65 results about "Mutation frequency" patented technology

The invention provides a calculation method and system for accurately identifying heterozygosity deficiency in antenatal diagnosis, and is applied to the technical field of medical data processing. According to the method, the bam file and the variation information are obtained through whole-genome library building and sequencing analysis and data processing. SNP sites are screened, allele mutation frequency is calculated, and effective sites are screened. Dividing a genome window, calculating a homozygous rate HR value, and constructing a nonlinear weight reference based on a normal sample; and finally, an improved CBS algorithm is combined with weight reference to identify a significant AOH section, a table containing position and HR mean information is generated, heterozygosity deletion identification is completed, extra CMA detection is not needed, and the method is accurate and efficient.

The invention discloses a phytolacca americana seed EMS mutation library establishment method, and belongs to the technical field of mutation library construction.The phytolacca americana seed EMS mutation library establishment method comprises the steps that concentrated sulfuric acid is used for pretreating phytolacca americana seeds, then germination accelerating is conducted on the seeds, and the seeds are treated with an EMS solution when the seeds are white, so that the problems that phytolacca americana seed coats are thick, and the sensitivity to EMS is low are solved; according to the mutagenesis method disclosed by the invention, high-density series allele point mutation can be induced and generated, the mutagenesis frequency is relatively high, and meanwhile, chromosome distortion is less caused. In the mutagenesis process, abundant genetic variation can be more efficiently created, and more possibilities are provided for screening mutants with excellent characters. Besides, the method has a remarkable advantage in the aspect of cost, the required chemical reagent is cheap, the experimental operation process is simple, and complicated equipment and technology are not required like common irradiation. The breeding cost is reduced, the working efficiency is improved, and wide popularization and application are facilitated.

The invention relates to the technical field of biomedicine, in particular to a liver cancer molecular subtype typing and prognosis model construction method based on glycometabolism and lactic acid metabolism related genes and application thereof. According to the invention, the typing and prognosis model of liver cancer subtypes is constructed for the first time on the basis of a coordinated regulation network of a glycometabolism gene and a lactic acid metabolism gene, the constructed liver cancer subtype subtypes comprise Cluster1 and Cluster2, and the total survival rates of different subtypes have significant difference; the clinical characteristics T.stage and TNM.stage of different subtypes are obviously different from each other; 12 kinds of immune cells have significant differences among different subtypes; iPS immune scores of different subtypes are significantly different; the TIDE scores of different subtypes have significant score differences; the genes with the highest mutation frequency in different subtypes comprise TP53, CTNNB1 and TTN. CLM scores of the prognosis model constructed on the basis of the collaborative regulation network of the glycometabolism genes and the lactic acid metabolism genes have no significant difference in different patient states; the 17 immune cells have significant differences between high and low risk groups. And a new way is provided for individualized treatment strategy formulation and survival outcome improvement of HCC.

The invention discloses a conjugational transfer-assisted orthogonal targeted mutation evolution system, which comprises a donor strain and a receptor strain, a donor strain intracellular muter performs targeted mutation on a target sequence to obtain a mutation sequence, a non-self-transferable conjugational transfer functional plasmid is introduced into the donor strain, and a non-self-transferable conjugational transfer functional plasmid is introduced into the receptor strain. The mutant sequence is unidirectionally and horizontally transferred from a donor strain cell to a receptor strain cell, and the complete orthogonality of the mutants in the initial donor strain and the mutant sequence in the receptor strain is realized through the orthogonality between the donor strain and the receptor strain. The results of a sacB-sucrose reverse screening experiment show that the mutation frequency of an ortho-MutaT7 system reaches 2.96 * 10 <-2 > at 96 hours, which is 1840.9 times of the natural mutation frequency of the sacB in donor cells, and the ortho-MutaT7 system generates mutation of 12 basic group change types in a target sequence. The Ortho-MutaT7 system developed by the invention realizes mutation diversity and also has orthogonality, leakage mutation and off-target mutation of mutants in host cells are fully avoided, and a direct relationship between a mutation sequence and a phenotype is established.

