57 results about "Enzymic digestion" patented technology

The invention relates to a method for rapidly detecting the cell adhesiveness of probiotics and application of the method. The method comprises the following steps: adding the probiotics into adherent culture cells, incubating, discarding liquid, digesting and collecting the cells by pancreatin, extracting total DNA (Deoxyribonucleic Acid), performing qPCR (Quantitative Polymerase Chain Reaction) amplification, and calculating the adhesion quantity of the probiotics on a single cell according to a Ct value. The invention provides a method for rapidly detecting the cell adhesion rate of probiotics by using a qPCR (quantitative polymerase chain reaction) technology, which comprises the following steps of: extracting total genome DNA (deoxyribonucleic acid) after the probiotics are adhered to cells on the basis of the gene expression difference of prokaryotic and eukaryotic cells, and detecting the specific gene expression level of the probiotics and the cells by using the qPCR technology. The cell adhesion ability of the probiotics is evaluated by calculating the ratio of the gene expression quantities of the two, and the method has the characteristics of high efficiency, rapidness, high accuracy and the like.

A system for targeted delivery of a medicament to the gastrointestinal tract (e.g., selectively to the duodenum) of a subject. The system includes a medicament, a mucoadhesive layer at least partially surrounding the medicament, and an enteric coating encasing the medicament and mucoadhesive layer. The enteric coating comprises a material degradable by a pH of the GI tract and a material digestible by a lipase. Also provided are method of making and using the system for targeted delivery of a medicament to the gastrointestinal tract. Also provided are a system for deploying a medicament-containing needle system using an expanding element. The system includes a medicament-containing needle system and an expanding element within a capsule. The capsule is initially coated with an enteric coating. As the enteric coating degrades the expanding element absorbs fluid that enters the capsule and expands to deploy the medicament-containing needle system from the capsule.

The present invention relates to methods for producing a protected deoxyribonucleic acid (DNA) product with enhanced resistance to nuclease digestion. The methods comprise digesting a double-stranded DNA molecule with an endonuclease to generate a digested double-stranded DNA molecule comprising a first strand and a second strand, wherein the first strand has a 5' overhang; and extending the second strand 5' to 3' using a polymerase in the presence of one or more nuclease-resistant deoxynucleotide triphosphates to generate the protected DNA product, wherein the protected DNA product comprises an extended second strand comprising at least one nuclease-resistant nucleotide incorporated into the extended second strand by the polymerase using the 5' overhang of the first strand as template. Also provided are protected DNA products and uses thereof.

The disclosure relates to methods of detecting Host Cell Proteins (HCPs) in biopharmaceutical samples containing a drug product. More specifically, the disclosure relates to methods of assaying HCPs in biopharmaceutical samples using enzymatic digestion protocols, liquid chromatography (LC) gradients, immunoaffinity capture protocols, and High-Resolution Mass Spectrometry (HRMS) to enhance the recovery, detection, and profiling of HCPs, which can impact the efficacy, shelf-life, and therapeutic response of drug products.

The invention discloses a culture medium for identifying gardnerella vaginalis, and relates to the technical field of bacterial culture media, the key points of the technical scheme are as follows: the culture medium comprises the following raw material components by concentration: 10.0 g / L of casein pancreatin digest, 3.0 g / L of cardiac pancreatin digest, 5.0 g / L of yeast extract powder, 1.0 g / L of corn starch, 5.0 g / L of sodium chloride and 13.0 g / L of agar, and the final concentration of defiberized sheep blood is 5%. The pharmaceutical composition is prepared from 1 mg / mL of sodium alginate, 1 mg / mL of sodium alginate, 1 mg / mL of polyanethole Sulfonate (SPS) and 10 [mu] g / mL of vancomycin. According to the invention, through optimal combination of specific components and dosages thereof, rapid identification and screening of gardnerella vaginalis can be realized, and the method has the advantages of accurate result, strong specificity and the like, and has practical popularization and application values.

