNA in a liquid phase system to form a target-probe complex; the complex is captured by a streptavidin magnetic bead, non-specific binding fragments are removed through gradient warming and washing, and target DNA is collected; the target DNA is subjected to PCR amplification, purification, construction of a sequencing library and double-end sequencing, unique alignment reads are extracted after alignment, and a genotype state is calculated according to the coverage depth distribution of the target site.

The invention provides a probe set for detecting the copy number of a TSPAN12 gene and application of the probe set. And the probe group comprises probes of No. 2 to No. 6 exons of the TSPAN12 gene. The probe with extremely high specificity is designed by avoiding a high GC content and a repetitive sequence region, so that a non-specific amplification phenomenon is effectively avoided, pathogenic variations such as point mutation, tiny insertion or deletion and the like of the TSPAN12 gene can be accurately detected, and the possibility of misdiagnosis and missed diagnosis is reduced. The method is especially suitable for TSPAN12 gene detection of familial exudative vitreoretinopathy (FEVR).

The invention discloses a method and a system for realizing DNA storage by aiming at multi-rule rotation coding of Chinese texts, and relates to the technical field of DNA storage. Encoding the Chinese text by using a five-stroke font input method; mapping is carried out according to the positive and negative code tables, and an interval balance and dynamic detection strategy is introduced in the mapping process to control GC content distribution in intervals and the probability of occurrence of homopolymers between the intervals. Scattering and recombining the sequence by using block coding, and compressing by using RLE coding, wherein the RLE coding generates a sequence file and a run-length file; performing GC content constraint on the sequence file by using cross coding; and br compression is adopted for the run-length file to further improve the compression ratio. And generating a DNA sequence through rotary coding. According to the method, five-stroke coding, positive and negative code table mapping and sequence reconstruction strategies are introduced, so that the information storage density is higher, the local GC content of the DNA sequence is more stable, the homopolymer length is smaller, the unexpected motif proportion is lower, and the data has higher reliability and safety in the storage process.

This invention relates to a kit and method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases. The kit includes a library hybridization reagent and a probe set for detecting XLIRD pathogenic genes, including capture probes that can simultaneously and specifically capture the RPGR, RP2, RS1, and CHM pathogenic genes. The capture probes for capturing the RPGR pathogenic gene include probes with sequences as shown in SEQ ID NO. 1-96, and capture probes targeting the high-GC region of the RPGR gene with sequences as shown in SEQ ID NO. 24-55. This invention targets the above four pathogenic genes, comprehensively analyzes their variation profiles and sequence characteristics, optimizes the amplification region range, and designs a high-density capture probe set including exons, introns, UTR regions, and upstream and downstream 20kb regions. Specifically targeting the high-GC content region of exon 15 of the RPGR gene, the probe sequence and capture strategy are adjusted to achieve efficient capture of the high-GC region. During the library PCR enrichment stage, the amplification enzymes in the system are screened and optimized. Combined with the probe set, multiple amplification enzyme systems are used, and finally, the most suitable amplification enzyme combination for this gene system is optimized.

The invention relates to the technical field of biological information, in particular to a methylation sequencing data processing method and system. The method comprises the following steps: acquiring local sequence characteristic data of a DNA sample, wherein the local sequence characteristic data comprises GC content data of 20 basic groups before and after each CpG site, ion concentration data and environment temperature data; performing conformation state prediction on the DNA molecules according to the local sequence characteristic data to obtain thermodynamic parameter matrix data containing melting energy, base accumulation energy and ion acting force; on the basis of the Brown kinetics theory, a motion track model of DNA molecules under thermal disturbance is constructed according to thermodynamic parameter matrix data, and conformation state transition data of all CpG sites are obtained. The sequencing deviation is simulated and corrected through Brown dynamics according to DNA sequence characteristics and thermodynamic parameters, so that the accuracy of methylation sequencing data is improved.

The subject application pertains to compositions and methods for enhancing reverse transcriptase (RT) activity and / or reducing the inhibition of RT by inhibitors, such as formalin, tannic acid and / or heparin. In some embodiments, RT inhibition is reduced by the addition of potassium glutamate, histidine hydrochloride monohydrate, poloxamer 188, or any combination thereof to a reaction mixture comprising a polymerase. In other embodiments, RT is enhanced through the addition of a polyvinyl sulfonic acid sodium salt (PVSA) to a reaction mixture. The subject application also provides oligonucleotide primers for use in the reverse transcription of target sequences and its enhancement. These primers have high GC content or low GC content. Methods of using a RT inhibition reducer or a RT enhancer in a composition with an RNA template and RT improves RT yield, RT sensitivity, or RT tolerance to various chemicals are also provided.

