381 results about "Enzyme digestion" patented technology

The invention discloses a dCAPS molecular marker for rapidly identifying the content of soybean protein and application of the dCAPS molecular marker. An upstream primer sequence of a primer pair for amplifying the dCAPS molecular marker is as shown in SEQ ID No. 1, and a downstream primer sequence is as shown in SEQ ID No. 2. According to the invention, the mispairing base primer is introduced to carry out PCR amplification and enzyme digestion on the to-be-detected soybean genome DNA, and the soybean protein content can be identified according to the band size of the enzyme digestion product. The dCAPS molecular marker disclosed by the invention can be used for identifying or assisting in identifying the soybean protein content, a high-protein soybean variety and a low-protein soybean variety are distinguished from the molecular level, and the dCAPS molecular marker has important significance on soybean molecular marker assisted breeding and high-protein soybean screening and cultivation.

The present application relates to a virus of reverse genetic operation construction of NADC34-like PRRSV2. Specifically, the full gene sequence of NADC34-like PRRSV2 BJ1805-2 isolated strain is segmented and connected to the vector by using PCR amplification, enzyme digestion and other methods with pACY177 plasmid as the carrier, and the BJ1805-2 full-length infectious clone recombinant plasmid (rBJ1805-2) is obtained. The infectious clone virus rBJ1805-2 is rescued in vitro, and the first NADC34-like PRRSV2 strain infectious clone platform in China is successfully built. The present application also relates to a modification method for constructing NADC34-like PRRSV2 strain which can adapt to Marc-145 cell passage culture. The infectious clone virus constructed by the present application has good in vitro and in vivo proliferation efficiency, and can cause specific viremia after inoculation in pigs; it has good safety, does not cause fever after inoculation in pigs, and will not cause piglet death. The modified strain can be used as a candidate strain for developing the first NADC34-like PRRSV2 specific vaccine in China, and is conducive to the prevention and control of PRRSV epidemic in China.

The invention provides a non-disease diagnosis purpose 5-hydroxymethylcytosine 5hmC detection method and a non-disease diagnosis purpose 5-hydroxymethylcytosine 5hmC detection kit. The detection method comprises the following steps: S1, glycosylation treatment; s2, carrying out enzyme digestion treatment on the GSREs; s3, carrying out APOBEC deamination treatment; s4, carrying out hydroxymethylation detection; the kit comprises a glycosylation reaction kit, a GSREs enzyme digestion reaction kit, an APOBEC deamination reaction kit and a PCR (Polymerase Chain Reaction) kit. The glycosylation reaction kit comprises a CutSmart buffer solution, UDP-Glucose, a T4-BGT enzyme and nuclease-free water, wherein the CutSmart buffer solution, the UDP-Glucose and the T4-BGT enzyme are used as raw materials; the GSREs enzyme digestion reaction kit comprises a GSREs enzyme, an enzyme digestion reaction buffer solution and nuclease-free water, the APOBEC deamination reaction kit comprises APOBEC, BSA (Bovine Serum Albumin), an APOBEC Reactive buffer solution and nuclease-free water; the PCR kit comprises a PCR reaction solution, a primer and a probe. According to the 5-hydroxymethylcytosine 5hmC detection method and kit for non-disease diagnosis purpose provided by the invention, precise quantification of extremely high specificity of 5hmC is realized, and the detection sensitivity is also improved while the specificity of a detection signal is improved.

The invention relates to the technical field of biology, and particularly discloses a fusion protein containing an SUMO tag and a preparation method and application thereof. The fusion protein provided by the invention comprises an SUMO tag and an antibacterial peptide, the antibacterial peptide can inhibit escherichia coli and staphylococcus aureus, and the amino acid sequence of the antibacterial peptide is as shown in SEQ ID No. 10, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 12, SEQ ID No. 15 or SEQ ID No. 17. The fusion protein can be efficiently expressed through a prokaryotic expression system, renaturation of the antibacterial peptide can be successfully achieved after enzyme digestion and purification, the antibacterial activity of the antibacterial peptide is recovered, and therefore efficient preparation of the antibacterial peptide is achieved.