A method of sequencing a nucleic acid characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one oligonucleotide used probe by reacting, in the presence of a polymerase, at least one of the single nucleoside triphosphates with a corresponding biological probe comprising (a) a first single-stranded oligonucleotide including an exonuclease blocking-site, a restriction endonuclease recognition-site located on the 5' side of the blocking-site and including a single nucleotide capture-site for capturing the single nucleoside triphosphate, and at least one fluorophore region located on the 5' side of the recognition-site arranged so as to render the fluorophore(s) quenched and (b) a second and optionally a third single-stranded oligonucleotide each separate from the first oligonucleotide and capable of hybridising to complementary regions on the first oligonucleotide flanking the 3' and 5' sides of the capture-site; (3) cleaving the first oligonucleotide strand of the used probe at the recognition-site with a restriction endonuclease to create a first oligonucleotide component bearing the fluorophores; (4) digesting the first oligonucleotide component with an enzyme having 3'-5' exonucleolytic activity to yield fluorophores in a detectable state and (5) detecting the fluorophores released in step (4). A corresponding method for use with enzymes having 5'-3' exonucleolytic activity as well as new biological probes and a kit derived therefrom is also provided.

This invention relates to the interdisciplinary field of cell biology, experimental oncology, and translational medicine, specifically to a cancer-associated fibroblast cell line, its construction method, and its applications. This invention fills the research gap in mouse-derived oral squamous cell carcinoma-associated fibroblast cell line models and provides a method for constructing such a cell line. Through the inverted adherent culture and enzymatic digestion methods of this invention, a mouse-derived oral squamous cell carcinoma-associated fibroblast cell line with a clear origin, high purity, and long-term phenotypic and functional stability can be obtained. This allows for the establishment of a cryopreservable and resuscitable mouse oral squamous cell carcinoma-associated fibroblast cell bank, providing technical support for research related to the tumor microenvironment and immunotherapy.

Described herein are methods for enriching, identifying, quantifying, and / or characterizing a protein of interest in a complex biological sample, such as a serum sample. The protein of interest may be enriched using a library of bead-based peptide ligands. The enriched protein of interest may subsequently be subjected to denaturation, reduction, and / or enzymatic digestion, to generate peptides which can be detected using liquid chromatography-mass spectrometry (LC-MS) techniques, and which are uniquely identifying of the protein of interest.

This invention discloses a method for producing single-stranded DNA using Cas nickase. This method solves the problem of limitations on the enzyme cleavage sites when using nicking endonucleases, and improves the nicking efficiency of plasmids by performing multiple nicking using multiple gRNAs in a single enzyme cleavage system, while also effectively improving the efficiency of subsequent digestion by exonucleases.

The invention relates to a propionibacterium acnes selective culture medium and a preparation method thereof, the culture medium comprises casein pancreatin digest, meat and stomach enzyme digest, heart and pancreatin digest, yeast extract powder, corn starch, sodium chloride, agar and defiberized sheep blood, and the volume is fixed with distilled water. Compared with an existing culture medium, the culture medium has the advantages of being reliable in detection and high in propionibacterium acnes separation rate and has a good application prospect.

Compositions and methods are provided, for preparing DNA libraries for high- throughput sequencing following methylation-sensitive and / or methylation-dependent enzymatic digestion of the DNA. The provided compositions and methods obviate the need to clean-up the DNA sample between the digestion step and subsequent library preparation steps. The provided compositions and methods are particularly advantageous for library preparation from small amounts of DNA, such as cell-free DNA from body fluid samples.

The invention provides inter alia a method for enzymatic digestion of an extracellular matrix protein in a tissue sample, comprising the steps of:—providing an aqueous medium that comprises (i) a matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of at least 2 mmol / L;—contacting said aqueous medium with a tissue sample that comprises an extracellular matrix protein under conditions that allow for enzymatic digestion of said extracellular matrix protein; wherein said tissue sample is a cartilage tissue sample.

Provided is a method for constructing a gE gene-deleted pseudorabies virus (PRV) strain using an adenine base editor (ABE) and use thereof. The method includes: designing an sgRNA sequence using the ABE with a start codon of the gE gene in a PRV as a target site, ligating an enzyme-digested fragment to a double-stranded DNA fragment with sticky ends to obtain a ligation product; and transforming the ligation product into a competent cell to allow plate screening and culture, selecting a resulting positive bacterial strain to allow expanded culture, and extracting a plasmid from a resulting positive bacterial solution; and transferring the plasmid into a target cell to allow the transfection for 24 h, collecting a resulting virus liquid, and centrifuging the virus liquid to collect a resulting supernatant.

