43 results about "Proteinase K" patented technology

The invention belongs to the technical field of molecular diagnosis, and particularly discloses a high-risk HPV16 rapid detection system based on RPA-CRISPR / Cas12a, which is prepared by the following steps: (1) mixing a nucleic acid lysis solution containing Tris (pH = 8.0), NaHCO3, polyvinylpyrrolidone and Tween-20 with Proteinase K to treat a sample, and performing pyrolysis at room temperature for 5 minutes to obtain a nucleic acid template; (2) an RPA reaction system contains an RNA inhibitor, a reverse transcriptase and an RPA reaction essential enzyme, and after the template and the starting agent in the step (1) are added, RPA amplification is carried out at 37 DEG C; (3) dropping the reaction liquid on a colloidal gold test strip subjected to position exchange and transformation of a sample pad and a water absorption pad, and carrying out result interpretation; and (4) adding the RPA product into a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reaction system containing the LbCas12a protein, the specific gRNA and the probe. According to the scheme, a rapid, high-sensitivity and cross-reaction-free detection system of the HPV16 is realized by optimizing a nucleic acid lysis solution formula, designing a specific primer and gRNA and modifying a test strip for detection.

The present invention provides a cfDNA extraction kit and method. The cfDNA extraction kit provided by the present invention includes proteinase K, a lysis buffer, magnetic microbeads, and a binding buffer. By limiting the composition of the lysis buffer and the binding buffer, it can not only effectively improve the extraction efficiency of cfDNA, but also significantly increase the purity of cfDNA and effectively enrich cfDNA of 100-200 bp. In addition, the kit components are easily available and low-cost, and the extraction operation is simple and suitable for high-throughput processing.

The invention provides an in-vitro induction and purification method for Giardia lamblia cysts. The induction method is characterized in that Giardia lamblia trophozoite is cultured in a TYI-S-33 culture medium containing pulvis fellis suis to obtain the Giardia lamblia cysts. According to the in-vitro induction method for the Giardia lancea cysts, the pig gall powder is added into the TYI-S-33 culture medium to induce the Giardia lancea trophozoite, and it is found that compared with common ox gall powder, the proportion of converting the Giardia lancea trophozoite into the cysts can be remarkably increased, and the generation rate of the induced cysts stably reaches 50%; according to the giardia lamblia cyst purification method disclosed by the invention, protease K is utilized for purification, so that pure cysts can be obtained, long-term stability and activity can be maintained, and the cysts can be stably stored for several weeks to several months at 2-8 DEG C and can be prepared into giardia lamblia cyst standard substances; the method is used for method verification, quality control, disinfection efficiency research, filtration efficiency evaluation and the like.

A method for extracting DNA from Kappa longicornis, relating to the field of molecular biology, includes the following steps: grinding a Kappa longicornis sample into powder; adding lysis buffer preheated to 60–65°C to the powder and incubating at 60–65°C; adding Proteinase K for protein digestion; adding RNase A for RNA digestion; extracting with a mixture of chloroform and isoamyl alcohol, centrifuging, and collecting the supernatant; adding pre-cooled isopropanol to the supernatant for precipitation, centrifuging, and obtaining the DNA precipitate; washing and drying the DNA precipitate; and dissolving the DNA in TE buffer or nuclease-free water. The beneficial effects of this invention are as follows: by optimizing the lysis buffer and combining it with stepwise enzymatic hydrolysis and organic extraction processes, polysaccharides and pigments of *Kappa longicornis* can be efficiently removed, and the extracted DNA concentration is >300 ng / μL, with a purity of A260 / A280≈1.76 and A260 / A230≈2.21, meeting the requirements of downstream molecular experiments; at the same time, reduced glutathione is used instead of 2-mercaptoethanol, making the operation safe and odorless, with low reagent costs, and suitable for batch sample processing.

This invention relates to a method and composition for dispersing biological tissues with the aim of obtaining single cells for analysis. More specifically, the composition of the present invention is an enzyme composition comprising proteinase K. The composition of the present invention can be used in a method for effectively dispersing tissues at low temperatures for applications in high-throughput single-cell omics research.

