37 results about "Nucleic acid amplification technique" patented technology

To provide high sensitivity and reliable data when performing PCR at high speed.SOLUTION: While applying thermal cycles at high speed reduces amplification efficiency and detection sensitivity, performing multiple thermal cycles to maximize an amount of amplified products in a short time also increases an amount of non-specific amplified products, causing a decrease in S / N and false positives, reducing the reliability of analysis results. For this reason, by changing the thermal cycle midway, it is possible to increase an amount of target products amplified at high speed and suppress non-specific amplification. More specifically, this disclosure proposes a nucleic acid amplification technique that includes a first phase in which a denaturation reaction is performed on a sample solution for a seconds, followed by an annealing and extension reaction for b seconds, and a second phase (b≠d) in which a denaturation reaction is performed on the sample solution after a first phase process is performed, for c seconds, followed by an annealing and extension reaction for d seconds.SELECTED DRAWING: Figure 2

In the present disclosure, during performance of PCR at a high speed, in order to provide data with high sensitivity and high reliability, application of a thermal cycle at a high speed reduces the amplification efficiency and reduces detection sensitivity, whereas performing multiple thermal cycles in order to maximize the amplification product amount in a short time also increases the non-specific amplification product amount, causes an S / N reduction and / or pseudo-positive determination, and reduces reliability of an analysis result. Thus, an increase in amount of amplification of the target product amount and suppression of non-specific amplification are achieved at a high speed by changing the thermal cycle midway. More specifically, the present disclosure proposes a nucleic acid amplification technology comprising: a first phase in which a sample solution is subjected to a denaturation reaction for a second(s) and then subjected to an annealing and extension reaction for b second(s); and a second phase in which the sample solution that has been subjected to the first phase step is subjected to the denaturation reaction for c second(s) and then subjected to the annealing and extension reaction for d second(s) (b≠d) (see fig. 2).

The present invention discloses a method for detecting Cronobacter sakazakii and its application. The method comprises: identifying a sequence rich in AT bases in the genome sequence of Cronobacter sakazakii as a target sequence; designing specific primers for the target sequence in an automated and high-throughput manner; designing a specific Tm value calculation formula to calculate the reaction denaturation and annealing temperature, and setting the nucleic acid amplification reaction conditions accordingly; and performing a nucleic acid amplification reaction under the condition of local melting of the template. The present invention targets sequences rich in AT bases that are usually avoided by traditional nucleic acid amplification methods, greatly broadening the candidate target region; through specific screening based on massive genomic data, and accurate calculation of the Tm value for AT-rich regions, local melting of double-stranded DNA is achieved, reducing the possibility of nonspecific amplification in nucleic acid amplification technology. This method effectively expands the scope of application of traditional nucleic acid amplification technology, while significantly improving reaction performance in terms of primer design flux, reaction specificity, etc.

The present invention discloses a method for detecting Shigella and its application. The method includes: identifying a sequence rich in AT bases in the Shigella genomic sequence as a target sequence; automatically and high-throughput designing specific primers for the target sequence; designing a specific Tm value calculation formula to calculate the reaction denaturation and annealing temperatures, and setting nucleic acid amplification reaction conditions accordingly; and performing nucleic acid amplification reaction under the condition of local strand separation of the template. The present invention targets sequences rich in AT bases that are usually avoided in traditional nucleic acid amplification methods, greatly broadening the candidate target regions; through specific screening based on a large amount of genomic data and precise calculation of the Tm value for AT-rich regions, local strand separation of double-stranded DNA is achieved, greatly reducing the possibility of non-specific amplification in nucleic acid amplification technology. This method effectively expands the application scope of traditional nucleic acid amplification technology and significantly improves the reaction performance.

