85 results about "Guanidinium thiocyanate" patented technology

The invention discloses a broad-spectrum and high-efficiency method for extracting RNA from various plant tissues and belongs to the technical field of biochemistry. In the invention, boric acid buffer solution is used as a buffer system, the plant tissue RNA is effectively separated and enriched first, the mixture of tris(hydroxymethyl)aminomethane (tris), hydrochloric acid (HCl), ethylenediamine tetraacetic acid disodium salt (EDTA-Na2) and sodium chloride (NaCl) is used as extraction buffer solution, hexadecyl trimethyl ammonium bromide (CTAB) is used as a detergent, 1 mol / L guanidinium isothiocyanate is used as a strong denaturant for inhibiting the activity of RNase, and thus high-quality RNA is extracted. The method can extract RNA from tissues such as ovules, anthers, blades and fibers of cotton and can extract high-quality RNA from recalcitrant plants such as arabidopsis thaliana, rice, draba matangensis, banana (peel and meat), masson pine and deodar. When the method is used for extracting RNA, the yield is high, the purity is high, and salt pollutant is avoided.

The invention discloses an excrement preserving fluid and applications thereof. Ethanol is taken as a fixing agent, sodium citrate is taken as an auxiliary fixing agent, EDTA-disodium is taken as an anti-coagulant, Tris-HCl is taken as a buffer solution, NaCl is taken as an ion strength maintaining agent, and the preserving fluid also comprises lauryl sodium sulfate. Through a reasonable formula,the number, composition, and abundance of microbes in an excrement sample are stabilized; the accuracy of sequencing analysis on human intestinal flora is guaranteed; the preserving fluid can preservean excrement sample at a room temperature; compared with a conventional preserving fluid, the preserving effect is better, moreover, provided preserving fluid does not contain any toxic component such as guanidinium thiocyanate, and the operation is safer.

The present invention is directed to a method for performing an RT-PCR for amplifying a target RNA comprising the steps of a) lysis of a cellular sample which is supposed to contain the target RNA with a lysis buffer comprising between 0.2 M and 1 M Guanidine Thiocyanate, b) diluting the sample to an extend such that Guanidine Thiocyanate is present in a concentration of about 30 to 50 mM, c) reverse transcribing in the presence of a mixture of first strand cDNA synthesis primers, the mixture consisting of oligo dT primers and random primers, and d) subjecting the sample to multiple cycles of a thermocycling protocol and monitoring amplification of the first strand cDNA in real time, characterized in that steps a) to c) are done in the same vessel.

An improved method of preserving a molecule in a bodily fluid comprises: (1) providing a preservative solution comprising: (a) an amount of a divalent metal chelator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), (ethylenebis(oxyethylenenitrilo))tetraacetic acid (EGTA), and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and salts thereof in the range of from about 0.001 M to about 2 M; and (b) an amount of at least one chelator enhancing component selected from the group consisting of lithium chloride, guanidinium chloride, guanidinium thiocyanate, sodium salicylate, sodium perchlorate, and sodium thiocyanate in the range of from about 0.1 M to about 10 M; and (2) adding the preservative solution to the bodily fluid, thus preserving the molecule. The molecule can be a protein or a small molecule, such as a steroid. The invention also encompasses preservative compositions suitable for preserving proteins or small molecules, and kits. Preservative compositions can further include at least one enzyme inactivating component selected from the group consisting of manganese chloride, sarkosyl, and sodium dodecyl sulfate in the range of up to about 5% molar concentration. Compositions and methods according to the present invention have many diagnostic and forensic uses, in addition to being suitable for preparing compositions usable by hunters for attracting animals.

