160 results about "Guanidine isothiocyanate" patented technology

The invention discloses a fluorescence quantitative PCR detection kit of hepatitis B virus and an application thereof. The kit is composed of the following independent components: DNA extraction solution I, DNA extraction solution II, DNA extraction solution III, DNA extraction solution IV, positive control interior label, PCR reaction liquid, probe HBV-SP, enzyme mixed liquor containing heat resistant DNA polyase and uracil DNA glycosylase, quantitative hepatitis B virus reference material, hepatitis B virus positive control serum and hepatitis B virus negative control serum, wherein DNA extraction solution I contains 0.2-1.0% of lauryl sodium sulphate (mass/volume), 1.0-4.0% of Triton (volume/volume) and 0.2-1.0mol/L of guanidinium isothiocyanate; DNA extraction solution II contains 100-300mmol/L of 4-HEPES, 100-300mmol/L of sodium chloride with pH of 6.5+/-0.2 and 100-400 mu g/ml of magnetic beads; DNA extraction solution III contains 0.1-1.0% of Triton (volume/volume) and 100-300mmol/L of sodium chloride; DNA extraction solution IV contains mineral oil. The fluorescence quantitative PCR detection kit of hepatitis B virus of the invention can be used for detecting the HBV-DNA concentration in samples of serum, blood plasma or latex and the like.

The invention provides an extration method of plant RNA by utilizing guanidinium isothiocyanate-chloroform extractive and Tris purification. The invention improves traditional guanidinium isothiocyanate method, adopts the two steps of chloroform extration after cracking and phenol-adding for purification, and respectively extract high-quality total RNA from the leaf blade and seed case of red-skinned pears, the leaf blade, fruit flesh and seed case tissues of grapes, the fruit of strawberries and the callus of arnebia euchroma. The extraction buffer liquid in the method contains guanidinium isothiocyanate, sodium dodecyl sarcosinate, sodium citrate, soluble polyvinylpyrrolidone and beta-mercaptoethanol, which can not only crack plant cells efficiently and release RNA, but also effectivelyinhibit the activity of RNA enzyme, and can also prevent the oxidation of phenolic compounds and suppress the interference of secondary metabolites. In the second step, Tris purification is utilized for crude extraction of RNA solution, which can eliminate the polysaccharides and protein which can coprecipitate with RNA. The invention is simple in operating steps, economic and practical, good in stability, and suitable for extracting high-quality total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites.

The invention relates to the field of molecular biology and discloses a kit and a method for extracting DNA from micro samples. The kit comprises a binding liquid, a regulated binding liquid and a deproteinized liquid, wherein the binding liquid comprises 3-8M of guanidinium isothiocyanate, 5-50mM of Tris-HCl with a pH value of 3.5-5.5, 5-25% of Ttiton-X 100, 5-20mM of Urea, 5-20mM of EDTA and 5% of Tween20, the regulated binding liquid is isopropanol, and the deproteinized liquid comprises 2-8M of guanidine hydrochloride, 5-50mM of Tris-HCl with a pH value of 6.5-8.0 and 5-50% of absolute ethyl alcohol. The DNA extracted by the kit and the method has high content and purity, the extraction speed is high, and no toxic reagent is used. The kit and the method are applicable for extracting DNA from various micro samples.

The invention discloses an animal nucleic acid sample normal temperature preservation reagent and an application thereof. The animal nucleic acid sample normal temperature preservation reagent comprises an acid cell lysis agent, nuclease inhibitors, a surfactant, sodium citrate and sodium acetate, wherein the acid cell lysis agent is guanidinium isothiocyanate, the used nuclease inhibitors are 8-hydroxyquinoline and beta-mercaptoethanol, and the used surfactant is lauryl sodium sulfate. On the other hand, the invention discloses a method for treating filter paper by using the animal nucleic acid sample normal temperature preservation reagent. The method comprises the following steps: 1) soaking the filter paper in the animal nucleic acid sample normal temperature preservation reagent; 2) baking the filter paper at 37-50 DEG C; 3) sterilizing the filter paper for later use.