The present invention discloses a method, detection system, and application thereof for simultaneously detecting multiple bacteria. The method comprises screening target sequences rich in AT bases in different bacterial genome sequences; designing primers that are both universal and specific for a single bacterial genome sequence; calculating the reaction denaturation temperature and annealing temperature based on the GC percentage, primer sequence length, and theoretical amplification product sequence length of the primer pair; screening suitable single bacterial primer pairs based on the sequence amplification length, reaction denaturation temperature, and annealing temperature to form a primer set for detecting multiple bacteria; setting nucleic acid amplification reaction conditions, performing a nucleic acid amplification reaction under conditions that partially melt the template, and obtaining an amplified product. The present invention achieves partial melting of double-stranded DNA through specific screening based on massive genomic data and accurate calculation of the Tm value of AT-rich regions, greatly reducing the possibility of nonspecific amplification in nucleic acid amplification technology.

The present invention discloses a T7 RNA polymerase mutant with high thermal stability and its application, relating to the field of biotechnology. The mutants include C125A, C216L, Q269M, C347L, M401K, W422F, Y457L, G464M, A468F, F481W, Q505A, C510E, C515P, V567P, V687E, M696A, A703T, H772K, P780K, R792M, H799G, K801G, E830G, C839N, P865L and / or A881F; the amino acid sequences of the mutants are shown in SEQ ID NO. 3 and SEQ ID NO. 28, respectively. Methods for selecting the mutation sites, expressing the mutants, measuring the melting temperature, and measuring the activity of the mutants are also disclosed. This mutant can undergo transcription reactions at high temperatures, reducing byproducts in the preparation of mRNA vaccines and drugs, and is suitable for isothermal nucleic acid amplification techniques. This invention utilizes deep learning to predict amino acid mutation sites, improve protein thermal stability, and rapidly screen for variants with high-temperature activity and stability. The new site has a significant effect on improving stability.

The invention discloses a T7 RNA polymerase mutant with high thermal stability and application of the T7 RNA polymerase mutant, and relates to the technical field of biology, the T7 RNA polymerase mutant comprises mutants C125A, C216L, Q269M, C347L, M401K, W422F, Y457L, G464M, A468F, F481W, Q505A, C510E, C515P, V567P, V687E, M696A, A703T, H772K, P780K, R792M, H799G, K801G, E830G, C839N, P865L or / and A881F; amino acid sequences of the amino acid sequences are respectively shown as SEQ ID NO. 3 to SEQ ID NO. 28. The invention discloses a mutation site selection method, a mutation site expression method, a melting temperature measurement method and an activity measurement method. The mutant can be subjected to transcription reaction at high temperature so as to reduce by-products generated in preparation of mRNA vaccines and drugs and is suitable for a nucleic acid isothermal amplification technology. According to the invention, mutation sites of amino acids are predicted by using deep learning, the thermal stability of proteins is improved, and variants with high-temperature activity and stability are rapidly screened out. The new point location has a good stability improvement effect.

Methods and kits for the rapid detection of active coronavirus infection by specifically targeting the negative-sense RNA produced during viral replication. The method utilizes nucleic acid amplification techniques, such as RT-PCR and qPCR, with primers specific to the negative strand, which serves as a marker for active viral replication. The present disclosure also encompasses a rapid, user-friendly lateral flow test based on CRISPR-Cas13a technology that targets the negative-sense viral RNA. This LFT employs guide RNAs specific to the negative strand and a reporter system to visually indicate the presence of actively replicating viruses, thereby distinguishing active infections from the presence of non-replicating viral remnants. The methods and kits offer a more accurate means of determining infectivity, guiding public health measures, and monitoring antiviral therapies.

The invention belongs to the technical field of in-vitro diagnostic reagents for rapid detection of human pathogenic microorganisms, and particularly relates to a kit for rapid combined detection of multiple pathogen nucleic acids related to a dyspnea pregnancy outcome and application of the kit. The pathogen is selected from one or more of escherichia coli, candida albicans, candida glabrata, group B streptococcus, streptococcus gonorrhoeae, chlamydia trachomatis or ureaplasma urealyticum, and the kit comprises a pathogen and lactobacillus rapid detection micro-fluidic chip. A primer group for detecting the pathogen and the lactic acid bacillus nucleic acid and an array consisting of an amplification control and a blank control are fixed on the micro-fluidic chip. According to the present invention, the microfluidic chip rapid detection technology and the nucleic acid amplification technology are adopted to achieve the purposes of rapid detection and multiple detection so as to rapidly and conveniently determine the poor prognosis risk of the pregnant woman at present, timely take the specific measures, and avoid the occurrence of the poor prognosis.

