152 results about "Dna amplification" patented technology

The invention discloses a method and device for photo-thermal amplification and rapid detection of multiple genes and application. An amplification system containing a photo-thermal amplification material is irradiated by a coaxial reflection LED light source outside 405-500nm, and isothermal amplification of a target gene is realized. According to the invention, a large number of single-stranded DNA amplification products are obtained by using asymmetric RPA, and then qualitative and quantitative detection is carried out through photo-thermal test paper. A detection area adopts a sandwich capture amplification product and a photo-thermal signal probe to generate a temperature change signal related to the content of the amplification product, and the gene is quantified according to the temperature change signal. The device comprises an LED light source, a spectroscope, a plano-convex cylindrical lens, a photo-thermal amplification system bracket, a test paper detection bracket and a light-shielding heat-insulating shell. According to the method, the photo-thermal conversion effect of a nano material is utilized, so that portable, rapid and low-cost multi-sample amplification is realized, and the portability limitation of traditional equipment is overcome.

The invention relates to the field of DNA amplification, in particular to an intelligent molecular diagnosis and analysis method, device and equipment and a storage medium. The method comprises the following steps: detecting multi-dimensional environmental parameters of the DNA amplification kit in real time based on a multi-dimensional sensing node; performing spatial topology layout mining and environment amplification perception fitting on the multi-dimensional environment parameters, and constructing a real-time amplification perception model; identifying a DNA amplification sample; performing layered temperature control analysis on the DNA amplification sample, and performing dynamic amplification constant-temperature control on the real-time amplification perception model, so as to construct a plurality of dynamic constant-temperature amplification models; carrying out regional DNA reaction degree evaluation on the plurality of dynamic isothermal amplification models one by one, and then carrying out regional flow velocity dynamic regulation, so as to construct a dynamic amplification reaction flow velocity regulation strategy; real-time amplification control is performed based on a dynamic amplification reaction flow rate adjustment strategy to generate a filter optimized amplification reaction signal. According to the method, the accuracy of molecular diagnosis analysis is improved through efficient and convenient DNA isothermal amplification.

The invention provides an intratumoral bacterium detection and analysis method based on circular DNA enrichment sequencing and related model construction, and belongs to the field of molecular biology. The intratumoral bacterium detection method provided by the invention comprises the following steps: extracting total DNA of cancer tissue or para-carcinoma tissue, carrying out linear digestion, purifying a linear digestion product, and enriching to obtain circular DNA; the method comprises the following steps: amplifying circular DNA, and breaking a DNA amplification product to 300-500bp; purifying DNA fragments, constructing a library, and sequencing; and performing genome comparison according to a sequencing result to obtain information of the intracellular bacteria. According to the method, complete bacterial genome and plasmid genome information can be captured, and the detection sensitivity of the intracellular bacteria and the richness of the bacterial genome information are improved. The invention further provides an intratumoral bacterium analysis method which can be used for screening generic cancer bacterial markers, and provides a cancer risk assessment machine learning model based on the intratumoral bacterium, so that the clinical application possibility of the intratumoral bacterium is improved.

The present disclosure relates to Taq DNA Polymerase mutants comprising at least three mutations which are capable of high levels of DNA amplification in the presence of several PCR inhibitors and compositions comprising the same. Also disclosed are methods of performing mutagenesis on wild-type Taq DNA polymerase to obtain the disclosed Taq DNA polymerase mutants, DNA amplification using the same, and cDNA amplification using the same.

The present document describes methods and systems for amplifying and quantifying amplification of a nucleic acid molecule, with a polymerase chain reaction (PCR) or a loop-mediated isothermal amplification (LAMP), by irradiating, with a heating activation light beam from a continuous wave laser a biological enzymatic reaction mixture in solution comprising a nucleic acid template, a polymerase enzyme, and chemically modified nanoparticles. Quantification of the amplification is achieved by irradiating the biological enzymatic reaction mixture during an annealing and / or elongation steps with an ultraviolet (UV) light source and measuring with a photodetector a transmission change in UV light transmission.

