89 results about "Nucleic acid amplification technique" patented technology

The invention relates to methods and kits for detecting and / or monitoring biological molecules in a test sample. For example, the invention relates to methods and kits for detecting and / or monitoring HIV p24 antigen in human body fluid, biological toxins such as ricin or botulism in an environmental or biological sample, and prion protein from human, deer or bovine, such as PrPSC, in a biological sample. The antigen detection signal is boosted by amplification of a polynucleotide linked to a detector molecule using methods for nucleic acid amplification technology.

The methods described herein, referred to as PCR-Activated Sorting (PAS), allow nucleic acids contained in biological systems to be sorted based on their sequence as detected with nucleic acid amplification techniques, e.g., PCR. The nucleic acids can be free floating or contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA. Systems and devices for use in practicing methods of the invention are also provided.

The invention discloses a nucleic acid composition for detecting novel coronavirus COVID-19 and application. The nucleic acid composition is prepared by the following steps: firstly, carrying out nucleic acid sample amplification based on a transcription-mediated amplification technology (TMA); then, specifically detecting amplified virus target nucleic acid in combination with activity of 'associated cleavage' of CRISPR-Cas13a protease; adding sgRNA, Cas13a protein, an ssRNA signal report probe and a reaction buffer solution into a reaction system containing target nucleic acid to be detected; and reading and detecting signals when the reaction is carried out so as to detect a target gene. By using the method disclosed by the invention, whether a sample contains a target nucleic acid sequence or not can be rapidly detected; and the nucleic acid composition is combined with the transcription-mediated nucleic acid amplification technology, so that the sensitivity of the detection methodcan reach a nanomole level, and the nucleic acid composition for detecting the novel coronavirus COVID-19 is suitable for rapidly detecting pathogenic microorganisms, gene mutation, specific target RNA and the like.

The invention provides a novel coronavirus visual constant-temperature (nucleic acid extraction-free) rapid detection kit. According to the invention, a specific nucleic acid sequence of a novel coronavirus (2019-nCoV) is used as a target gene; LAMP amplification primers are designed; a novel coronavirus constant-temperature rapid nucleic acid amplification technology detection method is established on the basis of the advantages of high specificity, high sensitivity, simplicity and convenience of a loop-mediated isothermal amplification technology; and a visual constant-temperature novel coronavirus rapid detection kit is constructed on the basis of the detection method. The kit provided by the invention can effectively detect standard plasmids with a concentration of 5 copies / [mu]l, andhas a reaction time of only 30 minutes. The kit provided by the invention can achieve the purpose of quickly and accurately detecting the novel coronavirus, and has good application prospect.

The invention discloses a recognition probe of cancer cells. The length of the recognition probe of the cancer cells is larger or equal to 70bp, and the recognition probe of the cancer cell comprises an nucleic acid aptamer which is combined with the cancer cells in a specific binding mode, and an extending sequence which is arranged at a 5' end or a 3' end of the nucleic acid aptamer, wherein the extending sequence is a single stranded nucleotide sequence which is not enabled to form a secondary structure by itself. The invention further provides a detection method of the cancer cells by using the recognition probe and a kit. The recognition probe of the cancer cells is simple in manufacture, free from immunogenicity, free from toxicity, and is good in chemical stability. The detection method of the cancer cells combines the recognition probe based on nucleic acid aptamer design with a nucleic acid amplification technology, is good in detection sensitivity, and is good in detection efficiency.

The methods and systems described herein provide an improved emulsion droplet based nucleic acid amplification method, which allows nucleic acids contained in biological systems to be detected, quantitated and / or sorted based on their sequence as detected with nucleic acid amplification techniques, e.g., polymerase chain reaction (PCR). The nucleic acids can be free floating or contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA.

An improved method of preventing carryover contamination of an amplification reaction involves treating uracil-containing DNA with uracil-N-DNA glycosylase and heating the DNA in the presence of polyamines, such as spermidine, spermine and the like. Alternatively, after treatment with uracil-N-DNA glycosylase, the reaction is further incubated with an enzyme having AP lyase activity.

The invention provides a same sequence primer transpositional nucleic acid amplification technology, which comprises the steps of: (1) linking the artificial sequence to the upstream of the target specific primer 5', and forming a chimeric primer; (2) taking the chimeric primer as an amplification target template to produce a secondary template with the two ends having artificial sequences; and (3) taking the artificial sequences with the same sequence of 65%-75% as the transpositional primer amplification secondary template. The technology is applicable to clinic molecular diagnosis, basically can utilize the primer design to prevent the false positive result from generating, and is particularly applicable to communicable disease nucleic acid detection and accurate diagnosis. The technology is particularly suitable to multiple PRC amplification technology, is nearly designed by aiming at the multiple PRC amplification technology, and the redundant and extraordinarily complicated primers of the multiple PRC amplification technology can only adopt the primer transpositional design to eliminate the nonspecific amplification of the primer polymer.