The invention discloses a lysis solution and an application thereof in tissue or cell preservation and RNA (Ribonucleic Acid) extraction. The lysis solution comprises a lysis solution A and a lysis solution B, wherein the lysis solution A comprises 2-4mol/L guanidinium isothiocyanate, 5-10mmol/L sodium chloride, 10-15mmol/L potassium chloride, the lysis solution B comprises 2-4mol/L guanidinium isothiocyanate, 1-5mmol/L spermidine, 30-40vt% of water saturated phenol, 0.4-0.5mol/L ammonium thiocyanate, 7-8vt% of glycerinum, 0.1-0.5mol/L sodium acetate and 0.2-0.5wt% of SDS (Sodium Dodecyl Sulfonate). When RNA is extracted by using the lysis solution A and the lysis solution B, tissue or cells do not need to be washed, and thus the extraction efficiency is remarkably improved; and due to synergistic effects of the components, the extracted RNA is high in purity and high in yield, and the purpose that animal RNA is extracted in laboratories rapidly with low cost and high quality in a large scale can be realized.

The invention discloses a kit for extracting and enriching free DNAs. The kit comprises a sample lysate, a binding solution, a primary washing fluid, a secondary washing fluid, an eluent and nano magnetic beads. The sample lysate comprises nuclease-free water, a reagent A and a reagent B, the reagent A is at least one of guanidine isothiocyanate, guanidine hydrochloride, sodium iodide and sodium perchlorate, and the reagent B is at least one of Tween20, Triton-X100, SDS, NP40 and a polyoxyethylene type nonionic surfactant. The binding solution comprises a reagent C in addition to the components of the sample lysate, and the reagent C is at least one of isopropanol and absolute ethanol. The primary washing fluid comprises the same components as the binding solution. The secondary washing fluid is the nuclease-free water, a sodium chloride solution with the molar concentration of 0.1-1M, a Tris buffer solution or a TE buffer solution. The eluent is the nuclease-free water, the Tris buffer solution or the TE buffer solution. The invention further discloses an extraction method for the free DNAs. The technical problem that organic solvents in the prior art have a certain toxicity is solved.

The invention provides a method for co-extracting DNA/RNA (Deoxyribonucleic Acid/Ribonucleic Acid) virus nucleic acid. The method comprises the following steps: cracking a sample, which is diluted with saline, with a guanidine isothiocyanate cracking solution containing an RNA precipitating aid agent; releasing and dissociating DNA or RNA of different nucleic acid viruses in the sample into a cracking solution; adding magnetic beads to form a magnetic bead-nucleic acid compound; washing to obtain a nucleic acid eluting solution. The novel method for co-extruding the DNA and the RNA from a respiratory tract sample is established based on the nano magnetic beads; in a whole process, toxic reagents including phenol, chloroform, beta-mercaptoethanol and the like are not used, and time and labor are saved. The method is used for commonly extracting the DNA and the RNA from the respiratory tract sample and is also applicable to nucleic acid co-extraction of other samples with different types, such as urine and blood; impurities including protein and the like are efficiently removed and the degradation of the nucleic acid is reduced; the aim of detecting DNA viruses and RNA viruses in the same tube at the same time is realized. The method is simple, convenient, rapid and safe, and is particularly suitable for fluorescent quantitative PCR (Polymerase Chain Reaction) detection and the like.

The invention discloses a method of simultaneously extracting animal DNA (Deoxyribonucleic Acid) virus and RNA (Ribonucleic Acid) virus nucleic acid in blood serum and double swabs. The method comprises the following steps of: carrying out lysis on a to-be-extracted substance by using guanidinium isothiocyanate lysate; adsorbing RNA by a silica gel membrane; removing impure protein by washing liquor I; removing impurities by washing liquor II; carrying out DEPC (Diethylpyrocarbonate) water-washing to remove nucleic acid, wherein the guanidinium isothiocyanate lysate comprises 3M-7M guanidinium isothiocyanate, 0.6%-1.0% TriTon-100, 30mM-50mM Tris-Cl, 5mM-15mM DTT (DL-Dithiothreitol), 60 mu g/mL-90 mu g/mL protease K, 10mM-30mM EDTA (Ethylene Diamine Tetraacetic Acid), and PH of the guanidinium isothiocyanate lysate is 4.3-4.6; the washing liquor I comprises 5M-6M guanidine hydrochloride, 53%-59% absolute ethyl alcohol, 70-90 mu g/mL protease K, and PH of the washing liquor I is 6.4-6.6; the washing liquor II comprises 70%-80% alcohol. The method disclosed by the invention has the advantages of being simple in extracting process, short in period, low in cost, and capable of simultaneously extracting RNA virus and DNA virus nucleic acid in an animal blood serum sample and a double-swab sample.