The invention discloses a multi-index fusion-based biotechnology product risk dynamic identification method and system, and relates to the technical field of biological safety management, and the method comprises the steps: S1, obtaining experimental data and environmental data from each stage of a transgenic crop life cycle, constructing a dynamic data set containing a molecular structure sequence, a function performance index and an ecological impact factor, and carrying out the construction of a dynamic data set; extracting the mutation frequency of the molecular structure sequence along with the time change, the abnormal fluctuation of the function expression index and the dynamic change trend of the ecological influence factor; s2, according to molecular structure sequence mutation frequency and functional performance index abnormal fluctuation, extracting a nucleotide replacement mode and a functional abnormality data set of the gene editing product in a research and development stage and an application stage, and determining a nucleotide replacement mode and a functional abnormality association mode; according to the biotechnology product risk dynamic identification method and system based on multi-index fusion, accurate prediction and dynamic management of ecological risks of transgenic crops are realized, and the environmental release safety is remarkably improved.

The invention relates to an islet repair ICK mode oral polypeptide optimization system taking VDAC1 as a target based on ESM2 and a genetic algorithm, and the system comprises a mutation module which is used for carrying out mutation and recombination by adopting the genetic algorithm according to original protein sequence data to generate an initial population; the prediction module is used for respectively inputting the protein sequence data of the initial population into a protein dimer dissociation energy prediction model, a protein toxicity prediction model and a protein cell penetrability prediction model to obtain a dissociation energy prediction value delta G, a toxicity probability value T and a cell membrane passing transmission probability Ed; and the optimization module is used for acquiring the optimal individual performance of the highest fitness score to output the Pareto optimal sequence index, and recording the hotspot residue distribution and the site mutation frequency at the same time. On the basis of keeping the biological activity of the original sequence, the key drug properties are improved in a breakthrough manner.

Provided are a high-throughput transcriptome sequencing-based IKZF1 gene exon deletion recognition system and method. The exon deletion recognition system is obtained by inputting the number of IKZF1 exon junctions, differential genes and expression quantities thereof, and IKZF1 exon and intron mutation frequency information of samples as features into a constructed random forest model and performing prediction, and can be used for accurately predicting and recognizing any exon deletion with high accuracy and high specificity, providing guidance for patient prognosis and treatment.

The invention discloses a megalurothrips megalurothrips methylamino abamectin benzoate resistance molecule detection method which comprises the following steps: (1) collecting megalurothrips megalurothrips female adults in a plurality of regions, and extracting DNA (deoxyribonucleic acid) in a single region in a mixed manner; (2) designing glutamic acid gated chloride ion channel gene specific primers of the megalurothrips megalurothrips, respectively adding specific tags at the front ends of the primers in all regions, and carrying out PCR (Polymerase Chain Reaction) amplification; mixing the PCR products of each region to obtain a library; (3) performing second-generation amplicon sequencing on the library to obtain off-machine data; and (4) analyzing offline data, merging the data in the same region, calculating the mutation frequency of each region by taking the ratio of the mutation (G232S) of the target sequence as an index, and judging the resistance of the megalurothrips usitatus to the emamectin benzoate. The method can be used for rapidly and massively monitoring the emamectin benzoate resistance condition of the field megalurothrips megalurothrips, and an important basis is provided for high-level emamectin benzoate resistance early warning and megalurothrips megalurothrips chemical treatment.

The invention discloses a group of KASP primers for detecting drug resistance mutation of bemisia tabaci and application of the KASP primers, and relates to the technical field of agricultural biology. The KASP primer disclosed by the invention comprises a primer combination which is used for detecting resistance mutations of deltamethrin, spirodiclofen and chlorpyrifos, such as BtabM918V (BtabM918V), BtabL925I (BtabL925I), BtabT929V (BtabA2083V) and BtabF392W (BtabF392W). One or more primer combinations are used for detecting the drug resistance mutation of the bemisia tabaci. The group of KASP labeled primers has good specificity and high sensitivity, and can accurately detect the resistance mutation frequency of bemisia tabaci to insecticides.

The application provides a calculation method and system for accurately identifying loss of heterozygosity in prenatal diagnosis, and is applied to the technical field of medical data processing. Bam files and variation information are obtained through whole genome library construction and sequencing analysis through data processing. SNP sites are screened, allele mutation frequency is calculated, and effective sites are screened. The genome window is divided, the homozygosity rate HR value is calculated, and the nonlinear weight reference is constructed based on normal samples. Finally, the improved CBS algorithm is used to identify significant AOH segments combined with the weight reference, generate a table containing position and HR average information, complete the identification of loss of heterozygosity, and do not need additional CMA detection, which is accurate and efficient.

