19 results about "Nucleic acid quantitation" patented technology

The invention provides a centrifugal micro-fluidic chip for a method for rapidly enriching nucleic acid by microbial biochar, and aims at a solid-phase carrier for adsorbing nucleic acid, and the centrifugal micro-fluidic chip capable of completing the whole process of nucleic acid elution, amplification and detection is designed. A quantitative cavity, an elution cavity and a detection area are arranged in the chip, the elution cavity is used for eluting nucleic acid to be detected from biochar, and the quantitative cavity is used for temporarily storing added eluent and quantifying the added eluent to accurately complete the elution process; the structure of the quantitative cavity, the structure of the elution cavity and the structure of the fluid channel are improved, and steps are additionally arranged in the fluid channel, so that a to-be-detected sample smoothly enters a detection area for detection through the capillary tube under the action of surface tension, and the capillary tube can be prevented from being blocked by solid-phase carriers used in pretreatment such as charcoal and magnetic beads; the accuracy of a detection result is ensured; the chip is simple in structure, low in cost, convenient to assemble and easy to layer and sterilize, can simultaneously realize high-throughput detection of more samples and more detection items, and improves the detection efficiency.

The current invention relates to a PCR detection system comprising a reaction mixture for analyzing, quantitating or detecting a target nucleic acid, wherein the reaction mixture comprises: a. a first primer, wherein said first primer comprises a region configured to selectively hybridize to said target nucleic acid under amplification conditions and a first 5'-universal tail which is not complementary to the target nucleic acid sequence; b. a first universal detection probe, wherein said first universal detection probe comprises a first fluorophore, characterized in that the first 5'-universal tail of said first primer comprises a nucleotide sequence, the complement of which allows selective hybridization to a nucleotide sequence comprised in said first universal detection probe. The current invention further relates to methods and uses of aforementioned PCR detection system.

The invention is suitable for the technical field of biological detection, and provides a single-tube integrated Cas12a-driven multiple DNA cyclic amplification three-mode signal detection platform and application thereof. According to the invention, a single-tube integrated CHA-RCA-CRISPR (CRC) multi-layer DNA cyclic amplification system is constructed, so that the experimental process is simplified, the complexity is reduced, and meanwhile, the risk of cross contamination of nucleic acid samples is reduced; the system is combined with an AuNPs-FAM probe to realize fluorescence, colorimetric and photo-thermal three-mode nucleic acid detection, detection is completed within 20-120 minutes, the sensitivity can reach an attomole (aM) level, and high-sensitivity, rapid and reliable SARS-CoV-2 nucleic acid quantitative detection is realized; complex instruments are not needed, and the requirements of on-site rapid detection and multi-scene actual detection are met; the modular design of the system can be expanded to nucleic acid detection of various pathogens, is suitable for the fields of clinical diagnosis, environmental monitoring, public health prevention and control and the like, and has wide application prospects.

The invention relates to the technical field of nucleic acid detection, and provides an ultra-high-speed qPCR instrument which comprises an air pressure generation component, a micro-fluidic chip, a liquid driving component, a temperature control component and a signal acquisition component, the air pressure generation component is used for generating at least one path of positive pressure and one path of negative pressure; a fluid pool, a fluid channel and at least two reaction cavities are arranged in the micro-fluidic chip, and the reaction cavities are used for accommodating PCR reaction liquid and carrying out temperature control reaction; the liquid driving part is respectively connected with the air pressure generating part and the micro-fluidic chip, and is used for driving the PCR reaction liquid to flow in the fluid channel and transfer among different reaction cavities through positive pressure or negative pressure; the temperature control parts are correspondingly arranged below the reaction cavities and are used for maintaining the reaction cavities at different set temperatures; the signal acquisition part is arranged at at least one reaction cavity and is used for acquiring a fluorescence signal when the PCR reaction liquid is positioned in the reaction cavity; the method meets the ultra-high-speed detection requirement, and improves the accuracy of a nucleic acid quantitative result.

The invention discloses a calibration method of a nucleic acid quantitative fluorometer, which adopts a methylene blue aqueous solution with stable chemical property as a standard substance to replace a biological nucleic acid reagent, and verifies the optical performance of the nucleic acid quantitative fluorometer by constructing a linear response relationship of a chemical standard substance. The method comprises the following steps: preparing a methylene blue mother solution and a gradient calibration solution, and carrying out light-shielding standing and thermodynamic stabilization treatment; collecting a fluorescence signal by using a nucleic acid quantitative fluorometer to construct a standard curve; selecting a verification sample for reverse measurement, and calculating an indication error, a channel range and measurement repeatability; and finally, judging whether the calibration result is qualified or not through hierarchical logic including linear validity verification and metrological characteristic subentry verification. The problems that an existing RNA kit is high in calibration cost, poor in linearity and unstable in result are solved, and the method has the advantages of being low in cost, easy and convenient to operate, high in result confidence coefficient and the like.