The present application relates to the field of DNA methylation modification detection, and particularly relates to nucleic acid sequence molecules and DNA methylation detection methods. The nucleic acid sequence molecules comprise at least a probe sequence, the probe sequence comprises at least one CpG site, at least one of which is a to-be-detected CpG site; the probe sequence comprises a methylation probe sequence and a non-methylation probe sequence, and the two probe sequences are modified with different fluorescent groups; and the probe sequence at least meets one of the following conditions: i) within the region of the to-be-detected CpG site and the 2 nt upstream and downstream of the to-be-detected CpG site, the overall GC content of the methylation probe is not more than 50%; ii) within the region of the to-be-detected CpG site and the 2 nt upstream and downstream of the to-be-detected CpG site, the overall GC content of the methylation probe is more than 50%, and the to-be-detected CpG site in the probe sequence is modified with LNA. The method of the present application can significantly improve the specificity and sensitivity of CpG site methylation detection.

The invention discloses a microdroplet type digital PCR (Polymerase Chain Reaction) premixed solution and application thereof in detecting a high-GC target, and belongs to the technical field of gene detection. The microdroplet type digital PCR premixed solution comprises 3 to 5 mM of MgCl2, 60 to 80 mM of KCl, 10 to 30 mM of Tris-HCl with the pH value of 8.3, 150 to 200 nM of dNTP, 0.1 to 0.2 U / l of hot start Taq enzyme, glycerol with the volume fraction of 0.25 to 1 percent, betaine with the volume fraction of 0.25 to 0.75 M, EDTA (Ethylene Diamine Tetraacetic Acid) with the volume fraction of 0.25 to 0.75 mM and formamide with the volume fraction of 0.5 to 2 percent. The premixed liquid has the advantages of good uniformity, high thermal stability, accurate amplification and good Taq enzyme activity maintenance, is suitable for a probe method, provides an efficient universal reagent for a droplet type digital PCR instrument, and realizes high-sensitivity detection of C228T mutation in a TERT gene high GC content (more than 70%) region.

This invention proposes a DNA data storage method with controllable GC content and homopolymers. The steps are as follows: performing LZW compression on the original text and converting the LZW-compressed text into a binary code stream; adding a Reed-Solomon error-correcting code to the binary code stream to generate a binary code stream with error correction; scrambling the binary code stream with error correction using a chaotic sequence generated by logical mapping to obtain a new binary sequence; and using a 64-ary encoding mapping table formed by selecting 64 mapping elements that meet the constraints from a base permutation set, transcoding the binary sequence into a DNA sequence using the 64-ary encoding mapping table, with each four bases being a mapping unit. This method fully considers data stability, decoding accuracy, and cost-effectiveness in the DNA storage process. Testing on Shakespeare's sonnets demonstrated its feasibility and effectiveness. Experimental results showed that the GC content of the encoded DNA sequence was maintained at 50%, and the homopolymer length did not exceed 2.

The invention relates to the field of bioengineering, in particular to CRISPR / Cas9 nucleic acid, a recombinant vector, a recombinant engineering bacterium, mRNA and a preparation method thereof, a recombinant protein, a composition, an expression and gene editing method and a product, and the CRISPR / Cas9 nucleic acid provided by the embodiment of the invention comprises a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. According to the present invention, by optimizing the codon ratio, the GC content, the sequence repeatability, the RNA secondary structure, the RNA free energy and the like in the DNA sequence, the DNA sequence capable of highly expressing the Cas9 protein version is obtained; the expression quantity of the CRISPR / Cas9 protein provided in the technical scheme is increased by 4-8 times compared with that of a wild type or other contrast, and a similar editing effect can be achieved with a lower dosage, so that the application value of the Cas9 protein in gene editing can be greatly improved.

The invention discloses a multiple PCR primer design method for drug-resistant mutation targeted detection and application thereof, and belongs to the field of biotechnology primer design, the method comprises the following steps: determining a drug-resistant gene of a target species and a detection site thereof; generating a primer set on the basis of one step length based on the target sequence position of the drug-resistant gene mutation site; the candidate primer is subjected to primer attribute evaluation such as TM value, primer length and GC content; meanwhile, a specific sequence is obtained through human-derived sequence non-specific amplification evaluation, dimer evaluation and comparison of a candidate primer and a nt library, the primer coverage degree is evaluated based on a species genome, a conservative primer sequence is obtained, and finally, a final target drug-resistant gene mutation site primer is obtained based on a mutation frequency result. The method is wide in applicability, parallel detection of multiple drug-resistant genes in a single cell can be achieved, and a new strategy is provided for developing design of multiple PCR system primers for drug-resistant mutation targeted detection.

The invention discloses a stable and high-efficiency high-fidelity DNA polymerase mutant and a preparation method thereof, relates to the fields of genetic engineering and minute biology, and aims to modify a high-fidelity DNA polymerase by rationally designing mutation sites and optimizing additives, enhance the thermal stability, extension efficiency and continuous synthesis capability of the high-fidelity DNA polymerase and maintain the high-fidelity characteristic of the high-fidelity DNA polymerase. The optimized DNA polymerase shows significantly improved amplification speed in the PCR amplification process, can efficiently synthesize longer DNA fragments, and is suitable for amplification of complex templates (such as sequences with high GC content or rich secondary structures). According to the method, the synthesis efficiency and accuracy of the long-fragment DNA are remarkably improved, and a more efficient tool is provided for genome sequencing, gene cloning, in-vitro diagnosis and the like.