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 present disclosure relates to a method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) reverse transcription of the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain a test cDNA fragment; (e) separating the test cDNA fragments so as to form a test cDNA fragment map; and (f) comparing the test cDNA fragment map to a control cDNA fragment map wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment map comprises the control cDNA fragment map. Also disclosed are methods of quantifying and / or assessing the integrity of at least one mRNA molecule in a composition, processes of making a composition comprising at least one mRNA molecule and a primer pair for use in the methods of the invention.

The invention discloses a rolling circle amplification method and application of the rolling circle amplification method in improvement of nanopore sequencing accuracy. In the construction process of the nanopore sequencing library, two ends of a to-be-detected target linear double-stranded DNA sample are connected with two joints with nick enzyme recognition sites, the joints are digested through the nick enzyme to generate two cohesive ends which are complementarily paired, and the two cohesive ends are connected, so that the target linear double-stranded DNA sample is obtained. According to the method, self-cyclization of a to-be-detected sample is realized to obtain a double-stranded circular DNA as a template for rolling circle amplification, a forward primer and a reverse primer are designed for the template to carry out rolling circle amplification reaction to obtain a nanopore sequencing library, and experiments prove that the nanopore sequencing accuracy can be improved to 99.9% by the method disclosed by the invention; and a longer DNA fragment product can be generated by utilizing rolling circle amplification, and the DNA fragment product is used for nanopore sequencing and the yield of a nanopore sequencing library is improved.

The present disclosure provides a method and composition for preparing a nucleic acid library.In some embodiments, the nucleic acid comprises cell-free DNA, including cfDNA that requires analysis, for example, by sequencing.The method can include restriction enzyme digestion, adapter ligation, and subsequent amplification, and can provide an improved approach to reduce the number of adapter-dimers generated during the process.In some aspects, the method for preparing a nucleic acid library can include: digesting DNA molecules with restriction enzymes to generate DNA fragments; ligating adapters to the DNA fragments by incubating with ligase to generate a mixture of adapter-ligated DNA fragments and adapter-dimers; amplifying the adapter-ligated DNA fragments to generate amplified adapter-ligated DNA fragments; and reducing the amount of adapter-dimers by distinguishing between the junction between adapter and DNA fragment and the junction between adapter and another adapter. TIFF2022530289000002.tif98128

A heme iron production process is disclosed. This process includes operations of separating red blood cells from whole blood via centrifuge, collecting the red blood cells, transferring the red blood cells into high speed centrifuges, centrifuging at a speed greater than 6000 rpm, combining effluent red blood cells with reverse osmosis water, increasing temperature and pH of the water, enzymatically digesting the effluent red blood cells into heme iron molecules and waste, and separating the waste from the heme iron molecules.

To enable high-sensitivity detection of peach that might cause allergies contained in food ingredients and products even if the amount is minute.SOLUTION: A method of detecting peach is provided, comprising extracting protein from a sample, obtaining an enzyme digest of the extracted protein using a protease, and analyzing the enzyme digest to detect at least one peptide selected from a group consisting of SEQ ID NOs: 1 through 3 using a mass spectrometer so as to qualitatively or quantitatively determine the presence or absence of peach protein in the sample.SELECTED DRAWING: Figure 1

The invention relates to the field of edible mushroom substrates, in particular to a treatment method for improving the utilization rate of an edible mushroom substrate. The method comprises the following steps: uniformly spraying a chemical reagent solution on lignocellulose, compacting and granulating, and carrying out stacking reaction to obtain a lignocellulose material; and mixing the lignocellulose material with other auxiliary materials to prepare the edible mushroom substrate for edible mushroom cultivation. According to the method, chemical reagents are added, densification granulation treatment is combined, and the enzyme digestibility of the lignocellulose material is remarkably improved by destroying a complex lignocellulose structure in the lignocellulose raw material, so that hyphae of edible fungi more easily intrude into lignocellulose. According to the method, the cooperative relationship between the density of densification granulation treatment and the stacking time is further optimized, the stability of the physical structure of the edible mushroom substrate is improved, the utilization rate of the edible mushroom substrate is further improved, and finally the yield and the biological efficiency of the edible mushrooms are remarkably improved.

Disclosed herein are compositions, devices, and methods for isolating human Adipose-derived stem cells (ASCs) without either electricity or enzymatic digestion. In particular, a paramagnetic immunobead (PIB) for isolating human ASCs is disclosed. In some embodiments, the PIB displays on its surface one or more antibodies that selectively bind ASC-specific antigens. Also disclosed is a method for isolating human ASCs using the disclosed PIBs.