The invention relates to a kit for extracting viral genome nucleic acid and a use method thereof. The kit is characterized by comprising protease K, silica-based modified magnetic beads, an extraction buffer solution, a rinsing liquid I, a rinsing liquid II, RNase-free water and the like, and further comprising a magnetic frame in a matched manner. The use method is characterized by comprising the following steps: adding protease K and the silica-based modified magnetic beads into the extraction buffer solution for one-time digestion pyrolysis of cells and adsorption pyrolysis of released virus nucleic acid; extracting guanidinium thiocyanate in the extraction buffer solution with the pH value of 5.5, so that the silica-based modified magnetic beads can adsorb more nucleic acid, that is, each gram of the silica-based modified magnetic beads can adsorb 10 mg or more of nucleic acid; utilizing hexadecyl trimethyl ammonium bromide contained in the extraction buffer solution for removing protein, polyose, phenols and other impurities, and utilizing polyvinylpyrrolidone K25 for removing pigments in the extraction buffer solution; conducting further purification on the silica-based modified magnetic beads adsorbed on the magnetic frame to obtain high-concentration genome nucleic acid. When the kit is utilized for extracting genome nucleic acid, independent digestion pyrolysis, washing and re-extraction on a source sample are not required in advance, so that the clinical test time is shortened, and the cost is greatly reduced.

An improved method of preserving a molecule in a bodily fluid comprises: (1) providing a preservative solution comprising: (a) an amount of a divalent metal chelator selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), (ethylenebis(oxyethylenenitrilo))tetraacetic acid (EGTA), and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and salts thereof in the range of from about 0.001 M to about 2 M; and (b) an amount of at least one chelator enhancing component selected from the group consisting of lithium chloride, guanidinium chloride, guanidinium thiocyanate, sodium salicylate, sodium perchlorate, and sodium thiocyanate in the range of from about 0.1 M to about 10 M; and (2) adding the preservative solution to the bodily fluid, thus preserving the molecule. The molecule can be a protein or a small molecule, such as a steroid. The invention also encompasses preservative compositions suitable for preserving proteins or small molecules, and kits. Preservative compositions can further include at least one enzyme inactivating component selected from the group consisting of manganese chloride, sarkosyl, and sodium dodecyl sulfate in the range of up to about 5% molar concentration. Compositions and methods according to the present invention have many diagnostic and forensic uses, in addition to being suitable for preparing compositions usable by hunters for attracting animals.

The invention discloses a method for extracting microbial total RNA in forest soil and litter, which comprises the following steps: 1, carrying out freeze-drying on a forest soil or litter sample, grinding, and uniformly mixing; 2, adding pyrocarbonic acid diethyl ester treating water into the powdered sample, and standing over night at -70 DEG C; 3, adding glass beads, hexadecyl trimethyl ammonium bromide buffer, lauryl sodium sulfate, diatomite and phenol / chloroform / isoamyl alcohol, and uniformly mixing; 4, severely shaking the mixed liquid, and centrifuging to obtain the supernate; 5, adding guanidinium isothiocyanate into the supernate, and centrifuging to obtain the supernate; 6, adding chloroform / isoamyl alcohol into the supernate, and centrifuging to obtain the supernate; 7, addingpolyethylene glycol 6000 into the supernate, standing, centrifuging, and removing the supernate; 8, washing, dissolving, and carrying out DNA enzyme digestion to obtain an RNA solution; and 9, detecting the integrality of the RNA through agarose gel electrophoresis, and using a nucleic acid protein determinator to detect the concentration and purity of the RNA. The RNA extracted by using the method has high yield, good integrality and better purity.

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 cervical cell preservation and DNA fast extraction integrated kit which comprises cell preservation liquid, a first cleaning solution, a second cleaning solution, eluent and a DNA purification column. The cell preservation liquid comprises 2-3mol / L of guanidine hydrochloride or guanidinium thiocyanate, 5-50mmol / L of Tris-HCl, 1-10mmol / L of EDTA and 5-7.5v / v% of Trixon-100. The cervical cell preservation and DNA fast extraction integrated kit has the advantages that cervical sample DNA can be preserved in the cell preservation liquid of the kit for more than a week under room temperature, transportation of collected samples is facilitated, subsequent DNA extract does not need complex splitting anymore, the whole extraction process can be completed in 10 minutes, and DNA extraction efficiency is increased evidently; in addition, the extraction operation only needs a liquid-moving device and a centrifuge, instrument dependence is low, and the extracted DNA is high in purity.