The present disclosure provides methods and systems for processing a nucleic acid sample. The present disclosure also provides methods and systems for detecting (e.g., optically detecting) a pathogen in a sample. The methods provided herein may use nucleic acid amplification.

The application relates to the field of nucleic acid amplification technology and provides a full-automatic nucleic acid amplification system integrated with microwave temperature control and a working method thereof.The system comprises a microprocessor, a temperature heating module, a nucleic acid amplification container, a microwave generator connected with the microprocessor and a thermocouple, the microwave generator is connected with the temperature heating module, the temperature heating module adopts a planar microwave resonator or a waveguide, the microwave generator is used for outputting microwave signals with specific frequency and power, the temperature heating module is used for directing the microwave signals to nucleic acid amplification reagents in the nucleic acid amplification container to heat the temperature of the nucleic acid amplification reagents, the thermocouple is used for monitoring the temperature of the nucleic acid amplification reagents and uploading the temperature to the microprocessor, and the microprocessor is used for controlling the frequency and power of the microwave signals according to the temperature of the nucleic acid amplification reagents.The temperature can be rapidly raised, the sample volume is reduced, and the nucleic acid amplification time is shortened.

The present invention provides a single-molecule detection and analysis method for detecting low-abundance biomarker proteins based on proximity ligation, which solves the technical problems of low sensitivity and low precision of existing real-time fluorescence quantitative PCR detection methods based on PLA. The present invention divides the reaction system into a plurality of micro-droplet systems, completes QPCR or PCR cyclic amplification reactions inside the micro-droplets, realizes signal amplification, and thus accurately quantifies the protein concentration. The present invention evenly distributes the reaction system in a plurality of droplet systems, performs signal amplification on them through nucleic acid amplification technology, thereby improving the signal detection rate of low-concentration proteins in the droplets, and can perform multiple protein detection quantification.

The invention relates to a suction-driven micro-fluidic device for real-time nucleic acid detection, provides an RNA (Ribonucleic Acid) detection system and application thereof, and in particular relates to a dual-nuclease-assisted isothermal cycle amplification integrated detection system and an RNA detection method based on the detection system. The detection system is composed of an initial reaction based on Cas13a nuclease and a cyclic amplification reaction based on a PDANSs biosensor. The initial reaction and the cyclic amplification reaction can be integrated in a single reaction cavity, the dependence on the nucleic acid amplification technology is eliminated, and the integrated RNA quantitative detection system is high in sensitivity and specificity.

The invention discloses a four-channel centrifugal micro-fluidic nucleic acid amplification chip with closed cavities, and relates to the technical field of micro-fluidic cores. Comprising a chip main body, a plurality of detection units are uniformly arranged along the circumference, each detection unit comprises a plurality of adhesive film valves, a plurality of amplification cavities, a plurality of quantitative cavities, a liquid injection cavity and a waste liquid cavity, the liquid injection cavities and the waste liquid cavities are communicated with the quantitative cavities, the adjacent quantitative cavities are communicated, each quantitative cavity is communicated with one adhesive film valve through a quantitative cavity liquid outlet channel, and each quantitative cavity is communicated with the other adhesive film valve through a quantitative cavity liquid outlet channel. The glue film valve is communicated with the amplification cavity through the amplification cavity liquid inlet flow channel, the cold aluminum with glue is arranged in the glue film valve, external force extrudes the glue film valve, glue in the cold aluminum with glue blocks a liquid outlet of the glue film valve, and therefore the amplification cavity is sealed. The device is low in manufacturing cost, can automatically close the chamber, ensures that liquid in the chamber does not leak in the amplification process, is particularly suitable for popularization and application in the field of instant inspection, and is suitable for two nucleic acid amplification technologies of isothermal amplification and variable-temperature amplification.

