48 results about "Digital polymerase chain reaction" patented technology

The identification of pre-defined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. The exponential, analog nature of the polymerase chain reaction is transformed into a linear, digital signal suitable for this purpose. Single molecules can be isolated by dilution and individually amplified; each product is then separately analyzed for the presence of pre-defined mutations. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.

The invention relates to a nucleic acid detection micro-fluidic chip, which comprises a substrate and a cover plate, wherein the cover plate is fixedly arranged on the substrate; the cover plate is provided with an oil phase sample-injection hole and a sample sample-injection hole; the surface, abutted with the substrate, of the cover plate is a structural surface; a first micro-flow channel connected with the oil phase sample-injection hole, a second micro-flow channel connected with the sample sample-injection hole and a liquid drop generation interface located in the joint of the first andsecond micro-flow channels, which are mutually communicated, are etched in the structural surface; the structural surface, in a liquid drop collection area, of the cover plate is fixedly provided witha thin plate; a gap is formed between the periphery of the thin plate and the boundary of the liquid drop collection area, so as to form a gas path; and the cover plate is also provided with a gas vent hole communicated with the gas path. The problems of the fusion and the demulsification of liquid drops are avoided; the homogeneity and the stability of the liquid drop are improved; meanwhile, the signal reading speed of the liquid drop is quickened during dPCR (digital Polymerase Chain Reaction) detection; and the detection period is shortened. The invention also relates to a preparation method of the nucleic acid detection micro-fluidic chip. The preparation method is simple and convenient; the cost is low; and the nucleic acid detection micro-fluidic chip not only can be produced in alarge-batch manner, but also can be produced in a laboratory in a small-batch manner.

The invention belongs to the field of image processing and in particular discloses a dPCR (Digital Polymerase Chain Reaction)-based novel full-automatic fluorescent signal acquisition and analysis method. According to the method, a fluorescent signal is extracted and analyzed by utilizing an idea of image region segmentation-image stitching-signal acquisition and analysis. Due to the method, the fluorescent signal acquisition and analysis method is applied to result detection at the later stage of dPCR in an engineered manner firstly, and due to a reasonable design, full automation of the whole system is realized, measurement errors caused by artificial interference such as manual marking, progressive scanning and the like are avoided, the accuracy is high, the operation and control are convenient, and the detection precision and the detection efficiency of the system are effectively improved. Due to the stitching step, the acquired scattered images can be perfectly stitched to be of the size of the original image during designing, the original image is highly reduced, convenience is brought to fluorescent signal analysis and detection at the later stage, and the detection accuracyis improved.

The invention provides an LED-based multi-window and multi-path detection method of digital polymerase chain reaction PCR. The method comprises the following steps: the LEDs are adopted as detection light sources, transparent windows are arranged at the tail end of a capillary pipeline in a segmentation manner after the digital PCR finishes polymerase chain reaction; each LED with one type of wavelength is responsible for detecting one window, a type of signal possibly sent by sample drops is detected. Since the drops of the digital PCR are ordered, multiple signals in each sample drop can be detected and accurately counted. Compared with the method of detecting the same drop by adopting multiple bundles of laser, the method is simpler and more feasible, has the characteristics of simple structure and low cost, and also can be used as an inserting piece to be combined with the existing detection equipment so as to increase the working speed of daily blood detection in a hospital.

A method for performing digital polymerase chain reaction (dPCR) is provided. The method includes partitioning a biological sample volume including a plurality of target nucleic acids into a plurality of partitions, where at least one partition includes at least one target nucleic acid. The method further includes determining a model for volume variation of the plurality of partitions and determining a number of partitions including at least one target nucleic acid. The method includes generating a concentration of target nucleic acids in the biological sample based on the model for volume variation and the fraction of partitions including at least one target nucleic acid.

The invention relates to a method for detecting a mutation in a microsatellite sequence locus of a target fragment from a DNA sample, comprising subjecting said DNA sample to a digital polymerase chain reaction (PCR) in the presence of a PCR solution comprising:

• • a pair of primers for amplifying said target fragment of the DNA sample including said microsatellite sequence;
  • a first MS oligonucleotide (MS) hydrolysis probe, labeled with a first fluorophore, wherein said first MS oligonucleotide probe is complementary to a wild-type sequence including the microsatellite sequence;
  • a second oligonucleotide reference (REF) hydrolysis probe, labeled with a second fluorophore, wherein said second oligonucleotide REF probe is complementary to a wild-type sequence of said target DNA fragment which does not include said microsatellite sequence. The invention also encompasses methods for the diagnosis and prognosis of cancer and a method for determining the efficacy of a cancer treatment.