The application discloses a siRNA sequence design method and an activity prediction device based on deep learning, and relates to the field of artificial intelligence assisted biotechnology. The method comprises the following steps: performing global scanning on a target mRNA, intercepting the target mRNA into RNA sequence fragments according to a predetermined step and a predetermined window width, and enumerating all available candidate siRNA sequence fragments; filtering out sequence fragments with GC content outside a predetermined range in the candidate siRNA sequence fragments, and excluding 15-mer sequences with a frequency greater than a preset threshold in a transcriptome; inputting siRNA sequence data, mRNA sequence and structure data and experimental information into a deep learning model, outputting an activity prediction value, analyzing siRNA sequences with high activity ranking, and outputting potential siRNA sequences. The application can more accurately predict the activity of siRNA in a real experimental scenario and efficiently design siRNA sequences.

The present invention provides a recombinant protein optimized based on the functional domain of HIC1, as well as its preparation method and application. The recombinant protein comprises the following structures, from N-terminus to C-terminus, in order: a cell-penetrating peptide, a BTB / POZ domain, a rigid linker, a zinc finger structure, a flexible linker, and a nuclear localization signal. The amino acid sequence of the recombinant protein is shown in SEQ ID NO. 4, and the nucleotide sequence encoding the amino acids is shown in SEQ ID NO. 3. The present invention replaces the coding sequences of the BTB / POZ domain and the zinc finger domain with codons preferred by Escherichia coli, reducing the GC content from 67% to 52%. The resulting recombinant protein can be efficiently expressed in Escherichia coli. Simultaneously, the cell-penetrating peptide and the nuclear localization signal are integrated into the same recombinant protein, achieving "transfection-free" tumor suppressor function and providing a breakthrough solution for targeted tumor therapy.

The present application relates to a DNA data encoding and decoding method, device, computer equipment and storage medium. The method includes: using a depth-first search algorithm to screen the generated basic base sequence to obtain a basic base sequence that meets preset conditions; selecting a first set number of basic base sequences from the screened basic base sequences as the basic base sequences used when encoding binary data and constructing a mapping table, selecting a second set number of basic base sequences as replacement sequences for replacing taboo sequences; encoding the binary data to be encoded into a base sequence of a DNA chain according to the mapping table, and using the replacement sequence to replace the taboo sequence in the base sequence. The embodiment of the present application can encode any binary data into a base sequence, and the generated base sequence does not contain overly long homopolymers and taboo sequences, and the GC content is balanced, which can reduce the probability of self-folding to produce secondary structures.

The invention provides a Pfu-MutS collaborative error correction PCR method and application thereof, and relates to the technical field of gene engineering. According to the method, high-fidelity Pfu DNA polymerase and heat-stable TaqMutS enzyme are synchronously introduced into a PCR reaction system, a mismatch-containing DNA chain is combined in real time through the mismatch recognition capability of MutS and the extension of the mismatch-containing DNA chain is inhibited, and meanwhile, mismatch bases are corrected by utilizing the activity of 3 '-> 5' exonuclease of Pfu, so that a dual error correction mechanism is formed. Complex protein fusion design is not needed, real-time error correction is achieved by directly utilizing the combination of commercially available independent enzymes, the development cost and the operation complexity are remarkably reduced, meanwhile, the additional purification step needed by traditional step-by-step error correction is omitted, and the method is suitable for high-fidelity requirements of conventional PCR amplification, long-fragment synthesis and high-GC-content templates and has wide application prospects. And an efficient and economic general solution is provided for the fields of DNA synthesis and gene editing.

The invention relates to the field of bioengineering, in particular to nucleic acid, mRNA and application thereof. The invention provides nucleic acid for coding luciferase. The nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. According to the artificial codon optimization method provided by the invention, a plurality of version DNA sequences for highly expressing firefly luciferase are obtained by optimizing the proportion of each codon in a DNA sequence, the GC content range, the sequence repetition condition, the RNA secondary structure, the RNA free energy and the like. Compared with wild-type firefly luciferase protein, the expression quantity of other contrasts in a cell experiment is improved by 2-201 times, the fluorescent brightness is obviously improved in a mouse experiment, and the application value of firefly luciferase serving as a reporter gene is greatly improved.

The present application relates to a kind of primer group, kit and method for detecting ATXN8OS and ATXN8 gene (CTA·TAG) n And (CTG·CAG) n Trinucleotide repeat number, belong to gene detection technical field.The primer group includes first primer pair and second primer pair;The forward primer sequence of the first primer pair is as shown in SEQ ID NO.1, and the reverse primer sequence is as shown in SEQ ID NO.2;The forward primer sequence of the second primer pair is as shown in SEQ ID NO.1, and the reverse primer sequence is as shown in SEQ ID NO.3.The present application is realized by the design of two primer pairs, complementary verification is realized, detection result is comprehensive and reliable, make up the technical deficiency of one generation sequencing and two generation sequencing in long fragment, high GC content repeat sequence detection, and detection cost is low, detection cycle is short.