The present invention discloses a type IV collagen detection kit and a preparation method thereof, relating to the technical field of in vitro diagnosis. The kit comprises a reagent R1 and a reagent R2, wherein the volume ratio of the reagent R1 to the reagent R2 is 3:1. The present invention improves the sensitivity and accuracy of detection while ensuring the stability and repeatability of the kit through a carefully designed reagent formula, in particular the precise proportion of the components in the reagent R1, such as ammonium chloride, sodium azide, and Brij‑35. In particular, the reagent R1 introduces a natural plant extract, astragalus polysaccharide, and a proteinase K inhibitor as key components. The unique preparation method and the activity-promoting effect on type IV collagen hydrolase significantly enhance the biological activity of the kit, making the detection results more reliable. In addition, the latex particles coated with two paired mouse monoclonal antibodies used in the reagent R2 have high specificity and affinity, further improving the accuracy and sensitivity of detection.

The invention discloses a kit and a method for extracting nucleic acid from an ophthalmic sample and application of the kit and the method in metagenome sequencing. A method of extracting nucleic acid from an ophthalmic sample includes extracting nucleic acid from the ophthalmic sample using a sample processing system comprising the ophthalmic sample, a carrier RNA, protease K, and a lysis reagent. According to the method, a carrier RNA is added to prevent loss of trace nucleic acid, high-concentration protease K is adopted to enhance digestion of protein, and a chemical reagent is adopted to lyse cells, so that excessive damage and degradation of nucleic acid and low-volume elution caused by severe oscillation of mechanical wall breaking are avoided, and the final concentration of nucleic acid is improved.

The invention relates to the technical field of virus extraction kits, in particular to a large-volume sample virus nucleic acid rapid extraction kit and method.The large-volume sample virus nucleic acid rapid extraction kit comprises a protease K solution, magnetic beads, a lysis solution, a rinsing solution 1, a rinsing solution 2 and an eluent; the magnetic beads are hydroxyl magnetic beads; the lysate is prepared from guanidine isothiocyanate, Tris alkali, Tris hydrochloric acid, triton X-100, isopropyl alcohol and polyoxyethylene ether; the rinsing liquid 1 is composed of a guanidine hydrochloride solution and isopropanol; the rinsing liquid 2 is composed of absolute ethyl alcohol and purified water; the kit provided by the invention can easily obtain viral nucleic acid from a large-volume sample, is simple to operate, can extract a plurality of samples at one time, realizes rapid diagnosis, and makes up the vacancy of a kit for automatically extracting viral nucleic acid from a large-volume sample at high flux in the current market.

Capture mixtures and activated capture mixtures are provided that are useful for nucleic acid separation and purification are provided. The mixtures comprise lithium lauryl sulfate, lithium hydroxide, a zwitterionic sulfonic acid buffering agent, and optionally, proteinase K, capture probes comprising a first specific binding partner (SBP), and a second specific binding partner immobilized to a solid support. Related combinations, methods, uses, and kits, are also provided.

The application discloses a DNA / RNA co-extraction method and kit based on a double-water-phase temperature-induced phase separation, which utilizes polyethylene glycol-sodium citrate to form a primary double-water-phase system at 18-25 DEG C, and by adding an ionic liquid modified temperature-sensitive polymer (IL-NIPAM) with a LCST of 32 DEG C plus or minus 1 DEG C, the IL-NIPAM selectively binds with RNA, and the separation of RNA and DNA in a biological sample is realized by temperature regulation. The DNA / RNA co-extraction kit includes a double-water-phase premix, and the IL-NIPAM is added in the double-water-phase premix in advance or during use, and the use concentration of the IL-NIPAM is 0.5-1.5% w / w. The method does not need proteinase K digestion and does not need to add organic solvents, and can quickly and efficiently recover DNA, mRNA and miRNA, and is suitable for the co-extraction of nucleic acids from trace samples such as fingertip blood, dry blood spots and FFPE sections, and is suitable for automation and large-scale production.

