129 results about "Deoxyribonucleoside triphosphate" patented technology

The invention provides a method and a system for digital quantitative analysis of nucleic acid amplification based on micro-droplet. The method comprises the following steps: preparing a to-be-detected nucleic acid amplification reaction liquid which includes a to-be-detected nucleic acid template, a reaction buffer water solution, deoxyribonucleoside triphosphate, a primer, polymerase and a product marking substance; loading the prepared to-be-detected nucleic acid amplification reaction liquid in a micro-pipeline of which the two ends are both provided with an opening, wherein the micro-pipeline is arranged above an open container, and the open container contains an oily liquid containing a surfactant; enabling the opening of one end of the micro-pipeline to do up-and-down reciprocating vibration on the surface of a liquid in the open container or do leftward-and-rightward reciprocating vibration below the liquid surface so as to generate a plurality of droplets which are flatly laid at the bottom of the open container; performing nucleic acid amplification reaction on the plurality of droplets in the open container; acquiring product signals generated after the nucleic acid amplification reaction is ended, and performing quantitative analysis on the nucleic acid template. The digital nucleic acid amplification analysis method can be used for implementing quantitative detection on low-concentration nucleic acid substances in a micro-system and is simple and convenient to operate, high in efficiency and low in cost.

The invention relates to methods for the amplification of ribonucleic acids, comprising the following steps: (a) a single stranded DNA is produced from an RNA by means of reverse transcription, using a single-stranded primer having a defined sequence, an RNA-dependent DNA polymerase and deoxyribonucleoside triphosphates; (b) the template RNA is removed; (c) a DNA duplex is produced by means of a single-stranded primer comprising a box sequence, a DNA polymerase and deoxyribonucleoside triphosphates; (d) the duplex is separated into single-stranded DNAs; (e) DNA duplexes are produced from one of the single-stranded DNAs obtained in step (d) by means of a single-stranded primer comprising a promoter sequence at its 5′end and the same defined sequence as the primer used in step (a) at its 3′end, a DNA polymerase and deoxyribonucleoside triphosphates; (f) a plurality of RNA single strands, both ends of which comprise defined sequences, are produced by means of an RNA polymerase and ribonucleoside triphosphates. The invention also relates to kits for amplifying ribonucleic acids according to one of said methods, said kits comprising the following components: (a) at least at least one single-stranded primer, which contains a promoter sequence; (b) at least one single-stranded primer comprising a box sequence; (c) an RNA-dependent DNA polymerase; (d) deoxyribonucleoside triphosphates; (e) a DNA-dependent DNA polymerase; (f) an RNA polymerase; and (g) ribonucleoside triphosphates.

The invention discloses a method for detecting fungaltoxin through multiple signals. The method comprises the following steps: 1) combining fungaltoxin with aptamer: adding a to-be-detected sample after the reaction of the fungaltoxin aptamer and complementary sequence thereof, thereby acquiring a mixed solution A; 2) amplifying a digestion auxiliary signal: reacting the mixed solution A with restriction enzyme, thereby acquiring a mixed solution B; 3) preparing a guanine tetramer structure: reacting the mixed solution B with tail-end deoxynucleotide transferase and deoxyribonucleoside triphosphate and reacting with ligand molecules, thereby acquiring a mixed solution C; and 4) detecting and analyzing: performing catalytic oxidation reaction on the mixed solution C and different substrates, generating ultraviolet, fluorescent and chemiluminescent signals and calculating the content of fungaltoxin in the to-be-detected sample according to the relation of the response strength of all the signals and the fungaltoxin concentration. The method disclosed by the invention has the characteristics of quick and simple operation and treatment, short detection time, marker-free effect, low cost, high precision, high sensitivity, and the like.

The invention relates to a gene diagnosis and detection core reagent transportable at normal temperature and of high sensitivity and high specificity. The reagent comprises reaction buffer, deoxyribonucleoside triphosphate (DNTP), heat resistant DNA polymerase, glycerin, bovine serum albumin (BSA), dithiothreitol (DTT) and tween-20. The reagent is characterized in that: proper amounts of polysaccharide and disaccharide are mixed into the core reagent; the reaction buffer of the reagent is composed of 5mM-50mM of Tris-HCL, 20mM-500mM of KCL, 1.0mM-5mM of MgC12 and 20mM-200mM of (NH4)2SO4; meanwhile 50uM-300uM of DNTP, 0.01U/ul-0.2U/ul of heat resistant DNA polymerase, 1mM-15mM of polysaccharide, 2M-100M of disaccharide, 3%-30% of glycerin, 10-300ug/ml of BSA, 0.5-10mM of DTT and 0.005-0.05v/v of tween-20 are mixed.