The invention relates to a method for detecting H1N1 influenza A viruses based on the LAMP (loop-mediated isothermal amplification of nucleic acid) technology, which comprises the following steps: extracting virus RNA (ribonucleic acid); carrying out reverse transcription and LAMP amplification; converting the LAMP amplification product into single-chain substances; and detecting the LAMP amplification product by using a nucleic acid nano-gold biosensor. The invention also provides a kit for detecting H1N1 influenza A viruses. By using the specially-designed LAMP inner primer to introduce an enzyme cutting site sequence segment into the amplification product in the LAMP amplification process, using the restriction endoenzyme TspR I to digest the amplification product and acquiring a large number of single-chain DNA (deoxyribonucleic acid) products with the length of 140bp or so by physical shock cooling, the combination of the LAMP technology based on the specific primer and the nucleic acid nano-gold biosensor with a specific probe ensures that the detection method and kit has high specificity and high detection speed.

Various methods and devices for spatial molecular analysis from tissue is provided. For example, a method of spatially mapping a tissue sample is provided with a microarray having a plurality of wells, wherein adjacent wells are separated by a shearing surface; overlaying said microarray with a tissue sample; applying a deformable substrate to an upper surface of said tissue sample; applying a force to the deformable substrate, thereby forcing underlying tissue sample into the plurality of wells; shearing the tissue sample along the shearing surface into a plurality of tissue sample islands, with each unique tissue sample island positioned in a unique well; and imaging or quantifying said plurality of tissue sample islands, thereby generating a spatial map of said tissue sample. The imaging and / or quantifying may use a nucleic acid amplification technique.

A method for extract nucleic acid includes such step as cracking nucleic acid sample, adding sample containing nucleic acid to cracking solution to form nucleic acid cracking solution, cracking nucleic acid sample to form nucleic acid cracking solution, cracking nucleic acid sample to obtain nucleic acid sample, cracking nucleic acid sample to obtain nucleic acid sample, cracking nucleic acid sample to obtain nucleic acid sample, cracking nucleic acid sample to obtain nucleic acid sample, cracking nucleic acid sample to obtain nucleic acid sample, cracking nucleic acid sample to obtain nucleicacid sample. 2, adsorb nucleic acid: adding that carry into the nucleic acid cleavage solution to obtain a carrier for adsorbing nucleic acid; 3, remove impurities: rin that carrier adsorbed with nucleic acid in a rinsing solution to remove impurities other than nucleic acid; 4, amplification reaction, wherein that rinse carrier adsorb with nucleic acid is directly added into a nucleic acid amplification system for amplification reaction. The carrier is cellulose filter paper; a simple, a method for extract nucleic acid based on cellulose filt paper is disclosed. Applicable to polymerase chain reaction amplification and recombinase polymerase isothermal amplification and other nucleic acid amplification technology, suitable for operating at room temperature, suitable for biological samples from various sources, can be loaded with nucleic acid cellulose filter paper directly into the nucleic acid amplification system for amplification reaction, the whole process is less time-consuming.

The invention discloses a nucleic acid amplification technology and an application thereof in detection of mosquito-borne viruses. The invention provides a method for detecting whether n kinds of target RNA sequences are contained in a sample to be detected or not. The method sequentially comprises the following steps: 1, extracting the total RNA of the sample to be detected; 2, carrying out reverse transcription recombinase polymerase amplification; and 3, carrying out recombinase polymerase amplification, wherein n is a natural number. The invention also provides a primer probe combination. The combination comprises primers and probes represented by sequence 1 to sequence 18 in a sequence table. A micro-fluidic chip, recombinase polymerase amplification and nucleic acid sequence dependent amplification are combined to effectively solve the problem of detection limit reduction of the micro-fluidic chip. The invention further provides a kit used for detecting the mosquito-borne viruses. The kit can simultaneously complete detection of six varieties of the mosquito-borne viruses one time, and has the advantages of high sensitivity, good specificity, fastness, good convenience, and wide application values.