The invention provides a hairpin probe combination for a self-hybridization chain reaction, a kit and application, and belongs to the field of DNA amplification and detection. According to the self-hybridization chain reaction SHCR provided by the invention, a traditional HCR system which must contain two DNA hairpin probes is simplified into an SHCR system which only needs one DNA hairpin probe through simplified design by designing a palindromic sequence at the stem part of the DNA hairpin probe, so that the number of sequences in the reaction system is reduced, the design is simplified, the number of basic groups is also reduced, and the cost is reduced. Although the sequence number is reduced, the detection effect and capability are improved compared with those of the existing hybridization chain reaction HCR; the probe can also be used for detecting ATP concentration in cells, can be further applied to detection of cell survival and the like, and has a wide application prospect.

The invention discloses a composition, a detection method and a kit for SpaceAS variable splicing space in-situ detection of nucleic acid. The composition comprises a pi-type probe, a U-type probe, an O-type probe, an S-type probe and a signal probe, and the O-type probe adopts an in-vitro cyclization strategy, so that the problem of low hybridization efficiency caused by dependence on enzymatic cyclization reaction in a traditional hybridization method is effectively solved; meanwhile, an efficient probe hybridization system, a high-sensitivity signal amplification mechanism and a multi-round iterative imaging process are combined, so that the detection sensitivity and specificity are remarkably improved. The detection method has the advantages of high efficiency, high sensitivity, high specificity, good repeatability, strong expandability and the like through the synergistic effect of five-step single-stranded DNA probe hybridization reaction and one-step DNA amplification reaction, and can be widely applied to space in-situ detection and analysis of variable splicing events.

The invention belongs to the technical field of DNA amplification, and relates to a mixed enzyme system capable of amplifying circular DNA as well as application and a product of the mixed enzyme system. The invention provides a mixed enzyme system capable of amplifying circular DNA (deoxyribonucleic acid). The mixed enzyme system comprises a polymerase III tau clip loader and a catalytic core compound. The mixed enzyme system provided by the invention is remarkably superior to a traditional method in the aspects of efficiency, cost, safety, applicability, sensitivity and the like, provides a brand new solution for efficient preparation of the circular DNA, and has a wide application prospect in the field of biotechnology.

The invention relates to the technical field of biochemistry, in particular to a lateral flow analysis test strip for detecting a vascular endothelial growth factor 165. In the test strip provided by the invention, a detection line and a quality control line are marked on a nitrocellulose membrane, the detection line is coated with a VEGF165 antibody, and the quality control line is coated with a conjugate of streptavidin and a biotinylated VEGF165 aptamer complementary chain. Compared with an existing VEGF165 detection method, the method has the advantages that the VEGF165 aptamer is introduced, compared with a whole antibody system, the cost is reduced, the limitation that the variety of VEGF165 antibodies in the market is limited and most of the VEGF165 antibodies need to be imported is overcome to a certain extent, signal amplification detection can be achieved without a complex and high-cost DNA amplification technology, visual detection is achieved, and the method is suitable for large-scale popularization and application. The detection limit can be as low as 0.12 ng / mL.

The present invention discloses a double-stage amplified DNA cascade circulation system and its application, which constructs a precise imaging system through CAR+CHA+HCR. The target is calculated by the "CAR" combination chain to determine whether to release miR-21, thereby achieving accurate detection of cancer cells. The amplification system consists of a "CHA+HCR" double cycle, where CHA consists of hairpin probes H1 and H2. When miR-21 is present, the CHA cycle is opened, and the formed H1 and H2 duplexes open the HCR cycle, thereby displaying a fluorescent signal. The HCR cycle consists of H3, H 3free The two cycles each perform their own functions and complement each other, ultimately forming a large CHA+HCR system, which further enhances the fluorescence signal.