The invention relates to the field of molecular biology and discloses mutant A type DNA (deoxyribonucleic acid) polymerase, and an encoding gene and an application of the mutant A type DNA polymerase. The mutant A type DNA polymerase is generated by amino acid site mutation of conservational motif of A type DNA polymerase in a dNTP (deoxyribonucleoside triphosphate) bond zone. Compared with the A type DNA polymerase not modified or mutated, the mutant A type DNA polymerase has increased dUTP (deoxyuridine triphosphokinase) doping speed; a dUTP doping effect of the mutant A type DNA polymerase is obviously better than control A type DNA polymerase; therefore, the mutant A type DNA polymerase is more applicable to some nucleic acid amplification systems using dUTP to substitute dTTP (deoxy- thymidine triphosphate) and allows the system to avoid nucleic acid amplification product contamination and not to lose amplification efficiency of a target product at the same time; and the mutant A type DNA polymerase meets application requirements of multiple PCR (polymerase chain reaction) fields of food, animal quarantine, human disease screening and the like, a forensic medicine field and a scientific research.

PCT No. PCT/JP95/02254 Sec. 371 Date May 5, 1997 Sec. 102(e) Date May 5, 1997 PCT Filed Nov. 6, 1995 PCT Pub. No. WO96/14434 PCT Pub. Date May 17, 1996Disclosed is a method for determining a nucleotide sequence of DNA product amplified by polymerase chain reaction not requiring removal of primers and/or 2'-deoxyribonucleoside-5'-triphosphates and/or derivatives thereof, which comprises reacting ribonucleoside-5'-triphosphates comprising ATP, GTP, CTP, UTP and derivatives thereof and one or more of 3'-deoxyribonucleotide-5'-triphosphates comprising 3'-dATP, 3'-dGTP, 3'-dCTP, 3'-dUTP and derivatives thereof in the presence of an RNA polymerase and a DNA product which has been amplified by polymerase chain reaction and contains a promoter sequence for the RNA polymerase to afford a nucleic acid transcription product, separating the obtained nucleic acid transcription product and determining a nucleic acid sequence from the resluting separated fractions.

The invention discloses a Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method. The detection kit comprises a PCR reaction solution, an enzyme mixed solution, a positive control, a negative control and an internal standard, wherein the PCR reaction solution comprises a PCR buffer solution, nucleic acid releaser, deoxyribonucleoside triphosphate, primers for target polynucleotide amplification and probes for target polynucleotide detection; the primers comprise a forward primer and a reverse primer. Or, the detection kit comprises a PCR reaction solution, an enzyme mixed solution and an internal standard, wherein the PCR reaction solution comprises a PCR buffer solution, nucleic acid releaser, deoxyribonucleoside triphosphate, a forward primer for target polynucleotide amplification, a reverse primer for target polynucleotide amplification and probes for target polynucleotide detection. The Pseudomonas aeruginosa nucleic acid fluorescent PCR detection kit shown in the embodiment of the invention with the advantages of no need of nucleic acid extraction, high detection sensitivity, wide detection range and high detection accuracy can quickly and accurately detect Pseudomonas aeruginosa DNA (deoxyribonucleic acid) in plasma, urine and other samples.

The invention provides a HBV nucleic acid quantitative detection system based on a micro-droplet digital PCR technology, the HBV nucleic acid quantitative detection system comprises an upstream primerand a downstream primer for amplifying hepatitis B virus DNA, a specific probe for detecting the hepatitis B virus DNA; a non-infected linearizing double-stranded plasmid quality control standard product and a probe for detecting the quality control standard product; and a buffer system comprising deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dUTP, and UNG. The HBV nucleic acid quantitative detection system is based on the micro-droplet digital PCR technology to realize the accurate absolute quantification of HBV DNA, faces the clinical actual demand, and has an important application prospect.