The present invention relates to methods and compositions for dissociating biological tissue with the aim of obtaining single cells for analysis. More specifically, the compositions of the invention are enzyme compositions comprising protease K. The compositions of the invention are useful in methods for efficiently dissociating tissue at lower temperatures for application in high throughput unicellular omics studies.

This invention relates to the field of nucleic acid release, specifically disclosing a rapid nucleic acid release reagent, kit, method, and application for animal tissue genotyping. The release reagent comprises a weakly alkaline reaction buffer, an enzyme-catalyzed reaction enhancer, a nonionic surfactant, a nucleic acid amplification enhancer, a first component composed of histidine, trehalose, and glutathione in a specific ratio, and a second component with a visual indicator function; a matching thermosensitive proteinase K formation kit is also included. The kit can be pre-packaged in multi-well reaction plates for improved ease of use. The release method of this invention achieves rapid release of nucleic acids from animal tissues through pre-incubation activation, mild-temperature lysis, and high-temperature enzyme inactivation steps. Simultaneously, the first component exhibits a strong synergistic effect with the thermosensitive proteinase K, effectively protecting the integrity of the nucleic acids.

The invention relates to the technical field of drug detection, in particular to immobilized protease K and application thereof in hair drug detection. The immobilized protease K comprises zirconia ceramic balls and protease K which is covalently connected with the zirconia ceramic balls through APTES amination; wherein the loading capacity of the protease K is 1-5mg / g. The specific detection method comprises the following steps: carrying out milling and enzyme digestion on to-be-detected hair and the immobilized protease K zirconia ceramic ball, and carrying out fluorescence immunochromatography on the obtained supernatant to detect the narcotics and metabolites thereof. According to the application, covalently solidified protease K is adopted, the operation stability is excellent, 50 DEG C high temperature and mechanical oscillation can be resisted, 85-95% efficient release of traditional difficult samples such as weak positive hair and dark hair can be realized, the detection rate and the detection accuracy of drugs and metabolites thereof are improved, and the false negative risk is reduced; the requirements of laboratory high-throughput detection and on-site rapid screening are met.

The invention relates to a preparation method of a narrow gadus bone peptide compound and chelated calcium of the narrow gadus bone peptide compound. The method comprises the following steps: by taking a walleye cod processing byproduct fish bone as a raw material, carrying out trypsin preliminary enzymolysis, ultrafiltration and protease K targeted enzymolysis to obtain the walleye cod bone peptide compound. The narrow gadus bone peptide compound comprises one or more active peptides of DFF, DIF, DNAF, DPF and DYF. Chelated calcium can be prepared by using the narrow gadus bone peptide compound or the narrow gadus bone active peptide. Waste resources are fully utilized, high value of low-value products is achieved, environmental pollution is reduced, high economic and social benefits are achieved, and the prepared chelated calcium with the high additional value can be applied to the fields of dietary nutrition, biological medicine products and the like.

This invention discloses a preservation solution for preserving sample DNA at room temperature and its preparation method, belonging to the field of biological sample preservation technology. The preservation solution comprises: a lysis system, a nucleic acid protection system, and a buffering and stabilizing system; the lysis system comprises a complex surfactant and an enzymatic hydrolysis aid; the complex surfactant comprises: polyether polyol fatty acid ester and cocamidopropyl hydroxysulfonate betaine; the enzymatic hydrolysis aid comprises lysozyme Lyso-V and proteinase K; the nucleic acid protection system comprises: nitrogen-heterocyclic polyamine-carboxylic acid derivatives and dextran sulfate; the nitrogen-heterocyclic polyamine-carboxylic acid derivatives comprise: 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''tetraacetic acid, 1,4,7-triazacyclononane-N,N',N''-triacetic acid, disodium ethylenediaminetetraacetate-nitrocyclic derivative, and diethylenetriaminepentaacetic acid-piperazine derivative; the buffering and stabilizing system comprises: an amphoteric buffer and a polymeric stabilizer; the amphoteric buffer comprises: 2-(N-morpholino)ethanesulfonic acid and N-tris(hydroxymethyl)methylglycine.