The invention relates to the field of molecular biology, and discloses a faeces sample stabilizing solution. The stabilizing solution comprises the following components: 0.02M-4M Guanidine thiocyanate, Tris-HCI of which the pH is 5-10 and the concentration is 2-20 M, 0.05-0.1 mM NaCl and 1-15 mM EDTA. According to the invention, the stability of the detected components in the faeces sample can be effectively kept, self-help fetch by a patient at home can be possible, the faeces stabilizing solution cannot be a hindrance to downstream detection (nucleic acid extraction and detection), the mixture of the faeces and the stabilizing solution can directly enter a detection process, greatly facilitates the flexibility of the follow-up treatment process of the faeces sample, and is beneficial to the detection and the treatment of digestive system diseases.

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 invention discloses a virus nucleic acid extraction kit based on nano magnetic beads. The lysis liquid comprises guanidine thiocyanate / guanidine hydrochloride, a nonionic surfactant, tris (hydroxymethyl) aminomethane hydrochloride and ethylenediamine tetraacetic acid, and the nonionic surfactant comprises one or more of triton X-100, Tween 20 and ethyl phenyl polyethylene glycol. The binding liquid is isopropanol, the volume ratio of isopropanol to pyrolysis is 1: 1, and the magnetic beads are silicon oxide hydroxyl nano magnetic beads. According to the kit, a nano magnetic bead extractionmethod is adopted, isopropanol is used as a binding liquid, making sample splitting, binding of nucleic acid and magnetic beads to be done in one step. The steps are simple, convenient and easy to implement, and the automatic extraction process of virus nucleic acid can be completed quickly at high flux within 9 min. The kit disclosed by the invention can be used for extracting RNA and DNA of viruses in sample, such as plasma, serum, nasopharynx swabs, urine, secretion liquid, virus concentrate, cell culture liquid supernatant and virus preservation liquid.

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 present invention is directed to a method for performing an RT-PCR for amplifying a target RNA comprising the steps of a) lysis of a cellular sample which is supposed to contain the target RNA with a lysis buffer comprising between 0.2 M and 1 M guanidine thiocyanate, b) diluting the sample to an extend such that guanidine thiocyanate is present in a concentration of about 30 to 50 mM, c) reverse transcribing in the presence of a mixture of first strand cDNA synthesis primers, the mixture consisting of oligo dT primers and random primers, and d) subjecting the sample to multiple cycles of a thermocycling protocol and monitoring amplification of the first strand cDNA in real time.

The invention relates to a nucleic acid recovery and purification kit which comprises a sol solution, a magnetic bead solution and a combination solution. The sol solution contains 3-4 mol / L guanidine isosulfocyanate, 20-30 mol / L Tris-HCl buffer solution and the balance of water. The magnetic bead solution contains 0.2-3 mg / mL hydroxy magnetic bead and the balance of water. The combination solution contains 40-60% isopropanol, 1-2 mol / L sodium chloride and the balance of water. The invention also relates to a nucleic acid recovery and purification method using the kit. The nucleic acid recovery and purification kit and method can quickly and efficiently recover and purify large-segment nucleic acids in a sepharose gel.

The invention relates to a bacterial lysate and a kit for extracting plasmid DNA and a method for extracting the plasmid DNA, and belongs to the technical field of biology. The bacterial lysate for extracting the plasmid DNA comprises lysis buffer, lysozyme and ribonuclease A(RNaseA); wherein100 mL of the lysis buffer comprises 1.5-10 g of sucrose, 0.7-2.2 g of ethylene diamine tetraacetic acid (EDTA), 0.3-0.9 g of tri(hydroxymethyl) amino methane hydrochloride, 2.7-5.4 g of NH4Cl, 0.1-2.0 mL of 5% Triton X-100, 0.2-0.6 g of CaCl2, 0.5-8 g of guanidine hydrochloride, 0.5-3 g of guanidinium isothiocyanate and the balance of water. The kit for extracting the plasmid DNA comprises the bacterial lysate, rinsing solution and eluant and is low in cost, high in purity of extracted plasmid and high in applicability.