The invention discloses a nucleic acid absolute quantification method based on capillary electrophoresis, and relates to the field of nucleic acid quantitation.The method comprises the steps that a purified nucleic acid calibration product with the same nucleotide sequence and length as each target nucleic acid target is prepared for the target nucleic acid target, and the calibration product is subjected to series dilution to obtain calibration working solutions with multiple concentration gradients; aiming at each target nucleic acid target, establishing a quantitative calibration curve based on the normalized electrophoresis signal obtained by each calibration working solution corresponding to the target nucleic acid target under the target cycle number and the initial concentration, and substituting the normalized electrophoresis signal obtained by the sample to be detected under the target cycle number into the corresponding quantitative calibration curve; therefore, accurate backstepping of the initial concentration of the target nucleic acid target in the to-be-detected sample is realized, and the problem that an existing fragment analysis technology cannot trace to an absolute copy number due to nonlinearity of PCR amplification is solved.

The present invention relates to the field of digital PCR, and specifically to a multi-layer three-dimensional digital nucleic acid amplification chip and a method for making and using the same. In the multi-layer three-dimensional digital nucleic acid amplification chip of the present invention, multi-layer superposition of microarray chips can be achieved, greatly increasing the number of reaction units for digital nucleic acid amplification, so that the dynamic range of nucleic acid quantification and the accuracy of the lower limit of detection are greatly improved, which is conducive to greatly increasing the reaction flux in actual detection. In addition, the present invention also provides a method for making and using the multi-layer three-dimensional digital nucleic acid amplification, which can further improve the accuracy of the quantitative results.

The invention relates to a kit and a detection method for synchronously detecting nucleic acid and protein markers of acute myocardial infarction, and the kit is used for carrying out specific modifier marking on a target nucleic acid amplification product, so that a'modifier 1-dsDNA-modifier 2 'nucleic acid amplification product is formed; nucleic acid amplification products and target protein are captured through different functionalized nano sensors, and the content of nucleic acid markers and protein markers in a sample is detected through fluorescence values of the nano sensors. According to the kit disclosed by the invention, complex steps such as nucleic acid extraction and purification required in a conventional nucleic acid marker detection process can be omitted; nucleic acid quantitative detection can be carried out without professional nucleic acid detection experiment equipment, personnel or site requirements, and home detection can be realized; the synchronous quantitative detection of the nucleic acid marker and the protein marker can be realized in a single reaction, the advantages of the nucleic acid marker and the protein marker are complementary, and quicker, more comprehensive, more accurate and more specific diagnosis information is provided for AMI diagnosis.

This invention belongs to the field of molecular biology, specifically relating to a method for quantitative detection of nucleic acids. It includes the following steps: S1 Designing a PCR amplification system containing the target nucleic acid sample and corresponding amplification primers, further adding the homologous exogenous reference sequence corresponding to the target nucleic acid, and also adding the internal reference sequence and its amplification primers, as well as the homologous exogenous reference sequence corresponding to the internal reference sequence; S2 Performing PCR amplification using a multiplex PCR reaction system, and constructing a library for high-throughput sequencing; S3 Counting the number of different types of sequences in the sample; S4 Quantitative analysis of the detected nucleic acid fragments can be performed by using the internal reference as a control; This invention also performs pairwise comparative quantitative analysis. By simultaneously adding the homologous exogenous reference sequence and the internal reference sequence for calibration, quantitative analysis of trace amounts of nucleic acid can be achieved, and the operation is simple, time-saving, labor-saving, and cost-effective.

Provided herein systems, compositions, and methods for nucleic acid quantification that combines the sensitivity and accuracy of digital PCR with the capability for high-throughput multiplexing. The methods integrate digital amplification in compartments with capillary electrophoresis or other methodologies as a readout mechanism.

Provided is a flexible thin film microfluidic chip, comprising a laminated film, at least one fixed bonding region, a plurality of functional chambers, fluid channels, at least one valve region and at least one liquid intake interface, wherein the fixed bonding region, the functional chambers, the fluid channels and the valve region are all distributed in the laminated film, the sizes and shapes of the functional chambers and the fluid channels are defined by the boundary of the fixed bonding region, and the liquid intake interface is connected to at least one functional chamber, and is sealed after liquid enters the functional chamber. The flexible thin film microfluidic chip is at least divided into three functional regions: a nucleic acid extraction region, a nucleic acid quantification region and a nucleic acid testing region, wherein the nucleic acid quantification region is located between the nucleic acid extraction region and the nucleic acid testing region; any functional region comprises at least one functional chamber; a functional chamber in the nucleic acid quantification region at least comprises a nucleic acid quantification chamber; and during a process in which liquid flows from the nucleic acid extraction region to the nucleic acid testing region, the liquid is temporarily stored in the nucleic acid quantification chamber and is quantified after the nucleic acid quantification chamber is fully filled, and a quantification range is adjustable.