The invention provides a fluorescent quantitation PCR (Polymerase Chain Reaction) detection kit for human papilloma viruses. The detection kit comprises a PCR reaction solution, wherein the PCR reaction solution comprises a PCR buffering solution, deoxyribonucleoside triphosphate, upstream and downstream primers used for amplifying target polynucleotide, and a probe for detecting the target polynucleotide. The fluorescent quantitation PCR detection kit for the human papilloma viruses, provided by the invention, has very good specificity and has the advantages that the operation is rapid, the method is simple and convenient and the sensitivity is high; meanwhile, an interior label, which is increased in a reaction system, can be used for effectively preventing detection from being false negative. The fluorescent quantitation PCR detection kit for the human papilloma viruses, provided by the invention, adopts a rapid nucleic acid releasing method when DNAs (Deoxyribose Nucleic Acid) are extracted; the method is simple, convenient and rapid, and heating is not needed.

The invention discloses a kit for quantitatively detecting vibrio parahaemolyticus in food and clinic sample. The kit comprises respectively packaged DNA extracting solution, PCR amplification reaction liquid, negative standard substance, positive reference material, quantitative standard substance and a plurality of sealed reagent bottles or test tubes. The invention is characterized in that the PCR amplification reaction liquid contains forward primer VP-F, reverse primer VP-R, oligonucleotide probe VP-P, heat-resistant DNA polymerase and deoxyribonucleoside triphosphate for target polynucleotide amplification. The vibrio parahaemolyticus pathogen in food samples and samples of puke, blood and dejection of patient with clinic gastrointestinal inflammation or visceral hemorrhage can be detected, thereby being capable of providing reliable experimental evidences for sensitively and quickly diagnosing vibrio parahaemolyticus pollution and infection and repetitive infection of vibrio parahaemolyticus at an early time; and simultaneously, the amount can be accurately fixed, thereby being capable of effectively monitoring clinic drug administration.

The invention discloses a kit for quickly examining loop SFTSV (severe fever with thrombocytopenia syndrome bunyavirus) mediated isothermal amplification of severe fever with thrombocytopenia syndrome bunyavirus. The kit consists of components including (1) 2.5 micro-liters of 10X amplification reaction liquid, (2) 2 micro-liters of 25mmol/l dNTP (deoxyribonucleoside triphosphate), (3) 5pmol of F3 outer primer, 5pmol of B3 outer primer, 40pmol of FIP inner primer, 40pmol of BIP inner primer, 20pmol of LF loop primer and 20pmol of LB loop primer, (4) I micro-liter of Bst (bovine somatotropin) DNA (deoxyribonucleic acid) polymerase, (5) 1 micro-liter of reverse transcriptase, (6) 1 micro-liter of fluorescent dye and (7) ddH2O. During usage, RNA (ribonucleic acid) of an examinee is added into the kit, change of colors of a reaction system is observed after amplification, and an examination result is judged accordingly. The examination kit and an examination method have the advantages that examination cost is low, operation is convenient, examination speed is fast, high specificity and high sensitivity identical to that of PCR (polymerase chain reaction) are realized, and scientific evidence for timely diagnosing and treating severe fever with thrombocytopenia syndrome is provided.

The invention discloses a method for measuring activity of terminal deoxynucleotidyl transferase. The method comprises the steps of: firstly, synthesizing a primer according to a single stranded template, annealing the single stranded template, then performing a 3' end tailing reaction catalyzed by TdT enzyme in the presence of one of four deoxyribonucleoside triphosphates, after the 3' end tailing reaction is completed, performing a polymerization reaction in the presence of enough DNA polymerase without 3'-5 'excision enzyme activity to generate double stranded DNA, finally measuring the relative quantity of the double stranded DNA or single stranded DNA in the reaction system after the polymerization reaction is finished, and deducing the activity degree of the Poly (A) polymerase according to the detection result, wherein the basic group at the first site on the used single stranded template in the 3' direction and the deoxyribonucleoside triphosphate used in the tailing reaction are not complementary. Compared with the existing method, the method disclosed by the invention is free of radioactive pollution, simple and quick to operate, low in reagent preparation cost and high in sensitivity.