The invention relates to the field of agricultural pest drug resistance detection, and provides a detection primer and method for chilo suppressalis ryanodine receptor I4758M mutation and application. According to the detection primer, a mutation recognition site is designed at the 3'tail end of an inner primer BIP, specific detection is achieved through the key base difference (A / G) of a wild type and a mutant type, and the primer group comprises an outer primer pair F3 / B3, an inner primer pair FIP / BIP and a loop primer pair LoopF / LoopB; the detection method is based on loop-mediated isothermal amplification (LAMP), the genotype is visually interpreted through color change of reaction liquid, a thermal cycler is not needed, and the detection method has the advantages of being high in accuracy and sensitivity, easy to operate, low in cost and the like. The matched kit comprises a rapid DNA extraction assembly and is suitable for field on-site detection. The gene mutation frequency of the specific site of the chilo suppressalis can be rapidly detected, and the technical support is provided for resistance monitoring, early warning and scientific treatment of bisamide insecticides.

The invention discloses a gene fusion detection method based on high-throughput single-ended sequencing. The gene fusion detection method comprises the following steps: comparing sequencing data to a reference genome; extracting a read sequence containing a soft shear fragment of more than 5bp, and comparing the read sequence to a first gene of the fusion gene; extracting read sequences of the soft shear fragments with the fragment length of more than 5bp again, comparing the read sequences to a second gene of the fusion gene, and filtering out comparison positions with the read sequences with the length of less than 20bp accounting for more than 80%; screening a true positive gene fusion breakpoint on the second gene; calculating the fusion mutation frequency on each true positive gene fusion breakpoint and sorting, and taking the true positive gene fusion breakpoint with the highest fusion mutation frequency as the fusion breakpoint. Gene fusion analysis is carried out based on single-ended sequencing data, the data volume required by detection is reduced, the cost is reduced, and meanwhile, the fusion detection sensitivity and efficiency are improved by optimizing the fusion analysis process.

This invention belongs to the field of radiation mutagenesis technology, and relates to a method for mutagenesis of lavender seeds, employing... 60 Lavender seeds are treated with Co-γ rays; the radiation dose is 50-500 Gy, and the dose rate is 1-3 Gy / min. This invention employs... 60 Radiation with Co-γ rays can efficiently induce mutations in the genetic material of lavender seeds, effectively breaking gene linkage and significantly increasing the mutation frequency. This provides a rich material basis for screening lavender mutants, allowing for the selection of seed half-lethal doses, and obtaining plants with variations in leaf color, leaf shape, plant height, and other traits. This provides technical support for lavender breeding using radiation mutagenesis technology.

The application discloses a molecular marker of citrus full mite resistance to ethyl acetazolamide and application thereof, and the molecular marker is SNP mutation A269G and / or T270A of succinate dehydrogenase SDHD subunit of the citrus full mite. A detection method of the molecular marker of the citrus full mite resistance to ethyl acetazolamide is also disclosed, total DNA of the citrus full mite to be detected is extracted, PCR amplification is carried out with the total DNA as a template, a succinate dehydrogenase SDHD subunit gene fragment containing a mutation region is obtained, and the obtained gene fragment is subjected to sequence comparison with the molecular marker, so that the resistance of the citrus full mite to be detected to ethyl acetazolamide can be judged. The molecular marker and the detection method can simultaneously detect a large number of samples, can quickly and effectively detect whether the resistance SNP molecular marker and the mutation proportion exist in a field population of the citrus full mite, and can evaluate the mutation frequency of the corresponding site in a certain geographical population.

The invention provides a deafness gene mutation cell line, a construction method and a detection quality control product, and belongs to the technical field of gene detection. The invention provides an sgRNA group for constructing a deafness gene mutation cell line, the sgRNA group comprises an sgRNA group designed for mutation sites of deafness genes GJB2, GJB3 and SLC26A4, and 35delG, 176-191del16, 235delC and 299-300delAT which carry the deafness genes GJB2 are obtained through gene editing; 538Cgt in GJB3; t and 547 Ggt; a; in SLC26A4, IVS7-2Agt is added; and extracting genome DNA of the editing cells, and mixing the genome DNA with wild type genome DNA to obtain the deafness gene mutation detection quality control product. The deafness gene mutation detection quality control product provided by the invention can simulate the deafness gene mutation frequency of a real sample, and can more accurately simulate real mutation occurring in vivo.