The invention discloses a microdroplet type digital polymerase chain reaction chip, which belongs to the field of digital PCR chips, and comprises a chip body and a collecting tube which are connected, a turbid phase channel flowing to the collecting tube, and an oil phase channel and a water phase channel which are converged at the inlet of the turbid phase channel are arranged in the chip body, the turbid phase channel is divided into a meandering channel and a straight channel according to the flowing sequence of the emulsion, the top surface of the chip body is provided with an oil phase channel inlet, a water phase channel inlet, a suction outlet and a hot melting column inlet, the suction outlet is directly communicated with a collecting pipe through the straight channel, and the hot melting column inlet is communicated with the meandering channel. An oil phase and a water phase enter the turbid phase channel through the oil phase channel and the water phase channel respectively to be mixed and flow into the collecting pipe, the turbid phase channel can be partitioned by the hot melting column during amplification, a part for generating emulsion is isolated, heating and pressurizing during amplification are facilitated, outward evaporation of a solution is avoided, outward loss of heat is reduced, and the amplification efficiency is improved. After amplification is completed, the solution can be directly sucked out from the suction outlet, and subsequent accurate detection is facilitated.

The invention provides a hepatitis B virus (HBV) DNA quantitative detection kit. The kit utilizes a digital polymerase chain reaction (dPCR) technology to quantitatively detect HBV DNA in biological samples and can be used for monitoring baseline level and change conditions of HBV DNA in patients suffering from hepatitis B and evaluating the response and therapeutic effect of antiviral therapy.

The invention discloses a digital polymerase chain reaction (PCR) detection kit and a digital PCR detection method for mutation detection of a gene mutation high-incidence region. The kit comprises apair of amplification primers, a wild-type specificity detection probe and a internal control probe; the amplification primers are used as a universal PCR amplification upstream primer and a universalPCR amplification downstream primer for detecting a plurality of mutation sites in a gene mutation high-incidence region, and an amplification region comprises a gene mutation high-incidence region to be detected; the wild-type specificity detection probe aims at the gene mutation high-incidence region; and the internal control probe aims at a gene conservative sequence in the amplification region of the gene mutation high-incidence region, wherein different fluorescent dyes are marked by the wild-type specificity detection probe and the internal control probe. The kit and the method can be used to distinguish the wild type and the mutant type of the gene mutation high-incidence region, can further determine the specific mutant type according to a positive signal position of the probes, and can realize quantification of various different mutant types to determine proportions of various mutations.

The invention discloses a method for detecting ARMS-ddPCR (amplification refractory mutation system-droplet digital polymerase chain reaction) gene site-directed mutation. The method aims at gene mutation of one site, primers comprise a wild type primer and a mutation primer, probes comprise a wild type probe and at least one mutation probe, a mutation site is detected by further combination witha ddPCR platform, and sample mutation site DNA is subjected to absolute quantification. The method is particularly suitable for amplification of small-fragment DNA samples such as plasma free DNA (cfDNA) and the like, with application of the method, gene mutation with quite low abundance can be detected from cfDNA, the method has the characteristics of good specificity and high sensitivity, can beapplied to absolute quantification detection of the gene mutation site by the ddPCR platform and has a quite important effect on guidance of clinic treatment and improvement of patient outcome.

The invention provides a nucleic acid combination for detecting pseudorabies virus and application of the nucleic acid combination, and a kit and a method for detecting the pseudorabies virus, and relates to the technical field of pseudorabies virus detection. The nucleic acid combination for detecting the pseudorabies virus comprises a first primer pair designed according to a conserved sequence of a gB gene of the pseudorabies virus, and a first probe, wherein the first primer pair comprises two primers, and the base sequences of the two primers are respectively as shown in SEQ ID No. 1 and SEQ ID No. 2; the base sequence of the first probe is as shown in SEQ ID No. 3. After the nucleic acid combination is adopted, rabies virus can be rapidly and conveniently detected on a digital polymerase chain reaction (PCR) platform, samples with low rabies virus content can be detected, and missed detection is avoided; the nucleic acid combination has the characteristics of being high in sensitivity, high in specificity, high in detection rate, and the like.