The invention relates to a novel orthobunyavirus fluorescence quantitative RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) detection kit and a novel orthobunyavirus detection method, and aims to provide the kit which is convenient to use and accurate in detection and the detection method which is high in accuracy and high in detection efficiency. The technical scheme is that: the novel orthobunyavirus fluorescence quantitative RT-PCR detection kit comprises a specific primer, a fluorescent probe, a PCR buffer solution, dideoxyribonucleotide triphosphate and a mixed preparation, wherein an upstream primer has a sequence of FP:5'-TGGCCCTGGCTCTATAAACAT-3'; a downstream primer has a sequence of RP:5'-AATGGCAGCCCAAATGAATC-3'; the specific probe has a sequence of P:5'-FAM-TCCAATGAYGCCAAGAAGTGGAA-BHQ1-3'; FAM is a fluorescent reporter group; and BHQ1 is a fluorescent quenching group.

The invention provides a fluorescent quantitative PCR detecting kit for a hepatitis E virus. The detecting kit comprises a PCR reaction solution, wherein the PCR reaction solution comprises a PCR buffering solution, deoxyribonucleoside triphosphate, a DNA polymerase, an upstream primer, a downstream primer and a probe, wherein the upstream primmer and the downstream primer are used for amplifying target polynucleotide, and the probe is used for detecting the target polynucleotide. The fluorescent quantitative PCR detecting kit for the hepatitis E virus is favorable in specificity, high in operation speed, simple and convenient in method and wide in detection range, and meanwhile an interior label additionally arranged in the reaction system can be used for effectively avoiding that the detected result is false negative. When the fluorescent quantitative PCR detecting kit for the hepatitis E virus is used for extracting RNA, a magnetic bead method which is good in adsorption effect and easy to purify is used, and the method can be used for effectively removing PCR inhibitors in complex samples, so that RNA with high purity and high yield is obtained, and detecting sensitivity, accuracy and stability are greatly improved.

Provided herein are methods and kits for isothermal nucleic acid amplifications that use a target nucleic acid template; a reaction mixture comprising a DNA polymerase having a strand displacement activity, a deoxyribonucleoside triphosphate (dNTP) mixture, a primer with a 3′ end and a 5′ end, a molecular crowding reagent, and a buffer solution for amplifying the target nucleic acid template. The buffer solution maintains a low salt concentration of the reaction mixture, and wherein the salt concentration results in a melting temperature (T_m) of the primer at least 10° C. below the reaction temperature. The amplification is effected under isothermal condition.

The invention provides a perylene excimer-based detection method for methylase activity and a screening method of a methylase inhibitor, and belongs to the field of biotechnology. The method comprises the following steps: firstly, preparing double-chain DNA, and then enabling the double-chain DNA to react with S-adenosylmethionine, restriction enzyme and different concentration of methylase, so as to obtain a mixed solution; enabling terminal deoxyribonucleotidyl transferase, deoxyribonucleoside triphosphate and TdT reaction buffer to react with the mixed solution, so as to obtain a reaction solution; finally, enabling the mixed solution of a perylene derivative probe and polycation to react with the reaction solution, and carrying out fluorescence detection on the methylase activity. The invention also provides a screening method of the methylase inhibitor. The activity of the methylase and the inhibitor is detected by using the change of ratio of a small molecule probe monomer to the excimer fluorescence intensity, the ratio of two given fluorescence signals is provided from the test, the fluorescence signals are not easily interfered in comparison with a pure fluorescence-intensified or weakened signal, and the sensitivity is higher.

The invention discloses a yak rotavirus detection kit based on a constant-temperature isolation type fluorescence PCR (Polymerase Chain Reaction) platform and application. The yak rotavirus detection kit is composed of a reaction buffering solution, a fluorescence quantitative PCR reaction solution freeze-drying pipe, a positive reference substance freeze-drying pipe and a negative reference substance freeze-drying pipe. The fluorescence quantitative PCR reaction solution freeze-drying pipe is formed through mixing and freeze-drying a Taq enzyme, reverse transcriptase, a primer for detecting, a probe and dNTPs (deoxyribonucleoside triphosphate). The kit has the advantages of short detection time (reaction time is only 42min), high specificity, high sensitivity and convenience for storage (preservation at 4 DEG C); the kit is matched with a constant-temperature isolation type PCR instrument, is very suitable for on-situ rapid detection of yak rotaviruses and can be widely popularized in grassroots.