The invention discloses application of a KASP molecular marker in identification of sweet melon peel color traits, and belongs to the technical field of molecular markers. The invention aims to promote gene localization and molecular-assisted breeding processes by utilizing molecular marker-assisted selective breeding on sweet melon pericarp color characters. The KASP molecular marker is a KASP marker RW2; the KASP marker RW2 is designed according to an SNP (Single Nucleotide Polymorphism) site at the position of Chr04: 526653. According to the application, the KASP molecular marker RW2 is used for performing genetic typing on the red-peel muskmelon and the white-peel muskmelon, the DNA of a target plant can be identified only through one-time PCR amplification, the whole detection process is simple and convenient, and complex steps such as enzyme digestion, electrophoresis and sequencing are not needed. Through the high-throughput genotyping system, a genotyping graph and a genotyping value can be rapidly obtained, so that rapid identification of the genotype of a target plant is realized. The KASP molecular marker RW2 provided by the invention can be more efficiently applied to the breeding work of sweet melon peel color germplasm.

The invention discloses a reconstructed respiratory syncytial virus F protein trimer, relates to the technical field of genetic engineering vaccines, and designs an RSV F protein trimer, a monomer of the trimer does not contain a p27 sequence, and a furin enzyme digestion peptide fragment aa110-aa136 is deleted; the F2 structural domain and the F1 structural domain are connected through a GS connexon; the invention also designs point mutation combinations, and the point mutation combinations can form an inter-chain disulfide bond, so that covalent connection of the trimer is realized, and the problem of depolymerization of the trimer is fundamentally solved; the trimer is stabilized in a pre-F state, a post-F sample structure does not exist, and an ELISA result shows that a neutralizing epitope is completely reserved; the trimer antigen structure is challenged by adopting a thermal acceleration experiment method, and a result shows that the purity of the trimer antigen reaches 95% or above after the trimer antigen is placed at 37 DEG C for 28 days, and the trimer antigen is very good in stability; when the trimer is used for preparing the vaccine, the standard required by laws and regulations can be met without freeze-drying.

The invention discloses a construction method of a human epidermal organoid, and belongs to the technical field of organoid cell culture. The method is mainly used for optimizing a culture method of human epidermal organoid, and particularly relates to an efficient epidermal organoid culture technology based on a specific culture medium and an enzyme digestion system. According to the method, a # imgabs0 # human epidermal organ complete culture medium is adopted for preparation, and a basic culture medium and an additive are mixed to form the complete culture medium suitable for cell growth. The primary specimens are treated, and the steps of cleaning, digestion, cell collection and the like are included, so that the activity and functions of cells are ensured. Through accurate inoculation and culture conditions, the formation and growth of the organoid are promoted, and the culture in a proper environment is ensured, so that the formation efficiency of the organoid is improved. The organoid is subjected to passage and cryopreservation treatment, so that the activity and functional stability of the organoid in the long-term storage and use process are ensured.

The invention provides a method and system for discovering antioxidant peptides from food and agricultural by-products and outputting enzymolysis recommendation, and relates to the technical field of bioinformatics and artificial intelligence crossing. The method sequentially comprises the steps of protein sequence collection and standardization, candidate peptide fragment interception, prediction of antioxidant activity through fusion of sequence and structural characterization, generation of releasable peptide fragments through enzyme digestion simulation, and construction of comprehensive evaluation indexes based on activity probability and process parameters. And an optimal enzymolysis scheme is searched in an enzyme combination and reaction condition space through multi-objective optimization, so that the method is suitable for high-valued utilization of food industrial residues and agricultural wastes, and has the advantages of high throughput, high accuracy and high implementability.