This invention belongs to the field of biotechnology and discloses a plasma cell-free DNA extraction kit, extraction method, and applications. The kit includes magnetic beads, lysis buffer, proteinase K, binding buffer, washing buffer, and elution buffer. The extraction method involves mixing the plasma sample, lysis buffer, proteinase K, binding buffer, and magnetic beads. The lysed plasma cell-free DNA (cfDNA) is incubated with the magnetic beads to form a magnetic bead-cfDNA complex. Under a magnetic field, the complex is washed to remove various impurities, and the plasma cfDNA is obtained after elution. The method of this invention is simple and rapid, with short sample lysis time and stable quality. Replacing isopropanol with anhydrous ethanol during lysis results in high nucleic acid recovery, making it suitable for plasma cfDNA extraction. The purity is suitable for subsequent sequencing and PCR / Q-PCR. The magnetic bead method is beneficial for large-scale use and has significant implications for research and clinical applications.

The invention relates to the technical field of molecular biology, and particularly discloses a kit for extracting viral nucleic acid and application of the kit. The kit comprises a lysis solution, a cleaning solution 1, a cleaning solution 2, an eluent, protease K dry powder, a protease K diluent and a magnetic bead solution. The kit adopts a magnetic bead nucleic acid extraction method to solve the problems of tedious extraction steps, complex operation, large harm to human and the like of a traditional method, and compared with an existing extraction kit, the kit has higher nucleic acid extraction efficiency.

The invention relates to the technical field of biology, in particular to a nucleic acid extraction reagent composition and application thereof. The invention provides a reagent combination simultaneously adaptive to common nucleic acid extraction and electrowetting platform adaptive nucleic acid extraction, according to the reagent combination provided by the invention, high-concentration guanidinium isothiocyanate is added into a lysis solution, heating is performed in a lysis step to assist lysis, lysis and nucleic acid adsorption are synchronously performed at the same time, and the extraction time is shortened; a surfactant with proper concentration is added into the lysis solution, the washing solution and the eluent, so that the reagent combination can be better driven on an electrowetting platform, the driving force is improved, and the uniform mixing effect is improved; meanwhile, protease K is added into the washing liquid, the common step of washing protein with high-concentration salt in a magnetic bead extraction method is omitted, a good washing effect can be achieved only through one-step washing, the extraction time is further shortened, meanwhile, residual protease K in the washing liquid is inactivated through high-temperature elution, follow-up amplification is not affected, and the method is suitable for application and popularization.

The invention discloses a liquid chromatography-tandem mass spectrometry determination method for altrenogest in pig tissue, which comprises the following steps: sample pretreatment: enzymolysis treatment: weighing 1.0 g of pig tissue sample which is pig liver, pig muscle or pig skin adipose tissue; a protease K solution is added into the pig tissue sample, pig liver or pig muscle tissue is subjected to enzymolysis for 1 h in a dark water bath at the temperature of 37 DEG C, and pig skin adipose tissue is subjected to enzymolysis for 30 min in a dark water bath at the temperature of 50 DEG C; performing ultrasonic treatment for 2 minutes after enzymolysis, and cooling to room temperature; according to the method, the purification efficiency and the specificity are greatly improved, and the problem of matrix interference is effectively solved. The MIP-MNPs prepared by taking altrenogest as a template molecule can specifically adsorb altrenogest and metabolites thereof through spatial structure matching and functional group action, so that non-specific interference of protein, fat and steroid impurities in pig tissues is eliminated, and the ionization inhibition effect of a matrix on mass spectrometry detection is remarkably reduced.

The application provides a method for improving the detection rate of norovirus in aquatic products, which comprises the following steps: first, using a proteinase K solution to enzymolysis the digestive gland of shellfish, then adding a TGBE solution to elute, then adding enzymolysis corn starch to block activated carbon to remove the RT-qPCR inhibitor in the detection sample, taking the supernatant to extract nucleic acid, and using a fluorescent quantitative PCR instrument to detect. The detection method of the application reduces the inhibitor in the shellfish sample which interferes with the fluorescent quantitative PCR by using enzymolysis corn starch. In combination with the proteinase K enzymolysis, the TGBE elution and the RT-qRCR, the norovirus in the shellfish sample is detected, and the detection rate of the shellfish sample is increased by 10-20 times in 4h compared with the national standard method. The virus recovery rate and the detection sensitivity are greatly improved, and the method is suitable for shellfish samples, and has the characteristics of simple operation, high detection sensitivity and short time consumption.