The invention provides a fluorescent sensing probe system and method for detecting ovarian cancer miRNA based on bidirectional primer exchange reaction, and belongs to the technical field of biological analysis and detection.According to the scheme, target miRNA and hairpin specific recognition-mediated bidirectional primer exchange reaction technology is creatively utilized, and through a double-primer system and a bidirectional strand displacement mechanism, the ovarian cancer miRNA can be detected through the fluorescent sensing probe system. The tandem G-rich repetitive sequence can provide hundreds of tandem binding sites for a signal molecule ThT, and a G tetramer-ThT tandem activation signal amplification system is constructed. According to the system, specific recognition of target miRNA and a hairpin and effective combination of a double-primer system and a two-way strand displacement mechanism mediated nucleic acid amplification technology are fully utilized to develop a serum-based'one-tube 'ovarian cancer specific miRNA accurate quantitative detection technology, and efficient, accurate and rapid detection of a novel ovarian cancer biomarker miRNA can be realized.

The invention relates to the technical field of immunodetection, and discloses an immunodetection method and system based on a piezoelectric biosensor, and the method comprises the following steps: under the condition that an antigen to be detected in the piezoelectric biosensor is combined with a capture antibody, adding a connecting antibody and DNA into the piezoelectric biosensor; dNA is amplified through a nucleic acid amplification technology; and analyzing the to-be-detected antigen according to an electric signal acquired by the piezoelectric biosensor after DNA amplification. According to the invention, the specificity of immunodetection and the ultrahigh sensitivity of nucleic acid detection are connected through the piezoelectric biosensor, and a direct signal which is originally weak and is easily submerged by noise can be converted and amplified into a strong output signal with extremely high specificity, so that the signal-to-noise ratio and the target substance detection sensitivity are greatly improved; and the detection limit of the piezoelectric biosensor is further reduced.

The present invention belongs to the technical field of genetic engineering, in particular to a method for detecting classical swine fever virus based on a G4-ThT biosensor and NASBA and a kit thereof. The specific method includes using the viral RNA extracted from a classical swine fever virus sample as the amplification target, performing isothermal amplification using an isothermal nucleic acid amplification technique, then adding a buffer solution containing Tris-HCl and KCl, and further adding thioflavin T to promote the folding of G-quadruplexes on the amplification products to form a complex, detecting fluorescence under the excitation of a light source with a specific wavelength, and making a judgment according to the intensity thereof. The present invention provides a probe that does not require special biotin labeling or thiol modification treatment, a detection method with significantly reduced detection costs, and a kit.

This invention belongs to the field of gene detection technology, specifically relating to a kit and detection method for rapid detection of gene variations based on click chemistry and a light-up click ligation probe. The light-up click ligation probe in the kit consists of two oligonucleotide fragments, head-to-tail linked and complementary to the target sequence: a 5'-terminal oligonucleotide fragment and a 3'-terminal oligonucleotide fragment complementary to the target sequence. Both the 3'-end of the 5'-terminal oligonucleotide fragment and the 5'-end of the 3'-terminal oligonucleotide fragment are labeled with click chemistry functional groups. When the target sequence is present, during nucleic acid amplification, the target sequence triggers a click chemistry reaction, and the light-up click ligation probe, complementary to the target sequence, releases a fluorescent signal, enabling real-time parallel synchronous detection of wild-type and mutant genes of the target gene in a single reaction tube or at a single detection point. This invention is applicable to various nucleic acid amplification technologies and possesses the ability to accurately identify single-base variations.

The invention relates to a method for detecting changes of nucleotide sequences by using an isothermal nucleic acid amplification technology, a primer set for detecting selected genetic variants and components of a reaction mixture.