The invention discloses an EmAGO2 truncated recombinant protein, an echinococcosis multilocularis indirect ELISA (enzyme-linked immuno sorbent assay) diagnostic kit based on the truncated recombinant protein and a use method of the kit. The method comprises the following steps: designing a specific primer by selecting a sequence of 1-738bp at the N end of an Em-AGO2 gene, carrying out PCR (Polymerase Chain Reaction) amplification by taking reverse transcription echinococcosis multilocularis DNA (Deoxyribonucleic Acid) as a template, connecting an amplification product with a pCE2-TA / Blunt vector, and then transforming into a competent cell for culturing, so as to obtain a pCE2-T-EmAGO2 plasmid; respectively carrying out double enzyme digestion on the plasmid and an expression vector pET-28a (+), and connecting enzyme digestion fragments to obtain a recombinant protein expression plasmid pET-28a-EmAGO2; the preparation method comprises the following steps: taking EmAGO2 as a template, transforming the EmAGO2 into competent cells, selecting positive clones for induced expression, and carrying out ultrasonication treatment, separation and purification, concentration and desalination treatment on expressed thalli to obtain the EmAGO2 truncated recombinant protein. Furthermore, a mouse anti-EmAGO2 truncated recombinant protein and goat anti-rabbit IgG are respectively used as an antigen and an antibody to establish an indirect ELISA diagnostic kit for the echinococcosis multilocularis, and the kit has high sensitivity, specificity and good repeatability, and can be used for early diagnosis of the echinococcosis multilocularis.

The invention discloses a recombinant III-type human-like collagen as well as a coding gene and an expression method thereof, and the method comprises the following steps: S1, carrying out sequence optimization by taking a human III-type collagen sequence as a template to obtain a recombinant III-type human-like collagen gene sequence; designing PCR (Polymerase Chain Reaction) amplification upstream and downstream primers according to the gene sequence, and amplifying the gene sequence of the recombinant III type human-like collagen; s2, carrying out double enzyme digestion on the recombinant III type human-like collagen gene and a plasmid, and connecting to obtain a recombinant plasmid; s3, linearizing the recombinant plasmid, transforming the linearized recombinant plasmid into agrobacterium, and screening; and S4, infecting lettuce with agrobacterium, culturing to express protein, freezing the infected lettuce with liquid nitrogen, grinding, extracting total plant protein, and purifying to obtain the recombinant III-type human-like collagen. The recombinant III type human-like collagen is expressed through lettuce, so that the method is safe and efficient, and has sufficient market value and important practical significance.

The invention relates to polysaccharide-based composite hydrogel as well as a preparation method and application thereof. The composite hydrogel is prepared from the following raw materials: hydroformylated modified ortho-hydroxyl-containing polysaccharide, an extracellular matrix enzyme digestive juice, tannic acid, cannabidiol, neomycin sulfate and borneol, by compounding multiple components, the use performance of the hydrogel can be remarkably improved, and the hydrogel is particularly suitable for a harsh environment. The composite hydrogel disclosed by the invention has excellent mechanical properties, good biological safety, certain self-repairing performance and relatively high anti-inflammatory, antibacterial and antioxidant properties, and can be applied to skin or mucous membrane tissue wound repairing.

The invention discloses modified hydroxytyrosol, a cosmetic, a preparation method and application. The provided preparation method of the modified hydroxytyrosol comprises the following steps: in the presence of an acidic solvent, mixing hydroxytyrosol, lipase and fatty acid ester for reaction, centrifugally layering a reaction product, and collecting components of a middle layer to prepare the modified hydroxytyrosol. According to the provided preparation method of the modified hydroxytyrosol, lipase and fatty acid ester are subjected to an enzyme digestion reaction, an active hydroxyl group in a product obtained through the enzyme digestion reaction and hydroxytyrosol are subjected to a dehydration reaction, so that an ester group molecular chain is grafted into hydroxytyrosol, and a modified hydroxytyrosol product with an ester group is prepared. And the obtained modified hydroxytyrosol has relatively high fat solubility and oxidation resistance.

The present invention discloses a kit for detecting residual total RNA of Escherichia coli, a detection method and an application thereof. By designing a pre-treatment system, a plasmid sample can be directly used for quantitative detection of residual total RNA of Escherichia coli by RT-qPCR after digestion. The detection method eliminates the influence of the enzyme digestion process on the detection result by setting a digestion quality control product, ensures the accuracy of the entire detection result, and has excellent specificity, accuracy, sensitivity and precision.