The invention relates to the field of biological detection, in particular to a nucleic acid extraction reagent, a kit and application thereof. The invention provides a cracking adsorption reagent. The cracking adsorption reagent comprises a component 1, a component 2 and a component 3, the component 1 comprises a magnetic bead mixed solution; the component 2 comprises protease K; the component 3 comprises a lysis solution with the pH value of 6-10; the lysate comprises chaotropic salt, salt ions, polyethylene glycol, a surfactant, a metal chelating agent, isopropanol and a buffer solution. According to the rapid nucleic acid extraction kit and the nucleic acid extraction method provided by the invention, the nucleic acid extraction efficiency is greatly improved, the application range is widened through technical innovation, the risk of cross contamination is reduced, the preservation capacity of nucleic acid is improved, and the rapid nucleic acid extraction kit can be adapted to automatic equipment for extraction; and a more efficient, convenient and universal solution is provided for multiple fields of biomedical research, disease diagnosis, epidemic prevention and control and the like.

The invention belongs to the technical field of molecular biology and in-vitro diagnosis, and particularly discloses a lysis solution composition and application thereof in preparation of a reagent for extracting genome DNA. The lysate composition comprises the following components: a pH regulator, 0.05-1.0% (v / v) of a nonionic surfactant, 0.1-2 mg / mL of bovine serum albumin, 0.5-5 mM of a reducing agent and 50-500 g / mL of protease K, and the pH value of the lysate composition is 7.0-8.0. The invention provides the genome DNA quick release lysate which is reasonable in composition and simple and convenient to operate, efficient release and stable storage of DNA in a complex biological sample are realized under the condition that a traditional purification step is not needed, and the obtained DNA can be directly used for nucleic acid amplification detection such as PCR (Polymerase Chain Reaction) and LAMP (Loop-Mediated Isothermal Amplification), so that the detection efficiency, repeatability and application convenience are improved; the actual requirements of rapid molecular diagnosis and on-site detection are met.

This invention provides a nucleic acid extraction and release agent for the detection of multiple pathogenic microorganisms, comprising the following components: Tween 20, proteinase K, EDTA, and KOH. The rapid nucleic acid extraction and release agent of this invention, after incubation with the sample, completes the nucleic acid extraction and release process, improving extraction efficiency, reducing reagent and sample usage, simplifying operation steps, and lowering labor costs. It has advantages such as high efficiency, low cost, and high quality.

This invention belongs to the field of food processing technology, specifically relating to an oat bran β-glucan extract, its preparation method, and its application. The preparation method involves obtaining the oat bran β-glucan extract from oat bran through multi-stage enzymatic hydrolysis. The enzymes used in the multi-stage hydrolysis sequentially include pentosanase, thermostable α-amylase, and proteinase K. This invention, under ultrasound assistance, allows pentosanase to efficiently break down cell walls first, significantly increasing the release rate of β-glucan. Subsequently, thermostable α-amylase and proteinase K precisely remove starch and protein impurities sequentially, greatly improving the purity of the product. This process route is mild, highly specific, and operates continuously, reducing impurity residue while improving extraction efficiency, providing a reliable and cost-effective solution for the large-scale preparation of high-purity, highly bioactive oat β-glucan.

This invention provides a nucleic acid extraction reagent, kit, and method. The nucleic acid extraction reagent includes a pretreatment solution, proteinase K, lysis binding buffer, magnetic beads, washing buffer 1, washing buffer 2, washing buffer 3, and elution buffer, all stored separately but used in combination. The pretreatment solution has a pH of 7.80-8.20 and is composed of 0.1%-1% sodium dodecyl sulfate, Tris-HCl solution, and EDTA-Na2 solution by mass. The lysis binding buffer has a pH of 7.80-8.20 and is composed of 3M-4M guanidine isothiocyanate, 11%-15% polyethylene glycol 6000 by mass, 5%-10% Triton X-100 by mass, Tris-HCl solution, and EDTA-Na2 solution. This invention achieves "high concentration, high purity, and high efficiency" nucleic acid extraction through innovative extraction reagent components (non-toxic lysis system), reagent kit design (high-throughput pre-packaging), method optimization (automated coupling), and application expansion (adaptation to frozen samples), providing a reliable template for molecular diagnostics.