The invention provides a virus sample inactivation cracking test kit. The virus sample inactivation cracking test kit comprises ammonium salt, guanidine isothiocyanate, lithium chloride, ethylenediamine tetraacetic acid and the like. The invention also provides a target nucleic acid magnetic capture reagent and a target nucleic acid capture test kit. The target nucleic acid magnetic capture reagent and the target nucleic acid capture test kit comprise magnetic polymer microspheres, an intermediate probe, a capture probe and the like. The invention also provides a primer pair and a real-time fluorescent nucleic acid isothermal amplification detection test kit for amplifying and detecting target nucleic acid, and a test kit used for virus inactivation, capture and real-time fluorescent isothermal amplification detection. The inactivation cracking test kit disclosed by the invention can realize quick inactivation at a room temperature and reduce RNA degradation, and is high in sensitivity; the target nucleic acid magnetic capture reagent has stronger specificity, and can obtain templates with good quality and large quantity; and a real-time fluorescent nucleic acid isothermal amplification technology enables reverse transcription and amplification to be carried out together, so the reaction time is shortened, and the sensitivity of a detection reagent is improved.

The present invention relates to a simple, effective and quick RNA extraction method for absorbing RNA by utilizing a silicon film column. The method includes the following steps: after cells are collected, denaturing solution and chloroform are added into the cells, uniformly mixed and centrifugated until the bacteria solution is divided into three layers in a tube; the top layer of liquid is extracted and transferred into the silicon film column to be centrifugated; the previous step is repeated; centrifugation is conducted again in order to ensure that no liquid exists in the silicon film column; double distilled water and eluent are added into the silicon film column; and after centrifugation, the eluent is collected into a clean centrifugal tube. The denaturing solution contains split agent, RNA extraction agent and water; wherein, the split agent is guanidinium isothiocyanate, sodium dodecyl sarcosinate or phenol; and the RNA extraction agent is sodium citrate, sodium acetate, sodium lactate, ascorbic acid, potassium malate, sodium oxalate or potassium tartrate. The extraction time of the method is not longer than 6 minutes, DNA pollution cannot be generated, the extraction process can be easily automated, and the purity of the extracted RNA is high.

The invention provides an extraction method of total DNA of metagenome of fish enteric microorganisms and a kit. The extraction method comprises the following steps: (1) fish sample sampling and fixing treatment of microorganisms on the surface of the fish; (2) acquisition of enteric microorganisms and collection of thalli; (3) embedding and cracking of cells; (4) extraction of DNA; and (5) DNA preservation. The kit comprises the following main components: a solution A which is a mixed solution of Tris and EDTA; a solution B which is a mixed solution of Tris, EDTA, NaCL, PVP, guanidinium isothiocyanate, Triton-X100, PMSF, and beta-mercaptoethanol; a solution C which is a mixed solution of Tris, EDTA, NaCl and DTT; a solution D which is a mixed solution of proteinaseK, a lysozyme, Tris, NaCl and SDS; a solution E which is a mixed solution of Tris saturated phenol and chloroform; F which is isopropanol; and G which is an ethanol aqueous solution. The DNA fragment obtained by the invention is high in purity, and the content of DNA of a host and the microorganisms on the surface of the fish is small.

The invention discloses a kit and method for rapidly detecting DNA methylation. Compared with traditional method, the method of the invention has the following four improvements: 1) DNA extraction and DNA modification are completed synchronously; 2) vulcanization accelerator MBT and guanidinium isothiocyanate are added in modifying solution so as to rapidly complete the DNA extraction and DNA modification processes; 3) the modification reaction adopts a short-time high-temperature processing method to ensure that the DNA can be in the desmolysis state, basic groups are fully exposed and cytosines which are not modified through methylation are converted to sulphonated uracils; and 4) the complicated dialysis desalination step after modification is simplified, namely the modified product is transferred in a nucleic acid purification column, then elution is performed after the column is processed with cleaning solution; and the DNA template of the methylated polymerase chain reaction (PCR) of the detected gene can be obtained. The method of the invention has the advantages of low cost, time saving and good repeatability and can be directly used to detect the methylation level of various tissue cell genes and has important use value in the biomedical detection technical aspects such as the early clinical diagnosis.

The invention relates to a stationary liquid for preserving human saliva, a preparation and an application, wherein the stationary liquid for preserving the human saliva comprises the following components: sugar, Tris-HCl, MgCl2, guanidinium isothiocyanate and water. The saliva stationary liquid provided in the invention has better preservation effect; the preservation effect is obviously better than the preserving saliva with single component; and with lower cost and convenient preparation, the stationary liquid for preserving the human saliva is hopeful to become the necessary and better stationary liquid for widely using the saliva as genome DNA (Deoxyribonucleic Acid) source.