The invention discloses a method for purifying recombinant protein. The framework of the fusion protein is characterized in that a series tag consisting of elastin-like protein (ELP) and SUMO is connected with a target protein. The fusion protein is purified by utilizing the reversible phase change cycle (ITC) property of ELP, and the separation and purification of the recombinant protein can be realized only through simple centrifugal operation, so that the fusion protein with higher purity is obtained, and the required recombinant target protein can be obtained subsequently through enzyme digestion and ITC. The technology is wide in application range, simple and convenient in purification method, low in cost and suitable for industrial amplification, and has a good commercial prospect.

The invention discloses a modular tumor microenvironment response polypeptide and a preparation method and application thereof. The modular tumor microenvironment response polypeptide is composed of the following functional modules: a targeting and chemotactic module used for targeting tumor cell surface p32 protein and tumor lymphatic vessels and promoting tumor tissue penetration and enrichment; the microenvironment double-response module is used for being cut by MMP-2 / 9 enzyme and broken by high-concentration glutathione GSH in a tumor microenvironment to trigger self-assembly; the self-assembly driving module is used for exposing after the microenvironment responds to activation and driving short peptides to be self-assembled to form a nano structure; a killing module: a pro-apoptotic peptide PAD used for targeting a mitochondrial membrane and inducing tumor cell apoptosis; and the stability optimization module is used for enhancing the degradation resistance of the oligopeptide. By integrating targeted delivery, enzyme digestion response, acid-sensitive release and stabilizing structures, the problems of insufficient targeting property, inaccurate release, poor stability, diagnosis-treatment splitting and the like of the anti-tumor drug and the developing agent are solved.

The invention belongs to the technical field of biological drug analysis, and particularly relates to a method and a system for quantitatively detecting antibody drugs by using a dimethyl-labeled modified peptide fragment as an internal standard. Sequentially carrying out dimethyl labeling on the enzymolysis peptide fragments of the antibody drugs to obtain a plurality of dimethyl-labeled modified peptide fragments which are used as internal labels; the method comprises the following steps: enriching antibody drugs in a biological sample, and carrying out enzyme digestion to obtain a plurality of unlabeled modified peptide fragments; and mixing the unlabeled modified peptide fragment with an internal standard, enriching the modified peptide fragment, and then carrying out quantitative detection on the modified peptide fragment by adopting liquid chromatography-tandem mass spectrometry. A one-to-one structural analogue internal standard is provided for the modified peptide fragment, and the matrix effect is eliminated. Meanwhile, the method is combined with a high-pH reversed-phase grading equal peptide fragment enrichment method, so that the detection sensitivity of the modified peptide fragments in the matrix is improved. The technology provides a sensitive and reliable quantitative analysis method for the modified peptide fragment, and can be used for quantitative analysis of antibody drugs in biological samples.

The invention discloses a nano antibody for resisting a listeria monocytogenes MurA protein and application of the nano antibody, and relates to the technical field of biology. The amino acid sequence of the nano antibody is as shown in SEQ ID NO.3. MurA protein of prokaryotically expressed listeria monocytogenes is used as an immunogen to immunize alpaca, total RNA of peripheral blood lymphocytes is extracted, VHH fragments are amplified through reverse transcription and two rounds of nested PCR, the VHH fragments and a pComb3Xss vector are converted into TG1 competent cells after enzyme digestion and connection, and a phage display technology is used for three rounds of elutriation, so that the total RNA of the listeria monocytogenes is obtained. The nanometer antibody for resisting the listeria monocytogenes MurA protein is successfully screened out. The nano antibody can specifically recognize MurA protein and whole listeria monocytogenes, and the immunocolorimetric sensor based on thiolated bacteriophage induced colloidal gold aggregation, which is established by utilizing the nano antibody, is strong in specificity, high in sensitivity and low in detection limit, and can greatly shorten the detection time.