A generic cancer prognosis evaluation system based on LAMA5 expression provided by the invention relates to the technical field of generic cancer prognosis evaluation, and comprises the following steps: acquiring multi-source molecular data, establishing a standardized data input path to obtain LAMA5 gene molecular information, and completing sample identification and field standardization by calculating mutation frequency and gene copy number variation of the LAMA5 gene molecular information to obtain a generic cancer prognosis evaluation result. And an LAMA5 generic cancer molecular database is formed. A standardized data input path and a unified field format are formed by establishing an LAMA5 generic cancer molecular database and integrating TCGA, GTEx, a human protein map and other multi-source molecular data, the database achieves generic cancer prognosis evaluation system calculation of LAMA5 mutation frequency and gene copy number variation, the data consistency and statistical reliability of multiple cancer species are improved, and the method is suitable for large-scale popularization and application. And a data basis is provided for pan-cancer level expression and variation analysis.

The invention belongs to the technical field of bamboo reed breeding, and discloses a mutation breeding method for obtaining a bamboo reed cold-resistant mutant based on EMS (Enhanced Mass Spectrometry), which comprises the following steps: step 1, inducing embryogenic callus; step 2, carrying out EMS mutagenesis treatment; step 3, performing EMS stress screening; 4, differentiation and rooting culture of adventitious buds; 5, screening the tissue culture seedlings at low temperature; the mutation obtained through mutagenesis treatment is a permanent change of a genetic structure of an organism and may appear in germ cells and somatic cells of a plant. Physical or chemical mutagenic agents are used to increase the frequency of mutations. In chemical mutagens, EMS is considered to be the most effective mutagen, the EMS can generate a large number of non-lethal point mutations in the whole genome, the mutation frequency is high, the chromosome abnormality frequency is low, the EMS is used for treating embryogenic calluses of the bamboo reeds, Hyp is used for screening, the low-temperature-resistant bamboo reed mutants are obtained, the planting area of the bamboo reeds can be enlarged, and the survival rate of the bamboo reeds is increased. And waste land in a low-temperature region is fully utilized.

The invention provides a sequencing data analysis method for low-frequency Indel mutation generated by CRISPR (clustered regularly interspaced short palindromic repeats) gene editing. The sequencing data analysis method comprises the following steps: acquiring double-end sequencing data, demultiplexing R1 and R2 data, merging the data, removing a linker sequence, splicing forward and reverse data, and finally calling crispresso2 software to carry out comparative analysis and data statistics. According to the method disclosed by the invention, the gene editing condition of the Indel mutation with low frequency and larger variation range generated by CRISPR gene editing can be quickly and accurately analyzed, particularly, the frequency and the mode of the Indel mutation generated by animal tissues or animal adults through CRISPR gene editing can be accurately evaluated, and under the condition that no control group is set and result correction is not needed, the method can be used for rapidly and accurately evaluating the Indel mutation frequency and the Indel mutation mode of the animal tissues or the animal adults through CRISPR gene editing. According to the method, the background noise is effectively controlled, the result is directly output, the statistical analysis efficiency is remarkably improved, and the efficiency and mode information of gene editing of multiple samples can be directly read under the same sequencing depth.

The application discloses a fusion protein, an in-vivo directed evolution system and application thereof, and the fusion protein comprises a Retron reverse transcriptase and an adenosine deaminase, and the adenosine deaminase is connected to the N terminal or C terminal of the Retron reverse transcriptase. The application firstly fuses the Retron reverse transcriptase with the adenosine deaminase TadA-8e, constructs a bifunctional fusion protein system with reverse transcription activity and adenosine deamination activity, realizes the functional coupling of Retron-mediated msDNA synthesis and adenosine deamination mutation introduction. The application significantly improves the mutation frequency and mutation diversity of a target gene, accelerates the generation, accumulation and fixation of beneficial mutations, and thus improves the in-vivo directed evolution efficiency.