The invention discloses a method for extracting total RNA from fermented grains used for Chinese liquor fermentation, and belongs to the biotechnical field. The total DNA is rapidly extracted from microbial communities in 2h against the particularity of a Chinese liquor fermented grain sample through combining a sodium laurate extraction technology and a guanidinium isothiocyanate-phenol-chloroform extraction technology. The microbial total RNA extracted through using the method has the advantages of high output, good integrity, good purity, and effective extraction of eukaryotic and prokaryotic microbes from the sample.

The invention provides a reagent for extracting pathogenic nucleic acid from an animal tissue sample. The pH of the reagent is 7 to 9, the solvent is water, and the solute contains the following substances with final concentration: 4 to 6 M of guanidinium isothiocyanate, 20 to 25 mM of sodium citrate, and 0.2 to 0.5 mass percent of sodium lauryl sulfate (SLS). The cost for extracting the nucleic acid by adopting the reagent is low; and the reagent is nontoxic, harmless, safe, quick and suitable for extracting the pathogenic nucleic acid in the animal tissue sample.

The invention is a method for separating RNA form organism sample, the method includes: dewaxes at first, the invention uses dimethyl benzene to wash olefin and packages the sample, then uses lower alcohol liquid to carry on rehydration and homogeneity. the rehydration method is carrying on wash with alcohol solvent whose thickness is decreased gradually, the sample and the alcohol are blended fully and centrifugated, then carries on homogeneity again by rhodanic acid guanidine liquid. The samples are heated to 90í½100íµ, then reclaims RNA, the method is carbonic acid chloroform extracting method in current technology. Then the invention carries on extraction, cataphoresis, chromatography, depositing or other proper technology.

Provided is a safe novel method for detecting Transmissible Spongiform encephalopathies (TSE). The method comprises: selecting a sample from a subject to determine whether the subject has transmissible spongiform encephalopathy; and detecting abnormal prion protein (Prpes) in the sample. The method detects PrPres without Proteinase-K treatment by disrupting the sample in guanidine thiocyanate lysis solution followed by phenol purification of proteins, and demonstration of the abnormal prion isoform by Western blotting using monoclonal antibodies against prion protein structure. Guanidine salts effectively kill TSE infectivity providing a laboratory safe environment and stabilize biomolecules so TSE samples can be procured in the field and transported to the laboratory in guanidine lysis solution for processing at a later date. This method provides for rapid detection of the abnormal prion isoform diagnostic of TSE and results are easily interpretable based upon very different Western blot patterns for abnormal prion isoform versus the normal prion.

A formulation containing guanidine thiocyanate together with acetamide, one or more acetamide derivatives, or a combination of acetamide and one or more acetamide derivatives is used to purify one or more nucleic acids contained in a medium. In particular, a medium containing at least one nucleic acid is combined with a binding matrix and the formulation in order to cause the at least one nucleic acid to separate from its in vivo cellular environment and to bind to the binding matrix. The binding matrix with at least one nucleic acid bound thereto then is separated from substantially the rest of the combined medium and formulation, after which the at least one nucleic acid is eluted from the binding matrix to obtain the at least one nucleic acid in a substantially purified form. If different nucleic acids are to be selectively purified from a single medium, multiple binding matrices, each compatible with a different nucleic acid, can be used.

The invention discloses a kit for easy and odorless extraction of fecal nucleic acid and an extraction method and relates to the field of molecular biotechnology. The kit includes a fecal stabilizingsolution containing Tris, guanidinium thiocyanate, ethylenediaminetetraacetic acid, sodium chloride, chlorobutanol, green tea extract, honeysuckle extract and an enzyme. The fecal stabilizing solutioncan store the DNA in the extracted fecal sample at a normal temperature of less than or equal to 40 DEG C for about 15 days, solve the problem of difficulty in transportation and storage and facilitate self-extraction and saving of the sample at home and is convenient and quick. The fecal stabilizing solution has the advantages of good deodorizing effect and use safety and can effectively removefecal odor gas such as ammonia gas and hydrogen sulfide thereby eliminating the odor and inhibiting the growth of bacteria.