The present invention relates to an in vitro method for identifying and/or characterizing one or more mutations in a hotspot mutation sequence of at least one ESR1 target fragment from a DNA sample,

said method comprising subjecting the DNA sample to a drop-off digital polymerase chain reaction (PCR) in the presence of a PCR solution comprising:

• • a pair of primers suitable for amplifying an ESR1 target fragment;
  • an oligonucleotide reference (REF) hydrolysis probe, labeled with a fluorophore, wherein said REF oligonucleotide probe is complementary to a wild-type sequence of the target fragment located outside of the hotspot mutation sequence;
  • an oligonucleotide hotspot (HOTSPOT) hydrolysis probe, labeled with another fluorophore, wherein said oligonucleotide HOTSPOT probe is complementary to a wild-type sequence of the hotspot mutation sequence of the target DNA fragment.

The invention discloses primers, a reagent kit and a method for detecting IDH1 (isocitrate dehydrogenase 1) R132H gene variation by the aid of ddPCR (droplet digital polymerase chain reaction) technologies. The method includes extracting cfDNA (cell-free deoxyribonucleic acid) in peripheral blood; designing two probes and a pair of primers for IDH1R132H mutant types and wild types; carrying out ddPCR amplification by the aid of the cfDNA used as a template; carrying out data analysis on obtained amplification products to obtain absolute quantities and proportions of IDH1R132H mutant genes. Thereagent kit for detecting the IDH1R132H gene variation is based on the method. The primers, the reagent kit and the method have the advantages that provided primer amplification fragments 83bp are particularly applicable to amplifying small-fragment DNA samples such as plasma free DNA and can be combined with the detection probes, accordingly, the IDH1R132H gene variation with extremely low abundance can be detected from the cfDNA, the primers, the reagent kit and the method are good in specificity and high in sensitivity, human IDH1R132H gene variation can be absolutely quantitatively detected by the aid of the primers, the reagent kit and the method, and important effects can be realized by the primers, the reagent kit and the method for guiding clinical treatment and improving patientprognosis; the IDH1R132H gene variation can be detected only by the aid of the peripheral blood without tissue specimen collection, and accordingly the method is particularly suitable for patients intolerant to tissue sampling.

The invention discloses a high-precision specific digital polymerase chain reaction (PCR) detection method for series infectious microbe contamination taking acetobacter aceti as priority in fermentedmilk. The method comprises the steps: firstly, to-be-detected infectious microbes are subjected to dead bacterium de-interference treatment, then bacterial DNA is subjected to low-impurity and high-purity extraction, a specific primer probe of the to-be-detected microbes is researched, developed and designed, a ddPCR system and procedure and the annealing temperature thereof are set, and thus thedetection method meets the requirements of specificity, sensitivity and quantification. The constructed method is good in specificity, has no non-specific amplification, and has good sensitivity andquantitative advantages.

The invention discloses a kit and a detection method for absolute quantitative detection of total vibrio cholerae and pathogenic vibrio cholerae. The present invention provides two sets of primers andprobes for the absolute quantitative detection of the total vibrio cholerae and the pathogenic vibrio cholerae. One set of the primers and probe is used for detection of the total vibrio cholera, thenucleotide sequences of the primers are as shown in SEQ ID No. 1 and SEQ ID No. 2, and the nucleotide sequence of the probe is as shown in SEQ ID No. 3. The other set of the primers and probe is usedfor detection of the pathogenic vibrio cholera, the nucleotide sequences of the primers are as shown in SEQ ID No. 4 and SEQ ID No. 5, and the nucleotide sequence of the probe is as shown in SEQ ID No. 6. The invention also provides a method for the absolute quantitative detection of the total vibrio cholerae and the pathogenic vibrio cholerae by use of a micro-droplet digital polymerase chain reaction. The method has strong specificity, high sensitivity, good amplification effect, strong operability and easy standardization.