The present invention discloses a primer and probe combination and a kit for detecting JAK2V617F mutation. The present invention obtains a primer and probe combination for detecting JAK2V617F mutation based on the sequence of the V617F site on the antisense strand of the JAK2 gene. Using the primer and probe combination of the present invention, the JAK2V617F mutation can be specifically detected, and the minimum mutation frequency detected is 0.01%. The minimum DNA input required is 100pg, which can meet the needs of detecting minor residual mutations. Compared with the detection of JAK2V617F mutation using the ordinary PCR-Sanger method, the present invention has the advantages of high detection sensitivity and good repeatability, which is conducive to the accurate detection of JAK2V617F minor residual mutations, and is also conducive to the formulation of diagnosis and treatment decisions for myeloproliferative tumors and the development of related therapeutic drugs.

The application discloses application of an RB1 mutant in a neurodegenerative disease. The application finds through experiments that juvenile fish and adult heterozygotes of a zrb1-KO mutant zebrafish (2 bases are deleted at positions 67 and 68 of the 2nd exon of an rb1 gene) exhibit motor and memory learning dysfunction, exhibit increased post-mitotic neuron apoptosis in the hindbrain, and have the characteristics of a neurodegenerative disease, and therefore, the zrb1-KO mutant zebrafish can be used to construct an animal model of a neurodegenerative disease. In addition, the application also statistically analyzes the mutation frequency and mutation type of RB1 of a neurodegenerative disease patient, and verifies the pathogenicity of R621S and L819V mutations of RB1 in neuron apoptosis through experiments, thereby providing an effective approach for diagnosis or treatment of a neurodegenerative disease.

The invention belongs to the technical field of gene engineering, and particularly relates to an SNP site related to autism spectrum disorder of children and application of the SNP site. According to the SNP site related to the autism spectrum disorder of the children, the SNP site is located at the 69599651bp position on a 7g22 chromosome, and A / G base mutation exists. In an autism spectrum disorder patient case group, the mutation frequency of the site is obviously higher than that of a healthy control group, and the site and autism spectrum disorder diseases are relatively strong in relevance, so that the site is possibly related to the ASD onset risk, and an auxiliary basis is provided for early diagnosis and early screening of ASD.

The invention discloses a lung cancer detection primer probe composition and application thereof in a diagnostic kit, and belongs to the technical field of biological detection. The technical problem to be solved is to provide a high-sensitivity and high-specificity primer probe composition for one-time detection of multiple lung cancer mutant genes. The invention provides a primer probe composition which is high in sensitivity (capable of detecting DNA mutation with mutation frequency as low as 0.5% and RNA fusion of 25 copies) and high in specificity and is used for detecting multiple lung cancer mutation genes at one time, and the primer probe composition is assembled into a kit, comprises EGFR, ALK, ROS1 and MET genes, covers 26 gene mutations and 26 gene fusion, and is used for detecting multiple lung cancer mutation genes. The detection result is high in sensitivity, strong in specificity, good in repeatability and simple, convenient and rapid to operate, and a rapid and reliable detection method is provided for clinic.

The invention provides a tongue squamous cell carcinoma assessment method based on targeted sequencing and clinical characteristic parameters, and relates to the technical field of tongue squamous cell carcinoma assessment computer processing programs, the method comprises the following steps: S01, obtaining a tumor tissue sample of a tongue squamous cell carcinoma patient, and detecting gene mutation data through a targeted sequencing technology; s02, screening mutant genes significantly related to clinical pathological characteristics of the patient to obtain first N genes with the highest mutation frequency; s03, constructing a prognosis risk scoring model, converting the first N genes with the highest mutation frequency into a binary mutation state, then taking the binary mutation state as a variable, analyzing the training model through multivariable Cox proportional risk regression, and calculating the risk score of the patient; and S04, judging a principle according to a risk median. According to the invention, an individualized risk assessment tool is provided for clinic, and a doctor is assisted to formulate a more reasonable treatment and follow-up visit scheme, so that the prognosis and survival outcome of a patient are effectively improved.