The invention discloses a preparation method and application of carassius auratus rahn aquatic OmpA gene cloning and prokaryotic expression as well as a polyclonal antibody. The preparation method comprises the following steps: culture and DNA amplification of rahn aquatic, design of an OmpA primer, gene cloning and prokaryotic expression, back multi-point subcutaneous injection of an immune rabbit, heart blood sampling and preparation of the polyclonal antibody. The invention further provides OmpA protein prokaryotic expression and protein purification, preparation of OmpA polyclonal antiserum and application of fish bodies. According to the invention, a rahnella aquatilis OmpA gene is cloned, a specific Hind III and EcoR1 double-enzyme digestion primer sequence OmpA-1F / OmpA-1R is designed, a prokaryotic expression vector pET32a-OmpA is constructed, high-immunogenicity OmpA protein is obtained through purification, OmpA specific multi-antibody serum is prepared, and the OmpA specific multi-antibody serum can be used for preparing the multi-antibody serum. The OmpA polyclonal antibody can be used for detecting and positioning the expression application of rahnella aquatica OmpA protein in crucian carp bodies through bidirectional agar diffusion, an immunoblotting method and an immunohistochemical method, and provides a reference basis for research and development and application of rahnella aquatica subunit vaccines and a molecular diagnosis technology as well as immune prevention and control of infectious diseases of the rahnella aquatica subunit vaccines and the molecular diagnosis technology.

The present application relates to a primer combination for amplifying whole genome DNA of sheep embryo cells and application thereof, and the primer combination is composed of 20 or 30 primers. The present application designs random primers based on sheep reference genome sequence, optimizes the reaction system based on MDA whole genome amplification technology, so that the whole genome DNA amplification product with high coverage and low mismatch rate can be generated by using 10 sheep embryo cells or as low as pg level genomic DNA, which can be directly adapted to downstream applications such as second-generation sequencing and targeted capture sequencing, and the detection cost is reduced.

The invention belongs to the field of detection, and discloses a method for detecting staphylococcus aureus in bulk ready-to-eat food through semi-quantitative developing, which comprises the following steps: designing a specific primer aiming at a tuf gene of staphylococcus aureus; dNA is extracted from a bulk instant food sample to be detected to serve as a template; s2, template DNA is added into an isothermal amplification reaction system for a recombinase-mediated isothermal nucleic acid amplification reaction, wherein the isothermal amplification reaction system comprises the specific primer in the step S1 and SYBR Green I fluorescent dye; after the amplification reaction is completed, a color change judgment result of a reaction system is observed: yellow green fluorescence is judged to be positive under an ultraviolet lamp, orange is judged to be negative under the ultraviolet lamp, and DNA amplification only needs 15 minutes; amplification and detection are completed in one tube and one step at the same time, and the detection result can be accurately, simply, conveniently and rapidly judged according to the presence or absence of an S-shaped curve and the change of the color of a reaction system.

The present invention provides a detection reagent for cardiac troponin I, comprising: a cTnI aptamer labeled with a first marker and a horseradish peroxidase covalent linker labeled with a second marker; the first marker and the second marker can be combined. The present invention also provides the application of the detection reagent, a detection kit and a detection method. The present invention uses a cTnI aptamer instead of a signal antibody to detect cTnI. As a molecular recognition element, nucleic acid aptamers have many excellent properties such as simple in vitro synthesis, good stability, and easy sequence design and modification, thereby reducing the difficulty of preparation while ensuring sensitivity. There is no need for complex and high-cost DNA amplification technology and nanomaterial synthesis and modification, and the method is simple and low-cost. Furthermore, the present application adds albumin as a supporting agent to the signal aptamer solution, which significantly improves the sensitivity, repeatability, applicability to complex samples and stability of the method.