The invention provides a nucleic acid extraction lysate for cast-off cells in human feces. The nucleic acid extraction lysate comprises 1-8 mol/L guanidinium, a 1-20 mM metal ion chelating agent and a100 mM Tris-HCl buffer solution with the pH of 7.5-8.0, wherein guanidinium is selected from at least one of guanidine hydrochloride, guanidinium isothiocyanate and guanidine thiocyanate; the metal ion chelating agent is selected from at least one edetic acid and water soluble salt of edetic acid. Preferably, the nucleic acid extraction lysate further comprises a surfactant with the volume of 1-20%, the surfactant is a non-ionic detergent, and the non-ionic detergent is selected from at least one of Tween-20, Tween-80, Brij-35, NP40 and Triton-100. The invention further provides a related preparation method and application. The nucleic acid extraction lysate can quickly and efficiently realize cell lysis, fully releases nucleic acid, meets the consumption demand for follow-up nucleic aciddetection and is suitable for large-scale popularization.

The invention provides a reagent for simultaneously liquefying phlegm and effectively protecting nucleic acid. The reagent contains guanidine, a cysteine derivative and a macromolecule inert particle,wherein guanidine is one or more of guanidine hydrochloride, guanidinium isothiocyanate, guanidine sulfate and guanidine carbonate; the cysteine derivative is one or more of cysteine, acetylcysteine,homocysteine, methylcysteine and cysteine hydrochloride; and the macromolecule inert particle is one or more of a polypropylene particle, a polyethylene particle, a polyvinyl chloride particle and apolystyrene particle. The reagent is a mixed solid of powder and particles and is capable of well permeating into a phlegm sample, and the phlegm sample is adequately mixed, so that phlegm can be adequately liquefied; and meanwhile, the reagent is capable of protecting nucleic acid in the phlegm sample from being degraded at a normal temperature for a long time. The reagent contains simple components and is low in cost, and the production process is simple, and the reagent is easy to use and operate and very suitable for wide clinical use.

The invention discloses a quantitative detection kit for human immunodeficiency virus HIV-1. The quantitative detection kit comprises sample treating agent, an upstream primer HIV-F used for target nucleotide amplification, a downstream primer HIV-R used for target nucleotide amplification, a Taqman probe HIV-P for detecting target nucleotide and RNA one-step reaction buffer, wherein the sample treating agent comprises guanidinium isothiocyanate, sodium citrate, dodecyl creatine sodium, beta-mercaptoethanol and proteinase K; a fluorescent group and fluorescence quencher are respectively combined to two ends of the Taqman probe HIV-P. When the quantitative detection kit is used for detecting, a to-be-detected sample is directly mixed with the sample treating agent and then is used for subsequent detection. Compared with a traditional quantitative detection kit for HIV virus, the quantitative detection kit has the advantages that the step of extracting and purifying nucleic acid molecules from the sample is omitted, and time-saving and simple operation is achieved.

The invention belongs to the field of molecular biology method and relates to a reagent composition for separating total RNA in a plant or a microorganism and a preparation method thereof. The high quality RNA can be obtained after a simple extraction by chloroform while guanidinium isothiocyanate and phenol are regarded as main components, positive ions are provided by NaCl, MgCl2 and the like, and a solution pH is stabilized by a sodium acetate-acetic acid buffer system. Glycogen serving as a nucleic acid precipitant is added in the RNA extraction reagent in the invention, so that the reagent disclosed by the invention, in comparison with reagents of the same type, greatly improves precipitation efficiency of the nucleic acid and also can efficiently separate the nucleic acid component which has a very low content in a tissue sample. Therefore, the nucleic acid precipitation process can be finished in a short period, the precipitation process at -20 DEG C for hours is avoided, and operating time is shortened greatly. The method is rapid as well as efficient and has wide applicable samples; and the reagent composition disclosed by the invention is cheaper than commercial TRIzol reagents. The disadvantages of high sample selectivity, fussy operation and relatively low efficiency of the traditional method are overcome. The operation is more flexible; and the sample after being homogenized can be stored at -20 DEG C and then used for the RNA extraction.

The invention discloses a kit for nucleic acid extraction with a magnetic bead method, magnetic beads and a preparation method of the magnetic beads. The kit for nucleic acid extraction with the magnetic bead method comprises a cell lysis solution, the magnetic beads and a washing solution, wherein the cell lysis solution contains guanidine isothiocyanate, Triton X-100, 3-[3-(cholamidopropyl) dimethylamino]-1-propanesulfonic acid and N-sodium lauroyl sarcosinate. According to the kit for nucleic acid extraction with the magnetic bead method, the components of the lysis buffer solution are optimized, so that cells can be quickly and fully lysed, proteins can be quickly and fully denatured, the proteins and nucleic acid are effectively separated, and the nucleic acid is released; and the working efficiency of the cell lysis solution is improved, and the nucleic acid extraction time is shortened.