The invention discloses a water environment DNA collection and preservation card and a preparation method thereof, and relates to the technical field of biology. The problems that due to the lack of DNA separation and purification conditions at the collection site in existing conventional studies of water environment DNA, water samples need to be brought back to a laboratory for environmental sample processing and DNA extraction, storage and transportation condition requirements are high, the cost is increased, and the storage time is limited are aimed to be solved, the water environment DNA collection and preservation card includes a material with the ability to lyse cells and adsorb the DNA, the adsorption material is internally adsorbed with salinity, and the salinity includes a cell lysis agent, a nuclease inhibitor, a nucleic acid stabilizer and an antioxidant, wherein the cell lysis agent is one or more combinations of Tris hydrochloride, NP40 (ethyl phenyl polyethylene glycol),sodium chloride, guanidine isothiocyanate and SDS (sodium dodecyl sulfonate); and the nuclease inhibitor is a metal ion chelating agent, and the metal ion chelating agent is one of EDTA and an EDTA sodium salt.

The invention provides a universal microbial pathogen lysate and application thereof. Specifically, the lysate comprises 30-60-mM Tris-HCl, a 3-6%(w / w) nonionic surfactant, 300-600-mM guanidine isothiocyanate and 20-80-mM KCl, wherein the pH of the lysate is 7.0-10.0, and the percentage is based on the total weight of the lysate. The lysate is applicable to pathogen microbes of various sources and has the advantages of high nucleic acid extraction speed and extraction rate and low price; surprisingly, the lysate can also improve the PCR amplification efficiency.

The invention discloses an extract which comprises an extract A and an extract B, wherein the extract A applied to tissue or cell preservation; the extract A comprises 1.2-2.0mol / L guanidine thiocyanate and 0.5-1.5mol / L guanidine hydrochloride; the extract B comprises 30-65vt% of water saturated phenol, 9-15vt% of glycerinum and the balance of a solvent in percentage by volume; the extract B further comprises 0.20-1mol / L guanidine thiocyanate, 0.4-0.8mol / L ammonium thiocyanate and a 0.0073-0.018mol / L surfactant by mole concentrations; the pH values of both the extract A and the extract B are 3.5-4.5; the extract B is applied to RNA extraction. Ratios of components of the extract disclosed by the invention are reasonably designed, the components have mutual synergistic effects, and extracted RNA is high in purity, high in yield and low in protein contamination.

The invention discloses an inactivated virus preserving fluid formula and a preparation method thereof, and the inactivated virus preserving fluid formula comprises the following components by weight: 5-20 g of a protein denaturant: one or a combination of more of high-concentration guanidine salt, guanidine hydrochloride and guanidine isothiocyanate; 0.5-2g of RNA (Ribonucleic Acid) enzyme inhibitor; a buffer stabilizer: 2-6 g of Tris; a surfactant: 1-4 g of sodium dodecyl sarcosinate; 0.001-0.01 g of an acid-base indicator; 0.2-1 g of an anti-freezing agent; the RNA enzyme inhibitor is one or a combination of more of EDTA (Ethylene Diamine Tetraacetic Acid) and guanidine isothiocyanate; the acid-base indicator is one of basic fuchsin and neutral fuchsin; the preserving fluid is safe and non-toxic, can keep a liquid state at a low temperature, is convenient to sample, can inactivate viruses and protect RNA enzyme degraded viruses, can resist growth of bacteria and fungi, is not easy to cause pollution, can be preserved for a long time and detect viruses, enables the viruses not to be infectious, and avoids the risk that transportation and detection personnel are infected.

The invention discloses an extraction reagent for exosome extraction. The extraction reagent is prepared from the following components: dithiothreitol, potassium chloride, potassium citrate, sodium formate, serum albumin, glycine and guanidinium thiocyanate, and has a pH value of 3.0-5.5. The extraction method comprises the following steps: a, centrifuging a body fluid sample through a centrifugal machine to obtain supernate; b, adding the extraction reagent into the supernate obtained in the step a, reacting for 2 hours, and centrifuging through the centrifugal machine; and c, suspending and re-dissolving the exosome deposit obtained in the step b with PBS to obtain exosome. The exosome extraction is simple in operation and short in time consumption, the operation procedure is simplified, the cost is saved, and clinical routinization of body fluid biopsy becomes possible.