A method for the quantitative monitoring of gene expression without either co-amplification of an added template or use of an endogenous constitutive transcript is provided. The process involves a duplex amplification reaction in which a single set of primers is used to amplify both genomic DNA and expressed mRNA from the same gene sequence. These primers are targeted for sequences flanking the splice junction / intron sequences for the mRNA / DNA respectively. By their use, any suitable nucleic acid amplification technology yields mRNA and DNA amplimers which are distinguishable by length and sequence heterogeneity. These amplimers are present in the final amplification reaction in ratios which are dependent upon the ratios of the expressed mRNA to the DNA in the sample, allowing the quantitation of mRNA in a sample which is normalized to the number of copies of genomic DNA since the genomic DNA acts as the internal quantitation standard, and in effect yields the amount of mRNA per cell. Any detection methodology which can detect amplimers of different lengths or sequences can be used for post amplification quantitation. This strategy may be employed for any gene system in which the mRNA sequence differs from the original genomic DNA sequence. The invention may be used, for example, in the determination of gene expression in both research and commercial applications.

The invention discloses a temperature control apparatus applied to a nucleic acid amplification technique and a control method thereof. The apparatus comprises a hot cover, test tubes, a temperature changing metal block, thermally conductive silicone grease, a semiconductor refrigeration sheet, a heat radiation metal sheet, a heat radiation apparatus, draught fans, a rubber pedestal, a full H-bridge driving module, a controller, a button, a display screen, a casing body and a temperature sensor. The temperature control apparatus of the invention is low in production cost, simple to operate and broad in application. Both the PCR amplification technique in the early stage and an emergent LAMP or RPA amplification technique can be switched and set in a customized mode on the apparatus through a man-machine interaction interface.

The invention discloses a method and a reaction device for visual detection of isothermal amplification of nucleic acid. The method comprises the following steps: in a special hermetic reaction device, adding a reagent capable of decomposing pyrophosphoric acid into phosphoric acid into a nucleic acid amplified reaction liquid in advance; after reaction, under the condition of not uncapping, mixing the nucleic acid amplified reaction liquid with other selected color reagents which are additionally stored in the device in advance to react and observing the color of the mixture after further reaction; and judging whether nucleic acid amplified reaction is carried out on a sample or not, wherein the selected color reagents are reagent combination capable of being in color reaction with phosphoric acid (radicals). The method disclosed by the invention is simple to operate and is quick and sensitive. The test result can be detected by virtue of naked eyes. The method is low in cost and makes up the defects in the existing technology for detecting nucleic acid amplification.

The invention discloses a PDCoV detection method based on a real-time fluorescence RT (reverse transcription) RAA (recombinase-aid amplification) technology. The method comprises experiment steps as follows: extracting RNA of PDCoV virus and designing Real-time RT-RAA primers and probes; establishing a Real-time RT-RAA reaction system, conducting a Real-time RT-RAA reaction on the RNA of the virusand obtaining real-time fluorescence detection results. The method has the advantages as follows: the PDCoV is detected simply and quickly at a low cost, the sensitivity of the Real-time RT-RAA detection method is consistent with that of a qPCR detection method, which is 102 copies / mu L, and the specificity is good.

This invention is directed to methods for the quantitative measurement of specific gene expression levels in biological samples. In one embodiment, methods for the quantitative monitoring of gene expression without either co-amplification of an added template or use of an endogenous constitutive transcript are provided. The former involves a duplex amplification reaction in which a single set of primers is used to amplify both genomic DNA and expressed mRNA from the same gene sequence. These primers are targeted for sequences flanking the splice junction and intron sequences for the mRNA and DNA respectively. By their use, any suitable nucleic acid amplification technology yields mRNA and DNA amplimers which are distinguishable by length and sequence heterogeneity. These amplimers are also present in the final amplification reaction in ratios which are dependent upon the ratios of the expressed mRNA to the DNA in the sample, allowing the quantitation of mRNA in a sample which is normalized to the number of copies of genomic DNA since the genomic DNA acts as the internal quantitation standard, and in effect yields the amount of mRNA per cell. Any detection methodology which can detect amplimers of different lengths or sequences can be used for post amplification quantitation. This strategy may be employed for any gene system in which the mRNA sequence differs from the original genomic DNA sequence. In another embodiment, methods for the quantitative monitoring of gene expression involving determining the ratio of genomic material to expressed mRNA without nucleic acid amplification and / or primer binding are described. This method includes the independent and direct detection of the genomic material and mRNA by complementary probe binding without prior amplification. Additional methods for quantitative measurement of gene expression rates without RNA transcription region introns are described. The invention is useful in gene expression determination in research and commercial applications.