The invention provides a kit for detecting IDH1 gene mutation based on a CRISPR (clustered regularly interspaced short palindromic repeats) technology and application, and belongs to the technical field of biological detection. The invention develops a new application of the CRISPR technology in detection of IDH1 gene mutation. The CRISPR-Cas12a protein specifically recognizes and cuts a target sequence under the guidance of crRNA designed for an IDH1 target sequence, and meanwhile, powerful trans-enzyme digestion activity is exerted to cut non-specific single-stranded DNA (ssDNA), so that visual detection of a target gene is realized. According to the invention, the crRNA is optimized, so that the detection efficiency of IDH1-R132H mutation is improved, and the application prospect is wide.

The invention discloses a preparation method for preparing a concrete shrinkage reducing agent from modified natural biomass chitin as well as a product and application of the concrete shrinkage reducing agent. Natural chitin is subjected to enzyme digestion, esterification, deacylation, alcohol amination and copolymerization to prepare the shrinkage reducing agent. According to the preparation method of the shrinkage reducing agent, the application range of chitin is widened, the production process is simplified, and the material cost and solvent use are reduced. The modified chitin shrinkage reducing agent has better water solubility, higher ether group amount, higher epoxy group amount, higher grafting rate, higher charge amount, higher stability and higher wetting effect, and the adsorption of the shrinkage reducing agent and the volatilization of water in concrete gaps are reduced. Compared with the commercially available shrinkage reducing agent, the modified chitin shrinkage reducing agent solution has higher foaming capacity and lower surface tension, and the concrete doped with the modified chitin shrinkage reducing agent has shorter setting time, higher mechanical property and crack resistance and lower shrinkage value and chloride ion permeability. The prepared shrinkage reducing agent is green, environmentally friendly and high in performance, has multiple benefits of economy, society and environmental protection, and is wide in application prospect.

The invention provides a construction method, system and product of a false positive judgment model of a mutation site obtained by sequencing after enzyme digestion fragmentation and related application. Specifically, the false positive judgment model is constructed on the basis of a plurality of characteristic parameters associated with mutation sites obtained by enzyme digestion fragmentation sequencing; the characteristic parameters comprise scores obtained by performing reverse complementary sequence alignment on the front and back extension 30bp of the mutation site, the sequence proportion of 15 basic groups at the tail end of the mutation site, the Bayesian conditional probability of the extracted mutation site, and the KS test statistic of the extracted mutation site. Furthermore, the invention also provides a method for constructing the model by utilizing the crowd detection rate and the corresponding false positive. According to the method, the data characteristics of the false positive mutation sites are introduced by utilizing digestion fragmentation, so that the reliability of the digestion false positive mutation sites is objectively evaluated under the condition that high depth and large data volume are not needed, and the detection accuracy of the mutation sites can be remarkably improved.

The invention belongs to the technical field of biomedical engineering, and particularly relates to a polypeptide preparation method for preparing a GLP-1 analogue through tandem expression. According to the method, a coding gene sequence of the GLP-1 analogue is repeatedly connected in series for a plurality of times, and then expression preparation is carried out on the basis of a genetic engineering technology. The inventor optimizes a series-connected polypeptide structure by adding a section of key peptide sequence. The key peptide serving as a leading peptide can greatly improve the expression quantity of the strain in the fermentation expression process; as a linked peptide, the key peptide sequence can effectively improve the recognition capability of enzyme digestion sites on one hand and can adjust the overall isoelectric point of the sequence on the other hand, so that the solubility, enzyme digestion yield and purity of a target product can be improved. Preliminary experiment results show that the expression quantity of the polypeptide prepared through expression is extremely high, the enzyme digestion efficiency is good, the good enzyme digestion efficiency can be achieved under the condition that the enzyme dosage is low, and the yield and purity of the final GLP-1 analogue are high.