The invention relates to a gene detection kit of staphylococcus aureus, and the detection kit belongs to the field of detection of food-borne pathogenic bacteria and is specially used for detecting the staphylococcus aureus (SA). The immunocapture PCR (polymerase chain reaction) detection kit of the staphylococcus aureus is mainly characterized by comprising 5 mu L of 10*PCR buffer, 2 mu L of dNTPs (deoxyribonucleoside triphosphates) (2.5 mmol/L), 0.25 mu L of Taq DNA (deoxyribonucleic acid) polymerase (5 U/L), 2 mu L of forward primer (10 pmol/L) which refers to primer 5'-GCGATTGATGGTGATACGGTT-3', 2 mu L of reverse primer (10 pmol/L) which refers to primer 5'-AGCCAAGCCTTGACGAACTAAAGC-3' and 99%-100% of double-distilled water, as well as a detection PCR tube coated with a staphylococcus aureus polyclonal antibody, a positive control PCR tube coated with the staphylococcus aureus polyclonal antibody, namely inactivated staphylococcus aureus bacteria liquid, a negative control PCR tube coated with the staphylococcus aureus polyclonal antibody, namely sterilized PBS (phosphate-buffered solution), a positive control 1 tube and a negative control 1 tube. The immunocapture PCR detection kit has the advantages of being capable of quickly, simply and accurately detecting the pathogenic bacteria.

The invention relates to functional genomics and a gene expression detection and analysis technology, in particular to a kit for quantitative evaluation for long-term recurrent risk of colorectal cancer. The kit is characterized by consisting of 18 pairs of primers, 18 specific taqman fluorescent probes, 10* reverse transcription-polymerase chain reaction (RT-PCR) buffer solution, deoxyribonucleoside triphosphate (dNTP) mixed liquid, reverse transcriptase, deoxyribose nucleic acid (DNA) polymerase, 10* PCR buffer solution and ribonucleic acid (RNA) enzyme inhibitor. By adopting the self-designed and optimized RT primers and integrating a RT-PCR technology and a taqman real-time fluorescent quantitative PCR technology, the kit is simple and rapid in operation, more stable in diction results and lower in detection cost.

A reaction mixture for a polymerase chain reaction (PCR) includes water, DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), potassium chloride, magnesium chloride, dithiothreitol (DDT), and an additive agent. The reaction mixture is capable of enhancing the sensitivity and specificity of PCR and increasing the yield of correct nucleic acid sequence copies when being used in PCR.

The invention relates to an isothermal PCR nucleic acid amplification method based on dI modified primer and endonucleaseV. The amplified target nucleic acid primer is completely matched with the template; 1 to 5 dI nucleic acids are on the 10 to 30 site at the 5' end; 10 to 30 nucleic acids are at the downstream of the dI nucleic acids. The reaction components of isothermal PCR comprise dI modified forward primer and reverse primer, target nucleic acid template, four kinds of deoxyribonucleoside triphosphates dATP, dTTP, dCTP and dGTP, DNA polymerase, endonucleaseV, single DNA chain binding protein and helicase. After the initial thermal denaturation and annealing hybridization, the mixture of isothermal PCR carries out the PCR reaction. During the amplification process, the circulation and regeneration of the primer-template compound is realized by endonucleaseV, which induces the DNA synthesis reaction; the single DNA chain binding protein and helicase can improve the efficiency of isothermal PCR. The amplification of the target nucleic acid of the invention does not rely on the thermal cycler; the invention has easy and convenient operation, which can be used for a large scale and high yield amplification of the target nucleic acid.

The invention provides a chikungunya virus isothermal amplification detection kit and a primer thereof. The kit comprises the primer, Bst DNA polymerase, a reaction liquid and SYBR Green I fluorescent dye, wherein the reaction liquid is 10mM deoxyribonucleoside triphosphate, 10*reaction buffer liquid and 150mM MaSO4; the sequences of the primer are sequences represented by SEQ ID NO:1-4 or sequences represented by SEQ ID NO:5-8 or sequences represented by SEQ ID NO:9-12 or sequences represented by SEQ ID NO:13-16. The kit provided by the invention has the advantages of short detection time, strong specificity, high sensitivity, high detection accuracy and programmed reaction, is easy and convenient to qualify and is widely applied to conventional detection and epidemiological survey in clinic and ports.