The invention provides an SERS (Surface Enhanced Raman Scattering) detection method for thyroid cancer BRAF V600E mutation, and belongs to the technical field of detection. A probe is composed of oligonucleotide of which the 5'end is connected with a cy3 fluorophore. During detection, a tissue sample is subjected to splitting decomposition and PCR amplification and then is hybridized with the probe, and uncombined probes are removed by adopting a wash buffer containing 95% formamide and 5% EDTA (Ethylene Diamine Tetraacetic Acid) and a DNA purification kit. A purified product is dropwise added to a KI aptamer modified silver nanoparticle coffee ring substrate, and an SERS spectrum is collected through 785 nm laser excitation. Mutation is judged through cy3 characteristic double peaks at 1188 cm <-1 > and 1393 cm <-1 >, and detection is completed within 4 hours. Clinical verifications show that the consistency rate with a gene detection result is 100%, the sensitivity reaches 0.1% mutation frequency, the limitations of long consumed time and high cost of traditional PCR / NGS are overcome, and the kit is suitable for intraoperative rapid diagnosis and targeted treatment guidance.

The application discloses a DNA polymerase III mutant and application thereof. The mutant contains an amino acid sequence shown in SEQ ID No. 1, and the mutant can be applied to improving the mutation frequency of a host strain genome. In the application, the Escherichia coli is used as a starting strain, a high-efficiency evolution auxiliary plasmid is introduced, and the mutation frequency of the host is improved; combined with competitive pressure directional screening of a tryptophan structural analog 5MT, finally, the Escherichia coli engineering strain with high yield of tryptophan is obtained. The auxiliary plasmid comprises a pBad24 plasmid vector skeleton and an engineered Escherichia coli DNA polymerase III danQ nucleotide fragment. The high-efficiency evolution auxiliary plasmid provided by the application can significantly improve the mutation rate of the host genome, combined with the directional screening of 5MT, the traditional mutagenesis cycle can be greatly shortened, the engineering strain with high yield of tryptophan can be efficiently screened, and the application has important application value.

The invention provides a method and a kit for non-invasive prenatal detection of deafness. The method comprises the following steps: step 1, collecting peripheral blood of a pregnant woman, and separating plasma and leukocytes; step 2, extracting cfDNA of the pregnant woman from the plasma, and performing quality control; 3, a specific primer is designed for a target mutation site, multiple PCR is conducted on the cfDNA through the specific primer, and free nucleotide of the cfDNA and the specific primer group are provided with UMI markers; and step 4, constructing a sequencing library, sequencing the PCR reaction result on a machine, counting alleles based on the UMI marker, calculating mutation frequency, and deducing the fetal genotype. Based on the cfBEST system, the genotypes of the maternal and fetus are accurately deduced, and non-invasive prenatal detection of the deafness monogene disease is achieved.

The application discloses a HIV-1 drug-resistant gene detection method based on NGS technology, belongs to the technical field of gene detection, adopts a series of processes such as NGS data quality control and filtration, genome alignment and extraction of effective information, construction of consistent sequences, construction of a pseudo-reference genome, HIVdb drug resistance detection, visualization of NGS sequencing results and HIVdb drug resistance detection results, a traceability evolution tree and final output results to complete HIV-1 drug-resistant gene detection. Compared with traditional first-generation Sanger sequencing technology, the system and method adopt the application, can detect drug-resistant sites with a mutation frequency of less than 10%, have higher detection sensitivity, and have important significance for early screening and detection of HIV-1 virus and doctors to formulate more effective drug use schemes after the onset.

The invention discloses a recombinant escherichia coli strain with high mutation frequency and a construction method and application thereof, and the construction method comprises the following steps: connecting a double-chain cytosine deaminase coding gene DddA from burkholderia to a plasmid pACYC Duet-1 through a seamless cloning technology to obtain a recombinant plasmid pACYC-tDddA-1416; the method comprises the following steps: on the basis of pACYC-tDddA-1416, introducing S1416P mutation by virtue of a cyclization PCR (Polymerase Chain Reaction) technology, so as to obtain a recombinant plasmid pACYC-tDddA-S1416P with high mutation frequency; the method comprises the following steps: carrying out chemical transfer to obtain escherichia coli BL21-pACYC-tDddA-S1416P; the method comprises the following steps: on the basis of pACYC-tDddA-1416, introducing S1416P mutation by virtue of a cyclization PCR (Polymerase Chain Reaction) technology, so as to obtain a recombinant plasmid pACYC-tDddA-S1416L with high mutation frequency; and the escherichia coli BL21-tDddA S1416L is obtained through chemical transfection. The recombinant escherichia coli strain with high mutation frequency provided by the invention can be used for adaptive evolution, and the method is simple and convenient.

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.