The invention provides a fluorescent RT-RAA (Reverse Transcription Recombinase Aid Amplification) primer for detecting MERS-CoV (Middle East Respiratory Syndrome Coronavirus), a fluorescent probe andapplication of the fluorescent RT-RAA primer and the fluorescent probe in detecting the MERS-CoV. The fluorescent RT-RAA primer and the fluorescent probe, disclosed by the invention, are capable of quickly and qualitatively detecting the MERS-CoV in a throat swab sample, detection can be carried out under the normal temperature of 39 DEG C, a diagnosis result can be obtained within 20 minutes, andthe detection time is greatly shortened; the fluorescent RT-RAA primer and the fluorescent probe are stronger in specificity and higher in sensitivity, quick diagnosis and whole-process monitoring onan epidemic situation can be completely met, and time is bought for early diagnosing and early treating the epidemic situation, reducing a case fatality rate and controlling the epidemic situation.

A method for the simultaneous detection of adenoviruses of subgenera A, B, C, D, E and F in a sample comprising contacting said sample with at least one forward primer and at least one reverse primer for a hexon gene of an adenovirus of subgenera A, B, C, D, E and F and subjecting the sample contacted with said primers to a nucleic acid amplification technique.

In recent years, nucleic acid amplification technology characterized by detection through the amplification of micro-scale target genes is adopted as a method for detecting infectious diseases and genes, however, because a dispensing nozzle has to be replaced while dispensing the reagent and samples, the arrangement space of consumables is increased or the consumables have to be erected frequently to result in the decreasing of automation. The invention provides an automatic analyzing device having higher usability. The automatic analyzing device allows the repeated use of the dispensing nozzle (6), by providing a tube set (8) having a separating plate (10) higher than the front end of the dispensing nozzle when a dispensing structure stops, without the need for considering the dripping of the liquid at the front end of the dispensing nozzle (6) after absorbing the reagent or the dispensing nozzle having left liquid after discharging the reagent and for considering the pollution of the dispensing nozzle (6).

The invention discloses a new deafness associated gene mutation multi-site detecting system. A multiplex PCR primer and a detecting probe are used for detecting whether mutation exists in deafness gene mutation sites; and the deafness gene mutation sites comprise 46 deafness gene mutation sites. According to the new deafness associated gene mutation multi-site detecting system, rapid detection for 46 mutation sites on 3 genes of a chromosome, one gene of an X chromosome and mitochondrial genes are carried out by improving an existing multiplex nucleic acids amplification technique, designing a specific detecting probe and setting special detecting system components and reaction conditions; the sites are obtained by arranging a plenty of data accumulated in clinical and scientific research work; besides non-syndromic deafness, common Alport syndromes also can be detected; compared with similar detecting products existing in the market, a reagent kit can detect more associated gene mutation sites of syndromic / non-syndromic deafness, the detection can be finished within one workday, and an exact detecting result can be obtained only by needing 20-30 nanogram DNA to the minimum.

The invention discloses an incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology and an application. The invention provides a pair of cross primers which contain restriction incision enzyme specific recognition sequences at an end 5', and are marked with fluorescent groups and quenching groups, a pair of displacement primers and 3-6 amplification primer constant-temperature amplification target gene segments; and the incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology can carry out visible detection, electrophoresis detection, real-time turbidity detection and real-time fluorescence detection. The method is convenient, quick, sensitive, specific and suitable for detecting various nucleotide fragments.

An improved method of preventing carryover contamination of an amplification reaction involves treating uracil-containing DNA with uracil-N-DNA glycosylase and heating the DNA in the presence of polyamines, such as spermidine, spermine and the like. Alternatively, after treatment with uracil-N-DNA glycosylase, the reaction is further incubated with an enzyme having AP lyase activity.

The invention provides an integrated digital nucleic acid amplification chip based on micro-droplets as well as a use method and an application of the integrated digital nucleic acid amplification chip. The integrated digital nucleic acid amplification chip comprises an upper cover and a bottom plate. The upper cover comprises an oil inlet, a sample inlet and an exhaust port. The bottom plate comprises a droplet generation area and a droplet storage area, wherein the droplet generation area comprises an oil inlet flow channel and a sample inlet flow channel; the oil inlet flow channel is connected with the sample inlet, the sample inlet flow channel is connected with the sample inlet, the intersection of the oil inlet flow channel and the sample inlet flow channel is a cross-shaped channel, and micro-droplets are generated at the intersection of the oil inlet flow channel and the sample inlet flow channel; the droplet storage area comprises a collection cavity flow channel and a droplet storage cavity; the collection cavity flow channel is connected with the cross-shaped channel, and the droplet storage cavity is connected with the collection cavity flow channel and the exhaust port. The processes of droplet generation, nucleic acid amplification, detection and the like can be completed on the chip, so that the detection efficiency and the detection accuracy are improved, and rapid development of an integrated digital nucleic acid amplification technology based on micro-droplets is promoted.