Preparation of a product comprising stromal vascular fraction cells includes washing human biological material comprising adipose in a container apparatus having an internal filter, which 5 divides an internal containment volume of the container apparatus into a tissue retention volume on one side of the filter and a filtrate volume on an opposite side of the filter, and a mixing device with at least one rotatable mixing member disposed in the tissue retention volume. The washing includes operation of the mixing device to rotate the mixing member through the human biological material within the tissue retention volume, and the washing is followed by digesting washed material within 10 the internal containment volume with added enzyme, centrifuging of the container apparatus to prepare a centrifuged pellet in the filtrate volume, selectively removing material of the pellet and preparing a product with a mixture of stromal vascular fraction cells of removed pellet material and an aqueous suspension liquid.

A method of sequencing a nucleic acid characterized by the steps of: (1) generating a stream of mononucleoside triphosphate by progressive enzymatic digestion of the nucleic acid; (2) producing at least one oligonucleotide-used probe by reacting at least one mononucleoside triphosphate with a corresponding biological probe in the presence of a polymerase, the corresponding biological probe comprising (a) a first single stranded oligonucleotide comprising an exonuclease closure site and (b) a second single stranded oligonucleotide comprising an exonuclease closure site; the present invention relates to a kit comprising (a) a single nucleotide triphosphate, (b) a closure site 5'side of the closure site and comprising a restriction endonuclease recognition site for capturing the mononucleotide triphosphate, and at least one fluorophore region located 5 'side of the recognition site and arranged to quench a fluorophore, and (b) second and optionally third single stranded oligonucleotides, their isolated from the first oligonucleotides, respectively, and capable of hybridizing to complementary regions on the first oligonucleotides flanking the capture sites 3'and 5 '; (3) cleaving the first oligonucleotide strand of the used probe at a recognition site with a restriction endonuclease to produce a first oligonucleotide component with a fluorophore; (4) digesting the first oligonucleotide component with an enzyme having 3 '-5' exonucleotide activity to produce a fluorophore in a detectable state, and (5) detecting the fluorophore released in step (4). Also provided are corresponding methods for use with enzymes having 5 '-3' exonuclease activity, as well as novel bioprobes and kits obtained therefrom.

The invention discloses a sequencing library for removing human nucleic acid and a construction method and application thereof, and the construction method comprises the following steps: fragmenting nucleic acid, adding a sequencing joint, constructing a DNA or RNA library, or co-constructing a library by RNA and DNA, taking the library added with the sequencing joint as a template, carrying out denaturation annealing on the library and a specific human hybridization probe, digesting by using DSN enzyme, and carrying out sequencing on the human nucleic acid. In the renaturation process, the human nucleic acid with higher content is firstly annealed with the human hybridization probe to form double strands, the double strands are degraded by DSN enzyme, the pathogenic microorganisms with low content and slow renaturation still keep a single strand state and cannot be digested by the DSN enzyme, the human nucleic acid is effectively removed, and the pathogenic microorganism nucleic acid is further enriched through subsequent PCR (Polymerase Chain Reaction). According to the method, the proportion of human nucleic acid in the library can be effectively reduced, and nucleic acid information of pathogenic microorganisms can be reserved to the maximum extent.

The invention relates to the technical field of modern biomedicine, in particular to a method for promoting differentiation of human umbilical cord mesenchymal stem cells based on an EVs composite material, which comprises the following steps: 1, preparing a double-layer composite scaffold; 2, preparation and characterization of EVs: primary cartilage cell culture, enzymatic digestion of cartilage tissue, digestion termination and cell collection, EVs extraction, and EVs characterization identification; 3, loading the EVs; 4, separation, identification and differentiation induction of the hUC-MSCs; and 5, differentiation effect detection. The method has the characteristics that the differentiation efficiency and stability are good, the EVs delivery efficiency is remarkably improved, the biocompatibility and safety are good, the operation is simple and convenient, the cost is controllable, and the application scene is flexible and wide.