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker of a rice pre-harvest sprouting related gene SD6 and application of the SNP molecular marker. The polymorphism of the molecular marker is C or T. The molecular marker and the rice pre-harvest sprouting phenotype are co-separated, the rice pre-harvest sprouting gene SD6 can be quickly and accurately classified by utilizing the molecular marker and combining a KASP technology, the pre-harvest sprouting ability of a plant is judged in the rice seedling stage, the breeding cost is reduced, and the breeding efficiency is improved; according to the method, tedious procedures such as enzyme digestion, electrophoresis and sequencing are not needed in the detection process, and efficient and environment-friendly application of the SD6 gene to commercial molecular breeding of rice is facilitated.

The invention relates to an shRNA (short hairpin Ribonucleic Acid) interference sequence of a targeted silence PCSK9 gene and a construction method and lipid-lowering application of a recombinant adeno-associated virus vector of the shRNA interference sequence. Hyperlipidaemia is a metabolic disease characterized by abnormal rising of cholesterol and triglyceride levels in blood, and the design of lipid-lowering drugs is the focus of attention to improvement of hyperlipidaemia. Proprotein convertase subtilisin / kexin type 9 (PCSK9) can be combined with a low-density lipoprotein receptor (LDL-R) and degrade the LDL-R, so that accumulation of LDL-C in blood is further promoted, and hyperlipidemia is caused. Aiming at the key target PCSK9, a specific shRNA interference sequence is designed, and a recombinant adeno-associated virus vector (rAAV) carrying the sequence is constructed by an enzyme digestion-connection method. In-vitro experiments prove that the vector can remarkably reduce the expression level of PCSK9 protein, so that the cyclic utilization of a low-density lipoprotein receptor (LDL-R) is promoted, and the concentration of low-density lipoprotein cholesterol (LDL-C) in plasma is reduced. The rAAV vector provided by the invention has the characteristics of low production cost, high transfection efficiency, lasting action time and the like, and provides a new thought for gene therapy of hyperlipidemia.

The invention discloses a bubble self-stratification digital detection method and system for miRNA quantification of extracellular vesicles, and belongs to the technical field of biomedical engineering and molecular diagnosis. The technical problem to be solved is to provide a simple and sensitive tumor extracellular vesicle miRNA detection method capable of simultaneously detecting various miRNAs. According to the scheme, a to-be-detected sample and immunocapture bubbles coupled with an anti-EpCAM antibody are incubated to enrich tumor extracellular vesicles, a to-be-detected miRNA sample is obtained through in-situ ultrasonic lysis, then the to-be-detected miRNA sample, a fluorescence / DNA double-coding magnetic bead conjugate and the like are used for constructing an enzyme digestion system for enzyme digestion, multifunctional click glass microbubbles are added to capture unreacted magnetic beads, standing is conducted, self-stratification is conducted, bottom magnetic beads are collected, and the EpCAM antibody is obtained. After fluorescence imaging, an image is input into the AI automatic fluorescence counting and decoding module for counting to obtain a result. The method is used for detecting miR-21 and miR-155 in tumor extracellular vesicles, and is suitable for noninvasive early diagnosis of tumors.

The invention relates to a method for detecting deletion in ITR to be detected contained in an AAV vector to be detected. The method comprises the following steps: cutting a fragment containing a sequence of the ITR to be detected from the AAV vector to be detected by using restriction enzyme, detecting the size of the fragment of an enzyme digestion product, and determining whether deletion exists in the ITR to be detected according to the size of the fragment.

The invention provides a method for improving the expression level of a meat toxin light chain. The method comprises the following steps: connecting a dissolution-promoting tag to an N end or a C end of meat toxin light chain protein to form soluble meat toxin light chain recombinant protein; the dissolution-promoting tag is a wild dissolution-promoting tag NT11 or a mutant protein mut7 of the wild dissolution-promoting tag NT11, enzyme digestion removal is not needed, the activity of a meat toxin light chain is hardly influenced and interfered, and the fusion protein can keep 93% of substrate enzyme digestion activity. The method further comprises overexpressing a molecular chaperone htpG in the genome of the host bacterium. According to the invention, a BoNT / A-LC high-efficiency expression system is constructed through multi-strategy optimization, so that the yield of BoNT / A-LC is increased by nearly 20% compared with that of wild type NT11-Lc, the yield reaches 647 mg / L through overexpression of a molecular chaperone htpG, and after combinatorial optimization, the yield is 8.5 times that of only expressed BoNT / A Lc, 1.79 times that of NT11-Lc and 1.43 times that of Mut7-Lc.

