11 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 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.

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.

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 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.