The invention discloses a microdroplet type digital polymerase chain reaction chip. The chip comprises a microdroplet structure, a microdroplet collection structure and a flow channel structure; the microdroplet structure forms a sample cavity and an oil storage cavity which are spaced, the microdroplet collection structure comprises a microdroplet collection tube arranged below the microdroplet structure, a collection cavity is formed in the droplet collection pipe, the flow channel structure is arranged on the droplet structure, an outlet of the flow channel structure is communicated with the collection cavity, inlets of the flow channel structure comprise a first inlet and a second inlet, the first inlet is communicated with the lower end of the oil storage cavity, and the second inlet is communicated with the lower end of the sample cavity; an inlet of the flow channel structure is respectively communicated with the lower end of the sample cavity and the lower end of the oil storage cavity, so that a sample and an oil product can enter the flow channel structure under the action of gravity, micro-droplets wrapped with the sample are formed under the action of the flow channel structure, and an outlet of the flow channel structure is communicated with a collecting cavity, so that the micro-droplets flow into the collecting cavity through the flow channel structure; and pipetting is not needed in the whole process.

The invention provides a digital polymerase chain reaction (PCR) system. The digital PCR system comprises a droplet formation assembly and a droplet nozzle assembly; the droplet formation assembly comprises a droplet collection tank; the droplet nozzle assembly is connected under the droplet formation assembly, and comprises a plurality of droplet nozzles; the droplet nozzles communicate with thedroplet collection tank; vaporizing components are arranged inside the droplet nozzles so as to be used for vaporizing part of the liquid of a digital PCR solution in the droplet nozzles, as well as quickly pushing the remaining liquid of the digital PCR solution into droplet forming oil in the droplet collection tank so as to form digital PCR droplets. By adopting a thermal bubble technology to form high-speed digital PCR droplets, the digital PCR system is capable of achieving a droplet formation rate of more than 1000 droplets per second; and moreover, the digital PCR system has highly efficient digital PCR oil utilization rate.

The invention discloses a composition, method and system for determination of a digital polymerase chain reaction (PCR). The composition comprises a water phase for generating water-in-oil liquid droplets, and the water phase contains water and fibroin nanofibers dissolved in the water. The composition particularly is an emulsion formed by the water phase and an oil phase which are mixed, whereinthe oil phase constitutes a continuous phase, and the water phase forms the multiple liquid droplets scattered in the continuous phase. The water phase contains the fibroin nanofibers, the water phasecan enter the liquid droplet interface to participate in liquid droplet formation, the liquid droplets formed by the water phase have the significantly higher reaction efficiency during the PCR, andthe stability of the liquid droplets is improved. The method for determination of the digital PCR has the high reaction efficiency and is suitable for determination of various target nucleic acids.

A microfluidic system (1; 1′) for dPCR of a biological sample and a respective method is provided by the present disclosure, the system (1; 1′) comprising at least one microfluidic device (2) having an inlet (23), an outlet (24), a flow channel (25) connecting the inlet (23) to the outlet (24), and an array of reaction areas (26) in fluidic communication with the flow channel (25), a flow circuit (3) connectable to the microfluidic device (2), for flowing liquid through the flow channel (25) of the microfluidic device (2), a sample liquid source connectable to the microfluidic device (2), for providing the microfluidic device (2) with a sample liquid (27), a primary sealing liquid source connectable to the microfluidic device (2), for providing the microfluidic device (2) with initial sealing liquid (28) for sealing the sample liquid (27) inside the array of reaction areas (26), a secondary sealing liquid source (4) connectable to the microfluidic device (2), for providing the microfluidic device (2) with additional sealing liquid (29), and a pumping device (31) connected to the flow circuit (3) and adapted to pump said additional sealing liquid (29) through the flow channel (25).

The invention relates to a method for detecting TCK (Tilletia controversa Kuhn) teliospores in soil through ddPCR (droplet digital polymerase chain reaction). The method comprises steps as follows: (1), total DNA (deoxyribonucleic acid) of a to-be-detected soil sample is extracted; (2), the total DNA of the to-be-detected soil sample serves as a template for ddPCR, and a PCR product is obtained; (3), the PCR product is put in a droplet read instrument, a signal is read, experimental data are analyzed, and the absolute content of TCK in the soil sample is obtained. A total DNA extraction method comprises steps as follows: the taken-back soil sample is sieved by a mesh screen for impurity removal and then is frozen; total DNA of the soil sample is extracted by adopting FastDNA<TM> SPIN Kit for Soil. With the adoption of the method, higher-quality DNA can be extracted from the soil, ddPCR is adopted for detection, accordingly, the absolute quantification of TCK in the soil is realized, control measures are taken in time, and economic losses are reduced.