The invention discloses a whole genome methylation detection method based on MeDIP-MSRE, and belongs to the technical field of epigenetics detection. According to the method, methylation immunoprecipitation sequencing and a methylation sensitive restriction enzyme technology are innovatively combined, firstly, a methylation specific antibody is used for conducting immunoprecipitation on sample DNA, and whole genome methylation fragments are enriched; then carrying out enzyme digestion on the enriched product by adopting methylation sensitive restriction enzyme, specifically removing an unmethylated DNA region, and reserving a complete methylation sequence; and finally, constructing a methylation map through high-throughput sequencing. According to the method, traditional hydrosulfite chemical conversion is not needed, DNA damage and base conversion deviation caused by the traditional hydrosulfite chemical conversion are avoided, meanwhile, high sensitivity of MeDIP and high specificity of methylation sensitive restriction endonuclease are fused, and the fidelity, sensitivity and specificity of detection are remarkably improved.

The invention belongs to the technical field of detection, and particularly relates to a nucleic acid detection method based on triple amplification and application of the nucleic acid detection method in ginseng identification. Mixing the liquid to be detected with the hairpin DNA, and assembling to form DNA enzyme; a hairpin substrate is added, enzyme digestion reaction is completed, and a trigger chain is generated; adding a circular DNA probe, carrying out rolling circle amplification, and collecting generated long single-stranded DNA; a G quadruplex structure in the long single-stranded DNA is utilized to catalyze a chromogenic reaction, and the concentration of miRNA in the to-be-detected liquid is analyzed through the RGB value of the color. According to the method disclosed by the invention, the low detection limit of the 651 fM is realized. The analysis of ginseng samples and related products verifies the effectiveness of the triple amplification platform, and a portable and reliable solution is provided for food safety supervision and quality control.

The invention relates to the technical field of biochemistry, and discloses a DNA chimera preparation and application method, which comprises the following steps: synthesizing phosphorylated linear annular L-rTDNA and template DNA, mixing in a 1 * T4 DNA ligase buffer solution according to an equal molar concentration of 10 [mu] M, heating at 95 DEG C for 5 minutes, and then carrying out gradient cooling to room temperature for renaturation for 120 minutes; adding T4DNA ligase to close a tail end notch, and carrying out enzyme digestion treatment to obtain annular rTDNA; the method comprises the following steps: synthesizing hairpin DNAHP (deoxyribonucleic acid), mixing rTDNA and HP according to an equal molar concentration of 2 mu mol / L, denaturating at 95 DEG C for 5 minutes, slowly cooling to 25 DEG C within 2 hours, and self-assembling to obtain the DNA topology limited chimera. According to the invention, a DNA chimera with dynamic response capability is constructed, an integrated detection process of enzyme-free assembly-target triggering-signal cascade amplification is realized, and meanwhile, the problems of anti-interference and ultra-sensitive detection in a complex matrix are solved.

The invention relates to the technical field of crop breeding science, in particular to a molecular marker closely linked with a wheat powdery mildew resistance gene PmNJ3930 and application of the molecular marker. The marker closely linked with the PmNJ3930 is a dCAPS molecular marker, and an upstream primer sequence and a downstream primer sequence for amplifying the molecular marker are provided. A product amplified by the primer is 354 bp after being subjected to NcoI enzyme digestion, which indicates that the wheat to be detected contains the PmNJ3930 gene and is a wheat variety with high powdery mildew resistance. When the primer pair provided by the invention is used for detecting the PmNJ3930 gene, whether the PmNJ3930 gene exists or not and the existence state of the PmNJ3930 gene can be determined, and the powdery mildew resistance of wheat is predicted, so that plants carrying the PmNJ3930 gene are quickly screened and are used for breeding disease-resistant varieties.