The invention discloses a preparation method of a self-powered biosensor for detecting patulin. According to the biosensor, a cathode and an anode of an electrochemical / colorimetric dual-mode sensor are constructed based on a gold nanoparticle / modified Prussian blue compound AuNPs / PB, carbon cloth, a DNA chain and a biological enzyme. By using the biosensor provided by the invention, a nucleic acid amplification technology is applied to sensitive and specific detection of patulin in bananas, and quantitative analysis of a target analyte is realized through signal amplification. The electric signal value and the RGB Blue value of the electrolyte are in linear correlation with the concentration of the patulin in the target detection object, so that sensitive, specific and accurate detection of the patulin in the banana is realized. The invention solves the problem of patulin detection. The biosensor does not need an external power supply in the detection process, and is low in cost, simple in instrument and easy to realize miniaturization.

The invention discloses a cadmium ion analysis method based on a feedback type HCR-Exo reaction, and belongs to the field of food safety detection. According to the method, the trigger chain T triggers the synergistic effect of HCR and Exo III, so that two nucleic acid amplification technologies are alternately carried out, and signal exponential amplification is realized. As the initiation chain T can be efficiently regenerated and recycled in the reaction process, the system can obviously enhance an electrochemical luminescence signal, thereby improving the detection sensitivity. Experimental results show that the detection limit of the system to cadmium ions is as low as 0.13 pM, and the system has ultra-sensitive detection performance. Besides, the system has the characteristic of high modularization, can be flexibly adapted to different recognition elements by replacing the recognition sequence of the hairpin probe, constructs a universal biosensing platform, and has a wide application prospect.

A method and system comprising a programmed computer that can be used to improve nucleic acid analyte detection using real-time amplification and monitoring, the method and system capable of discriminating closely related nucleic acid sequences from one another. In one embodiment, the detection of target sequences in which single nucleotides are different from each other was discriminated by applying a mathematical transformation. In another embodiment, two target sequences in which single nucleotides are different from each other were detected and discriminated from one another using a labeled probe specific to only one of the two target sequences.

The present invention provides a purpose of a thermostable Cas protein and a detection system and a kit for a target nucleic acid molecule. Specifically, the present invention provides a reaction system for detecting a target nucleic acid molecule, wherein the reaction system includes: a guide RNA, Cas12b (formerly known as C2c1), and a nucleic acid probe, which is detected after the reaction is completed. In addition, by combining with nucleic acid amplification techniques (such as LAMP, etc.), the sensitivity of the detection method can be greatly improved. The detection system provided by the present invention can be used for rapid detection of pathogenic microorganisms, gene mutations, single nucleotide polymorphisms, and specific target DNA, etc., and nucleic acid samples can be quantified.

Provided herein are compositions and methods for accurate and scalable single cell multiomics methods, and their applications for mutational analysis in research. diagnostics, and treatment. Further provided herein are multiomics methods for parallel analysis of DNA, RNA, and / or proteins from single cells using Primary Template-Directed Amplification (PTA) nucleic acid amplification.

The invention relates to a nucleic acid amplification method based on a monodisperse framework material and application thereof, and the nucleic acid amplification method comprises the following steps: mixing the monodisperse framework material with a PCR reaction system, and carrying out amplification according to a PCR program; wherein the monodisperse framework material comprises any one or a combination of at least two of a metal organic framework material, a mesoporous silicon material or a mesoporous carbon material, and the aperture of the monodisperse framework material is 3-7nm. The method provided by the invention can be suitable for various nucleic acid amplification technologies, especially for accurate quantification of low-abundance nucleic acid samples, and effectively prevents amplification products from diffusing outwards and the environment from being polluted.

The invention discloses application of a synovial fluid source exosome miRNA marker in an osteoarthritis diagnosis product, and belongs to the field of biological medicine detection. The synovial fluid source exosome miRNA marker is miR-3196, and the reagents comprise reagents for detecting the expression level of the synovial fluid source exosome miRNA marker miR-3196 by a sequencing technology, a nucleic acid hybridization technology and a nucleic acid amplification technology. Experimental verification finds that miR-3196 is remarkably low in expression in a synovial fluid exosome of an OA patient, after the miR-3196 low in expression in the exosome is taken by cartilage cells, related signal channels are activated and degradation of the cartilage cells is promoted by regulating MAPK15 in the cartilage cells in a targeted mode, and the miR-3196 can be applied to early clinical diagnosis of osteoarthritis.