The invention discloses a microRNA detection method based on Hg<2+> assistance and a double-polymerase isothermal nucleic acid amplification technology, and belongs to the technical field of optical bio-sensing. On the basis of a combined catalytic action of a polymerase Klenow fragment and terminal deoxynucleotidyl transferase, microRNA is effectively extended into a polythymine base sequence which is relatively long; Hg<2+> is added, so that a T-Hg<2+>-T double-stranded structure is formed; an SYBR Green I fluorescent dye is interpolated into the T-Hg<2+>-T double-stranded structure, so that the fluorescence of the SYBR Green I is enhanced; and a fluorescence enhancing degree is positively related to the concentration of the microRNA; therefore, the sensitive detection of the microRNA is achieved.

A device for in-line blood screening and testing using biochips is disclosed. The screening methods include nucleic acid amplification techniques and antibody / antigen assays to detect target molecules and agents indicative of infectious diseases or metabolic diseases.

The invention discloses a method for quickly obtaining molecules with characteristics from a complicated nucleic acid library. The method comprises the following steps: firstly, respectively amplifying a single molecule of the library on a single micro-bead A by an emulsion nucleic acid amplification technique, so that only a nucleic acid molecule with a specific sequence is formed on each micro-bead A; coupling a target molecule on another micro-bead B (such as a magnetic bead) with different properties, mixing the two micro-beads together to combine, and then cleaning uncombined micro-bead A, and separating the micro-bead B with the target molecule, so that the micro-bead A combined with the target molecule is separated together; the nucleic acid molecule which is specifically combined with the target molecule on the micro-bead B on the micro-bead A is a required single sequence nucleic acid molecule. The method is fast and convenient; the single nucleic acid molecule with high affinity can be obtained without cloning and sequencing. The technique can be widely applied to screening of promoters, and screening and identifying of aptamers.

A PSA (Prostate Specific Antigen) high-sensitivity detection method based on nanotechnology is a novel biological high-sensitivity detection method which combines the magnetic bead probe technology, Au-nanoparticle double-mark probe technology, enzyme-linked immunosorbent assay technology and the nucleic acid amplification technology together. According to the detection method, an antibody corresponding to a protein to be detected (such as PSA) is cross-linked with a magnetic bead, and after the protein to be detected in a sample is captured, an Au-nanoparticle dual-mark probe marked by another antibody and nucleic acid is added to the captured protein. After magnetic separation and purification are carried out, nucleic acid undergoes release amplification, a visual result is obtained by means of agarose gel electrophoresis, and the grey level value scanning is subsequently performed. Compared with the conventional ELISA (Enzyme-Linked Immunosorbent Assay), the detection sensitivity of the detection method disclosed by the invention is increased by six orders of magnitude, and the PSA high-sensitivity detection method based on nanotechnology is fairly significant for the early diagnosis of caners and the detection of trace important substances, nucleic acids and proteins.

The invention discloses reverse complementary fluorescent PCR at 5' ends of primers and belongs to the technical field of molecular biology-nucleic acid amplification. The reverse complementary fluorescent PCR is characterized in that a segment of sequence reverse complementary fluorescent PCR is added at the 5' ends of a pair of primers for amplifying, so that tail ends of amplified products arecomplementary; 3' ends of target products also can be templates and primers with each other, so that amplification efficiency is increased; the sensitivity is improved by 50 times on the basis of enlarging a conventional PCR index by 109-12, or 5 cycles of amplification can be reduced under the condition of same sensitivity; on the other side, complement of the 5' ends of the primers cannot extendrather than inhibit polymerization nonspecificity between 3' ends of a primary primer pair or 5 cycles of amplification are reduced, so that an anonspecific amplification region can be kept off. Themiddle same sequence primer taking the reciprocal first to second base adenine A at the least significant end of 3' as a tail and UDG enzymolyzed dU are combined to replace an amplification product ofdT; in addition, by closing mineral oil, no primer dimer PD is produced in a PCR reaction cycle, and cross contamination of product aerosol adhesive is avoided.