The present invention relates to the field of nucleic acid extraction, particularly to a nucleic acid extraction method and a kit. The kit comprises a nucleic acid chemical cracking solution and an inhibition component removing solution, wherein the nucleic acid chemical cracking solution comprises guanidinium isothiocyanate, N-lauroyl sarcosine sodium salt and PBS; and the inhibiting component removing solution comprises a mixed solution of an aluminum ammonium sulfate dodecahydrate solution and cross-linked polyvinylpyrrolidone (PVPP) and ammonium acetate. The nucleic acid extraction methodcomprises the following steps of chemically cracking a sample by adopting the nucleic acid chemical cracking solution, removing impurities, adsorbing the nucleic acid, and performing washing, eluting,and collecting to obtain the nucleic acid. According to the method, comprehensiveness and unbiased property in nucleic acid extraction are fully considered, purity and integrity of nucleic acid are also considered, loss in the process is reduced to ensure concentration, nesting can be formed for different types of biological or environmental samples, and broad-spectrum applicability of the extraction method is guaranteed.

The invention discloses an extraction method of the genome DNA of isolated soy proteins, comprising the following steps of: firstly sufficiently mixing isolated soy proteins and a TE buffer solution to prepare a TE mixed solution; then adding a guanidinium isothiocyanate lysis solution with volume twice larger than that of the TE mixed solution, sufficiently uniformly mixing, and lysing at room temperature; adding isometric phenol and chloroform/isoamylol to extract proteins; centrifugalizing, and then adding isometric chloroform/isoamylol to a supernate to extract the proteins; centrifugalizing, and then adding isometric chloroform to the supernate to extract the proteins; centrifugalizing, and precipitating the supernate by using isopropanol; centrifugalizing, and washing by using ethanol for precipitation; airing at the room temperature; and dissolving in the TE buffer solution so as to obtain the TE solution of the genome DNA. According to the extraction method, the genome DNA which has high quality and is suitable for PCR (Polymerase Chain Reaction) detection is extracted from the isolated soy proteins, the concentration of the genome DNA is 1558 micrograms/ml, and the value of OD260/OD280 is 1.666; and in addition, the invention has the advantages of easy operation and short consuming time and is beneficial to fast detection.

The invention discloses an extracting method for the total RNA (Ribonucleic Acid) of galangal. The method is improved on various aspects on the basis of the conventional guanidine thiocyanate-phenol-chloroform extraction method according to the characteristic of rich secondary metabolism product of the galangal and particularly rich polyhydric flavonol, i.e., acetone, water-soluble polyvinylpyrrolidone (PVP) and beta-mercaptoethanol are added in an RNA extracting solution. The acetone can be used for dissolving colored matters such as flavonols and the like; the water-soluble PVP can be sufficiently combined with phenol substances to form chelates; the beta-mercaptoethanol as a strong reducing agent can be used for providing a reducing condition so that polyphenol substances are not easy to oxidize; the three substances synergistically take effects, so that the interference and obstruction of the polyphenol substances are effectively inhibited. According to the invention, a crudely-extruded sediment of the RNA is treated by using Tris saturated phenol and high-concentration potassium acetate after being obtained, and part of proteins and polysaccharides can be simultaneously removed.

The invention discloses a nucleic acid extraction kit and a nucleic acid extraction method, and belongs to the field of molecular biology. The nucleic acid extraction kit comprises a lysis solution, awashing solution I and a washing solution II, and the lysis solution comprises Tris, EDTA, Tween20, dithiothreitol and guanidinium isothiocyanate. The nucleic acid extraction method comprises the following steps: mixing the lysis solution, a nucleic acid attachment and a sample to be extracted to obtain the nucleic acid attachment adsorbed with the nucleic acid, cleaning the nucleic acid attachment adsorbed with the nucleic acid by using the cleaning solution I, the cleaning solution II and an eluent in sequence, carrying out solid-liquid separation after each cleaningprocess, and collectingthe supernatant to obtain the nucleic acid. According to the nucleic acid extraction kit and the nucleic acid extraction method provided by the invention, cells can be fully and effectively cracked, the cracking efficiency is high, nucleic acid with higher concentration and higher purity is obtained, the accuracy of subsequent operation is ensured, operation such as centrifugation is not needed, the operation is simple, one-time extraction is carried out within 1 hour, and the extraction efficiency is improved.