The utility model provides an amplification device for nucleic acid detection, and relates to the technical field of nucleic acid amplification, the amplification device comprises a frame body, the right side of the upper end of the frame body is provided with a water cooling head, the upper end surface of the water cooling head is provided with a Peltier, the top of the Peltier is provided with a fluorescent quantitative plate, and the middle part of the upper end of the frame body is movably provided with a movable frame; a film sealing hot cover is arranged at the bottom of the inner side of the movable frame; and the lifting driving assembly is used for driving the film sealing hot cover to do vertical lifting motion, the lifting driving assembly comprises a servo motor, the servo motor is fixedly installed at the left end of the inner side of the movable frame, a gear is arranged at the output end of the servo motor, and the side end of the gear is connected with a rack in a meshed mode. The amplification device provided by the utility model realizes film sealing and PCR functions, and has the advantages of stronger instrument compatibility, simpler experimental operation, more reliable PCR result and better stability; the film has a heat sealing function and is matched with an aluminum foil heat sealing film, so that the effects of high efficiency, low cost, tight film sealing and evaporation prevention are achieved.

The application discloses a primer 5' end reverse complementary fluorescent PCR, belongs to the field of molecular biology-nucleic acid amplification technology, and is characterized in that a pair of primers are added with a sequence reverse complementary fluorescent PCR amplification at the 5' end, which brings about end complementarity between amplification products, and the 3' end of a target product can also be used as a template and a primer to increase the amplification efficiency. 9‑12 On the basis of the conventional PCR, the sensitivity is increased by 50 times, or 5 cycles of amplification can be saved under the condition of the same sensitivity; on the other hand, the 5' end complementarity of the primer cannot be extended, but can inhibit the polymerization of the original primer to the 3' end, and 5 cycles of non-specific amplification can be avoided. The primer with the same sequence in the middle and the primer with the A adenine at the last 1-2 bases of the 3' end + UDG enzyme dU instead of dT of the amplification product, and mineral oil sealing can prevent the generation of primer dimers PD in the PCR reaction cycle and eliminate the cross contamination of product aerosol glue.

The application discloses a double-detection self-powered sensor for detecting sugarcane diseases, application and a detection method thereof, and comprises a biological anode based on nucleic acid amplification product M, a biological cathode based on Au@MB and a self-powered sensor constructed by an electrolyte; wherein the electrolyte comprises glucose, methylene blue PBS buffer and a photothermal test solution. The self-powered sensor can efficiently amplify the nucleic acid of the pathogen of sugarcane smut and shoot rot through a nucleic acid amplification technology, so that the sensor can detect the pathogen at a low concentration, and the missing detection rate is effectively reduced. Meanwhile, multi-mode synergistic detection (electrochemistry, colorimetry and photothermal) further improves the accuracy and specificity of detection, avoids the misjudgment caused by signal cross interference in the traditional method, and ensures the reliability of the detection result.

The invention provides a gene, a primer probe combination and application for species identification of giant bumblebee, and belongs to the technical field of biology.The gene, the primer probe combination and the application have the advantages that conserved sequence fragment COI genes of giant bumblebee are screened as targets through massive DNA (deoxyribonucleic acid) sequencing and sequence comparative analysis, and optimal primers and specific fluorescent probes are selected; the method has the advantages that double control on a target in a real-time fluorescent PCR reaction process can be realized, the specificity on species identification of the giant bumblebees is very high, cross reaction with other bumblebee species is avoided, and the sensitivity is high and can reach about 2.5 copies / mu L. Compared with a recombinase polymerase isothermal nucleic acid amplification technology, the method has the advantages that the requirement on the length of the real-time fluorescent PCR primer is short, a primer dimer is not easy to generate, the amplification effect is better, and the repeatability is better. Meanwhile, data are collected through real-time fluorescence, the amplification condition of nucleic acid does not need to be detected through electrophoresis, and the whole reaction can be completed within one hour. The operation is simple and fast.