The application provides an alcohol-free and proteinase K-free nucleic acid extraction reagent and a nucleic acid rapid extraction method, and relates to the technical field of molecules.The extraction reagent comprises a lysis solution, and the lysis solution comprises guanidine isothiocyanate with a molar concentration of 2-4 M, a thiol reducing agent with a volume fraction of 1%-5% v / v, sodium erythorbate with a mass fraction of 0.02%-0.5% w / v and a surfactant with a mass fraction of 1%-10% w / v; and the lysis solution has a pH of 7-8.5.The lysis solution can be stably stored at room temperature for a long time; in application, only one washing solution is matched, one-step washing is performed, the complexity of nucleic acid extraction can be reduced, and the extraction time is shortened; and the lysis solution does not contain alcohol reagents, and there is no need to dry before nucleic acid elution.The technical problems that, in the prior art, the lysis solution has complex components, proteinase K needs to be matched, many impurities are introduced, multiple washing is needed, the steps are complicated, and a long time is consumed are solved.

Capture mixtures and activated capture mixtures are provided that are useful for nucleic acid separation and purification are provided. The mixtures comprise lithium lauryl sulfate, lithium hydroxide, a zwitterionic sulfonic acid buffering agent, and optionally, proteinase K, capture probes comprising a first specific binding partner (SBP), and a second specific binding partner immobilized to a solid support. Related combinations, methods, uses, and kits, are also provided.

This application relates to the field of biotechnology, specifically disclosing a method for extracting trace DNA from soil. This application utilizes extraction buffer and proteinase K to gently incubate and lyse soil samples, releasing DNA adsorbed on the soil and intracellular DNA into the extraction solution. Protein removal is performed using phenol-chloroform-isoamyl alcohol nucleic acid extraction buffer, followed by centrifugation, filtration, and elution using ultrafiltration centrifuge tubes to obtain an extract containing the target DNA. This method can efficiently extract trace amounts of environmental DNA or metaDNA from soil, and is particularly suitable for DNA extraction in extreme environments where biological survival is difficult, providing technical support for DNA-based biological research in extreme habitats.

The invention relates to the technical field of cell culture and biological materials, and discloses a morphology-adjustable three-dimensional cell culture scaffold and a preparation method and application thereof.The preparation method comprises the following steps that oligopeptide Fmoc-FF is dissolved in an organic solvent to prepare an Fmoc-FF solution, the Fmoc-FF solution and a protease K aqueous solution are mixed, and a three-dimensional cell culture scaffold is obtained; the self-assembly process of the Fmoc-FF is initiated, and hydrogel is formed; after the Fmoc-FF forms hydrogel, protease K in a system is removed by utilizing the shear thinning characteristic of the hydrogel, and the Fmoc-FF nanofiber which is free of protease K residues and has a specific microstructure is obtained. According to the method, the diameter and length of the self-assembled nanofiber are adjusted by changing the adding concentration of protease K under the enzymatic hydrolysis effect of protease K on Fmoc-FF dipeptide. The method avoids the problem of high coupling of micro-morphology and macro-mechanical properties in a traditional collagen hydrogel system.

The present application relates to a kind of extraction free DNA reagent based on suction filtration method, kit and extraction method.The kit contains reagent and adsorption column;The reagent includes lysing solution, proteinase K, protease solution, CarrierRNA, binding liquid, washing liquid and eluent;The adsorption column is the silica gel membrane adsorption column that can be used by suction filtration.The present application does not need centrifugal equipment, and the extraction and purification of DNA can be completed by suction filtration, with the characteristics of simple operation, low cost, high extraction rate, low safety risk, suitable for the extraction of free DNA in serum, plasma and other acellular samples.