Amplification device comprising a collection compartment connected to a reaction compartment by at least one linking element comprising at least one channel or cavity, for the duplication, in large numbers, of a DNA or RNA sequence of a sample mixed with a solution, part of which is transferred from the collection compartment to the reaction compartment by means of the external actuation of the linking clement

Every year, approximately 94 million cases of Salmonella gastroenteritis, with 155000 deaths, are reported each year and 85% of them reported to be food-borne. Investigation of the foods whether they are clean for Salmonella and sensitivity, easy applicability, absence of false positivity and negativity and the speed are the features sought in the analysis method for this investigation. It is not desirable for analysis to detect the presence of dead bacteria in food. Although the final product does not contain microbiologically harmful live bacteria during the food process, the detection of dead bacteria transmitted before the process causes the food product to be unfairly diagnosed as harmful. To prevent this situation, the analysis kits depending on molecular methods, increase their microorganism detection levels up to to 104 while reducing their sensitivity. Since the molecular methods cannot discriminate dead and live organisms, a confirmation test is required to prove that the positive result of the analysis belongs to the live bacteria in the food, which results in additional cost and time loss. In the same way, it is necessary to verify whether the colonies that grow in the gold standard culture method, belong to Salmonella bacteria. In the developed system; 105 dead bacterial DNA is eliminated in the food to prevent false positive results and the minimum detection limit is 10 bacteria. Also, in developed system, 4 primers specific to 6 regions of DNA are used. Therefore, the specificity of the method is very high (99.9%) and no verification test is needed. Since PCR systems require a device with complex temperature control units, they can make analysis in a laboratory-dependent manner. In the proposed system, DNA is amplified at constant temperature; no temperature cycle is required, therefore no complex instrument and laboratory infrastructure are required. All the procedures can be easily performed outside the laboratory on a portable mini-heater where pre-enrichment, DNA isolation from the sample and PCR steps are performed. For molecular analyses, the device is required to display the result of imaging or analysis. In the developed method, DNAs amplified by the loop-mediated isothermal DNA amplification method, are hybridized and combined with the labeled probe and then can be read by lateral flow method with the naked eye. As the results are visible by eye, no additional device is required. The classical culture method is accepted as the gold standard, but the duration of analysis is 7 days for positive samples, 3 days with verification test, for the molecular methods, and 5.5 hours including pre-enrichment time in the developed system.

The invention discloses a reagent and a method for improving the tolerance of a PCR inhibitor and application, and belongs to the technical field of molecular biology. According to the invention, the thermosensitive uracil DNA glycosidase and the deoxyuridine triphosphate are combined and used as the PCR antiinhibitor to be applied to DNA amplification of a whole blood sample, and experiments prove that the composition can improve the endurance capacity of a PCR reaction system to a whole blood inhibitor, reduce the false negative rate and improve the accuracy and reliability of detection. Meanwhile, the method for carrying out PCR reaction by using the composition is simple to operate, low in cost and high in nucleic acid detection sensitivity. Therefore, the reagent and the method have a good application prospect in detection of nucleic acid extracted from a whole blood sample.

The invention discloses a Phi29 DNA polymerase mutant with improved thermal stability, a construction method of the Phi29 DNA polymerase mutant and a genetically engineered bacterium. The mutant is obtained by performing single-point mutation on serine at the 527th site on an amino acid sequence shown as SEQ ID NO.2. The invention further discloses a construction method of the Phi29 DNA polymerase mutant. The mutant is realized through a single-point mutation design, and compared with a wild type Phi29 DNA polymerase, the mutant shows better thermal stability in a temperature interval of 40-70 DEG C. The Phi29 DNA polymerase mutant obtained by means of the construction method provided by the invention has better thermal stability. When being used for DNA amplification operation at a relatively high temperature, the fluorescent probe can present relatively high thermal stability, so that the fluorescent probe has relatively high application value and development potential in multiple fields of molecular biology research, clinical diagnosis and the like.