The present invention discloses a method for amplifying double-stranded circular nucleic acids, comprising: mixing and incubating a CRISPR RNP complex with a double-stranded circular nucleic acid, wherein the double-stranded circular nucleic acid includes a first strand and a second strand, wherein the sgRNA can specifically bind to a target site on the first strand, and wherein the Cas9 nickase can cleave the second strand; adding a nuclease exonuclease to digest the second strand to obtain a nicked strand; separating the CRISPR RNP complex from the first strand; and using the nicked strand as a primer and the first strand as a template, using a polymerase to extend a nucleic acid complementary to the template from the primer. The double-stranded circular nucleic acid amplification method of the present invention has strong specificity and simple operation, can obtain a long single-stranded rolling circle amplification product, and has broad application prospects.

The present application discloses a method for obtaining maternal-derived mesenchymal stem cells from the placenta. The method for obtaining maternal-derived mesenchymal stem cells from the placenta in the present application includes obtaining placental tissue from a position within a radius of 5 cm from the umbilical cord in the placenta and more than 0.5 cm from the fetal surface; subjecting the placental tissue to enzymatic digestion treatment with an enzymatic digestion solution, and then culturing the product of the enzymatic digestion treatment to obtain maternal-derived mesenchymal stem cells. The method of the present application solves the technical problem of separating maternal-derived mesenchymal stem cells from the placenta. The prepared maternal-derived mesenchymal stem cells are large in quantity, high in purity, and not easily contaminated with neonatal cells, and can meet the clinical application requirements; the method of the present application solves the problems of low quantity of maternal-derived mesenchymal stem cells obtained from the decidua basalis and easy loss of the decidua basalis, and provides a new solution and approach for obtaining maternal-derived mesenchymal stem cells from the placenta.

The invention discloses application of cornua cervi pantotrichum tissue-derived exosome-like nanoparticles in preparation of a medicine for preventing and treating senile cardiovascular diseases. A sterile, stable and high-purity cornua cervi pantotrichum tissue exosome is obtained by adopting an enzyme digestion-differential centrifugation-ultra-high-speed centrifugation combined method, the physiological efficacy of the cornua cervi pantotrichum tissue exosome in heart protection is studied, and it is found that the cornua cervi pantotrichum tissue exosome can significantly improve cardiac dysfunction of acute aging mice induced by D-galactose (D-Gal). The cardiac ejection function, the cardiac fibrosis and the inflammation invasion level are remarkably improved. The compound has treatment potential in preparation of medicines for preventing heart protection and treating heart diseases, and provides a new strategy and a treatment method for treating cardiovascular diseases of old people.

The invention discloses a co-culture-based tils cell amplification method, which comprises: S1, tumor tissue treatment: obtaining a fresh tumor tissue, and avoiding cell activity reduction caused by long-time storage; s2, tumor cell culture: carrying out enzyme digestion on one part of tumor tissue, separating out tumor cells, carrying out primary culture, and establishing a tumor cell line; s3, TILs separation: carrying out mechanical separation and density gradient centrifugation on the other part of tumor tissue to separate out TILs; s4, TILs pre-stimulation, wherein the separated TILs are subjected to pre-stimulation in a culture medium containing IL-2, and activation and proliferation of the TILs are promoted; s5, co-culture: mixing the pre-stimulated TILs and tumor cells according to a ratio of 1: 1 or 2: 1, and carrying out co-culture; and S6, TILs amplification: after co-culture is finished, separating out TILs, and further amplifying in a culture medium containing IL-2 to obtain high-activity TILs. According to the invention, the activity of TILs, the tumor antigen specificity and the amplification efficiency can be effectively improved.

A system for the molecular detection and genotyping of Toxoplasma gondii, consisting of: a DNA extraction unit configured to extract and purify DNA from stool and tissue samples; a PCR amplification unit configured to amplify T. gondii -specific DNA sequences using the nested PCR technique with primer sets comprising: external primers sag2Fext and sag2Rext, which target the non-coding region of the SAG2 gene to generate 1000 bp amplicon and nested inner primers sag2Fint and sag2Rint targeting the coding region of the SAG2 gene to generate a 500 bp amplicon; a gel electrophoresis system configured to visualize PCR amplification products on agarose gel; a restriction digestion unit configured to digest the nested PCR products with BsrDI and BspMI enzymes in a single reaction for genotyping; and a genotype analysis unit configured to classify the genotype of T. gondii into three clonal lineages and non-clonal genotypes based on restriction fragment profiles.