The present invention relates to a nucleic acid quantification method comprising the steps of: (i) providing a sample comprising a plurality of nucleic acid molecules; (ii) amplifying at least two loci in the plurality of nucleic acid molecules wherein the at least two loci comprise at least one multi-copy locus and at least one single-copy locus, and wherein the amplification method is a digital polymerase chain reaction method; (iii) detecting at least two amplification products; and (iv) quantifying the amount of the plurality of nucleic acid molecules present in the sample; wherein the quantifiable nucleic acid concentration ranges of the at least one multi-copy locus and the at least one single-copy locus overlap. The invention also relates to a kit for use in the method of the invention.

In some aspects, the present disclose provides methods for amplifying and quantifying nucleic acids. Methods for amplifying and quantifying nucleic acids comprise isolating a sample comprising nucleic acid molecules into a plurality of microchambers, performing a polymerase chain reaction on the plurality of microchambers, and analyzing the results of the polymerase chain reaction. In some aspects, the present disclosure provides devices consistent with the methods herein.

The present invention discloses a flexible thin film microfluidic chip, which includes a laminated membrane, at least one fixed binding area, multiple functional chambers, a fluid channel, at least one valve area and at least one liquid inlet interface, wherein the fixed binding area, the functional chamber, the fluid channel and the valve area are all distributed in the laminated membrane, and the size and shape of the functional chamber and the fluid channel are defined by the boundary of the fixed binding area, the liquid inlet interface is connected to at least one functional chamber and is closed after liquid is introduced; the flexible thin film microfluidic chip is divided into at least three functional areas: a nucleic acid extraction area, a nucleic acid quantification area and a nucleic acid detection area, wherein the nucleic acid quantification area is located between the nucleic acid extraction and capture area and the nucleic acid detection area; any functional area includes at least one functional chamber; the functional chamber in the nucleic acid quantification area includes at least a nucleic acid quantification chamber, and during the process of liquid flowing from the nucleic acid extraction area to the nucleic acid detection area, it needs to be temporarily stored in the nucleic acid quantification chamber and filled with the nucleic acid quantification chamber. The present invention can realize the liquid quantification function of non-fixed volume without changing the physical size of the nucleic acid quantification chamber, thereby meeting the actual use requirements under different conditions.

Disclosed in the present invention are a nucleic-acid absolute-quantification system and nucleic-acid absolute-quantification method based on nucleic-acid isothermal amplification. In combination with a nucleic-acid synchronous isothermal amplification method and digital micro-droplet quantification technology, the nucleic-acid absolute-quantification method comprises: preparing a reaction system which is based on nucleic-acid isothermal amplification; segmenting the reaction system into several micro reaction units, and performing an isothermal amplification reaction on the micro reaction units; and after the isothermal amplification reaction, performing fluorescence signal detection on each of the micro reaction units, and on the basis of the number of negative micro reaction units after detection and the number of positive micro reaction units after detection, performing absolute quantification on the copy number of nucleic acids in a sample. The nucleic-acid absolute-quantification method provided in the present invention has the advantages of realizing a stable system and a high compatibility, involving low device requirements, and realizing high sensitivity and quick detection, is applicable in particular to the design and development of a fully automatic digital nucleic-acid quantification platform, and is applicable to the quantitative detection of nucleic acids in clinical tests and basic research.

The application belongs to the field of gene sequencing, and relates to a standard substance for calibrating a high-throughput sequencer, a preparation method and application thereof. 97 DNA fragments of 320 bp in length are selected from a genome. The DNA fragments are optimized through the principle of chirality, reverse design, GC content homogenization, structural stability control, series modularization integration, and are added with tags and primers at two ends. The candidate fragment library with a length of 456 bp is prepared through gene fragment synthesis, cloning and identification, and target fragment amplification and purification. After correction, the above-mentioned fragments are mixed according to the correction ratio to obtain a multi-fragment DNA mixed standard substance. Nucleic acid quantification, sequencing, GC content calculation, uniformity test and stability research are carried out on the standard substance, and value assignment and uncertainty evaluation are completed, so that the metrological traceability is ensured. The high-throughput sequencer calibration standard substance containing 97 456 bp fragments is successfully developed, and the measurement characteristics are systematically confirmed.

This invention discloses an integrated digital nucleic acid detection chip based on vertically perforated membrane emulsification and its application. The chip's main structure consists of three layers: upper, middle, and lower. The upper layer is the main part, including a nucleic acid extraction and purification zone, a vertically perforated membrane emulsification droplet generation zone, and a digital isothermal amplification and detection zone. The chip uses a vertically perforated membrane to prepare uniformly sized microdroplets, reducing the requirements for chip manufacturing processes and improving the uniformity of microdroplet size. This reduces the complexity of subsequent image recognition and artificial intelligence algorithm development, improving the scientific rigor and accuracy of the detection results. In this chip, nucleic acid quantification uses absolute quantification, eliminating the need for plotting working curves and avoiding errors caused by standards. Furthermore, by integrating nucleic acid extraction, purification, amplification, and digital detection onto a single chip, it achieves integrated nucleic acid detection with a "sample in - result out" process.