The present invention discloses a method for detecting 5-hydroxymethylcytosine in the whole genome and its application, which relates to the technical field of biochemical detection. The method comprises the following steps: 1: Blocking the 3'-OH end of the DNA to be detected; 2: Preparation of glycosylated 5hmC DNA; 3: TdT-mediated DNA amplification; 4: Fluorescence measurement. The present invention constructs a homogeneous, template-independent signal amplification method for detecting 5-hydroxymethylcytosine in the whole genome. This method is based on specific enzyme-catalyzed reactions and multiple signal amplification strategies, and has the characteristics of high sensitivity, good specificity, no need for separation steps, sequence-independent amplification, and the ability to detect 5hmC at any position in the whole genome. Moreover, the present invention can achieve accurate and sensitive detection of actual clinical samples.

The present invention discloses a primer-probe composition for detecting the heat laelaps of honey bees and a detection method thereof. Specifically disclosed is a primer pair for detecting the heat laelaps of honey bees, the primer pair being a pair of primers for specifically amplifying the COX1 gene or containing the COX1 gene fragment, and the nucleotide sequence of the COX1 gene being SEQ ID No.1. The present invention utilizes multiple strains of heat laelaps of honey bees separated from domestic popular bee colonies for DNA amplification and sequence analysis comparison, screens the conservative nucleic acid sequence as the target, and develops a primer-probe composition for detecting the heat laelaps of honey bees and a real-time fluorescence PCR detection method. The primer-probe composition and the detection method of the present invention have high sensitivity, a minimum detection limit of 2.0 copies / μL, and good specificity, and have no cross reaction with other bee mites, and can be used for the detection of the heat laelaps of honey bees and the identification of the heat laelaps of honey bees from other bee mites, as well as the quarantine of imported and exported bees and bee products.

The invention discloses specific DNA (deoxyribonucleic acid) amplification primers for detecting large-fragment translocation of three rice varieties and application of the specific DNA amplification primers. The three varieties are respectively an indica type rice variety BANDI, an indica type rice variety LUDI GOCHYA and an Aus variety Kasalath. The total number of the specific DNA amplification primers is four, and the specific DNA amplification primers comprise TG7-CHR12, TG58-CHR8-1, TG58-CHR8-2 and TG87-CHR5. On the specific primers, the 2251bp band, the 4207bp band, the 3813bp band and the 4113bp band can be detected in sequence, and the genotypes are chromosome translocation genotypes. The four specific primers are used for amplifying translocation type and non-translocation type varieties, and the result shows that the markers can accurately identify whether the rice chromosome translocation exists or not. The DNA marker designed by the invention is high in accuracy and strong in specificity, and has relatively strong practicability in molecular breeding.

The present application describes a method for effectively amplifying and detecting certain nucleic acid sequences in a population. The selected population can be further characterized, for example, by sequencing. The method includes combined liquid and solid phase amplification.

The invention discloses a material of light-triggered amplification-photo-thermal detection test paper for tacA, tacB and tacC. A sample is irradiated by a coaxial reflection LED light source outside 504nm, a photosensitizer CsYF4 (at) Yb2O3 provides photoelectrons and heat for aRPA through light triggering, a target gene in a positive sample is used as a template to initiate an amplification reaction, three single-stranded DNA amplification products are obtained, and the three single-stranded DNA amplification products are used for detecting the tacA, the tacB and the tacC; a photosensitizer CsYF4 (at) Yb2O3 is modified by three nucleic acid chains to obtain three signal probes, a detection region captures an amplification product and the signal probes in a sandwich form, a photo-thermal temperature change signal positively related to the content of the amplification product is given, and tacA, tacB and tacC in a sample are quantified according to the temperature change signal. Photoelectron and photothermal effects are generated by using the photoactivity of the photosensitizer CsYF4 (at) Yb2O3, the temperature is rapidly controlled, the photosensitizer is in direct contact with an amplification system on a molecular level, photothermal detection test paper detection is carried out, and the photothermal efficiency is remarkably improved.

The present invention provides an in vitro method for detecting cell-cell interactions and related kits and uses, wherein the method comprises: (a) at least two primary binding agents, wherein a first primary binding agent binds to a first molecule on a first cell and a second primary binding agent binds to a second molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically different; (b) at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; The first and second secondary binders are conjugated or fused to DNA sequences, wherein the DNA sequences are different and connected to form a circle and are amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or (iii) the first secondary binder is labeled with a FRET donor and the second secondary binder is fused to an enzyme, which reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich portion on the surface of a molecule adjacent to the enzyme; wherein the method comprises: i. contacting an isolated sample containing cells with the at least two primary binders; ii. contacting the sample with the at least two secondary binders; iii. performing a washing step; iv. detecting the interaction between the secondary binders.

The application belongs to the technical field of DNA amplification, and has a mixed enzyme system capable of amplifying circular DNA, application and product thereof. The application provides a mixed enzyme system capable of amplifying circular DNA, wherein the mixed enzyme system comprises a polymerase III tau clamp loader and a catalytic core complex. The mixed enzyme system provided by the application is significantly superior to traditional methods in terms of efficiency, cost, safety, applicability and sensitivity, and provides a brand-new solution for efficient preparation of circular DNA, and has a wide application prospect in the field of biotechnology.

The invention belongs to the technical field of microorganisms, and particularly relates to a specific detection method for survival of fusarium pseudograminearum in soil. The method comprises the following steps: adding PMA into a soil sample, carrying out mixed incubation and exposure treatment, extracting DNA, carrying out qPCR detection, preventing DNA amplification of dead cells in total DNA, and determining the number of viable bacteria of fusarium pseudograminearum in the soil. According to the detection method, the optimal PMA concentration is 50 [mu] mol L <-1 >, the optimal exposure time is 15 min, dead and surviving fusarium pseudograminearum spores are distinguished, and the lowest detection limit is 628 spore g <-1 >. The invention provides a rapid, sensitive and reliable method which is used for quantitatively detecting the surviving fusarium pseudograminearum in the soil, and the method has the advantages of high sensitivity, high sensitivity and high sensitivity. Accurate and reliable data are provided for monitoring and early warning, early diagnosis and prevention and control measures of the wheat basal stem rot.

The present disclosure relates to Taq DNA Polymerase mutants comprising at least three mutations which are capable of high levels of DNA amplification in the presence of several PCR inhibitors and compositions comprising the same. Also disclosed are methods of performing mutagenesis on wild-type Taq DNA polymerase to obtain the disclosed Taq DNA polymerase mutants, DNA amplification using the same, and cDNA amplification using the same.

The invention relates to the technical field of plant breeding and biology, in particular to a loquat transgenic method mediated by a magnetic nano material. According to the method, a red fluorescent plant expression vector containing a loquat EjIPUT gene is constructed, and the full length of the gene sequence is as shown in SEQ ID No.1. EjIPUT genes and magnetic nano Fe3O4 particles form a compound through electrostatic force, the compound and loquat pollen are mixed in a pollen culture solution by standing on a magnetic frame, pollen precipitates containing the compound are obtained after standing, the precipitates are dried and pollinated to castrated stigmas, and reproductive isolation is performed. Finally, DNA amplification identification, fluorescence identification, expression quantity identification and the like show that a loquat transgenic plant over-expressing the EjIPUT gene is obtained.

Disclosed herein are DNA amplification methods for quantifying DNA fragments of a target DNA in a sample by size. This can be used, for example, to detect tumor-derived viral DNA in blood sample and distinguish it from larger viral DNA from non-tumor sources. In particular, disclosed herein are methods of detecting, monitoring or treating a human papilloma virus (HPV)-associated malignancy in a subject that involves detecting a presence or absence of at least one circulating tumor-derived HPV DNA in a sample from the subject. Kits for accomplishing the same are also provided.