143 results about "Asymmetric PCR" patented technology

The invention belongs to the technical fields of biochips and diagnostic reagents, and discloses a method for detecting various respiratory viruses, and primers and probes thereof. In the invention, nucleotide sequences of 14 respiratory viruses, namely adenovirus, human metapneumovirus, influenza virus A, influenza virus B, respiratory syncytial virus, bocavirus, rhinovirus, coronavirus (HKU1, NL63 and SARS), and parainfluenza virus (type I, type II, type III and type IV) are analyzed, and corresponding reverse transcription primers, PCR primers and specific probes are designed. Specific gene segments are amplified by reverse transcription and multiple asymmetric PCR methods; a fluorescence-coded microsphere group coupled with the virus specific probes and the PCR amplification product are incubated and hybridized by liquid phase chip technology; and finally the Bio-PlexTM200 is used for detection. The detection method has the advantages of high flux, high specificity and sensitivity, stable results and good repeatability, the detection method is easy to operate, and the detection speed is high.

The invention discloses a kit for quickly detecting 15 pneumonia pathogenic bacteria. The kit can detect streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae, mycoplasma pneumoniae, pseudomonas aeruginosa, baumanii, enterococcus faecalis, enterococcus faecium, klebsiella pneumoniae, escherichia coli, enterobacter cloacae, stenotrophomonas maltophilia, burkholderia cepacia, legionella pneumophila and chlamydia pneumoniae which cover clinically common pneumonia pathogenic bacteria difficult to culture. 16S rDNA and specific gene sequences corresponding to the pneumonia pathogenic bacteria are detected by combining gene chips with multiple asymmetric PCR reactions, and the categories of the bacteria in a to-be-detected sample are identified in genus and species. The kit makes up for the defect that current clinical detection of pneumonia pathogenic bacteria is not in time or comprehensive and a novel detection means for early diagnosis and early treatment of patients suffering from pneumonia is provided.

The present invention discloses an asymmetric PCR amplification method, its special primer and application, aims to provide a simple, effective PCR amplification for preparation of single-stranded product. The asymmetric PCR primer of the invention comprises some PCR primer pairs, in which an unrelated nucleic acids sequence to target sequence to be detected is added onto 5′-terminal of one primer. The asymmetric PCR amplification provided includes the steps: 1) preparative denaturing; 2) repetitiously denaturing, primers annealing, extending cycles as the first stage of PCR amplification; 3) repetitiously denaturing, primer extending cycles as the second stage of PCR amplification, wherein an unrelated nucleic acids sequence to target sequence to be detected is added onto 5′-terminal of one PCR primer of each pair in extension. With the asymmetric PCR amplification of the invention, high throughput of single-stranded products can be obtained, single PCR amplification or multiple PCR amplification can be carried out. And the method can be widely used in detection of nucleic acids.

The invention belongs to the field of a detection reagent, and relates to a detection and identification kit for MTBC (mycobacterium tuberculosis complex) and an identification method of the MTBC. According to the method, fluorescence probes and amplification primers for detecting the MTBC are designed through sequence alignment, mycobacterium and the MTBC specific SNP (single nucleotide polymorphism) loci on an rrs gene are detected based on a real-time fluorescent quantitative PCR (polymerase chain reaction) platform and with an asymmetric PCR amplification technology and a probe melting curve analysis technology, so that mycobacteria are identified, and the MTBC and NTM (nontuberculosis mycobacteria) are further identified. The method has the characteristics of convenience in operation, short detection time and high specificity and sensitivity.

A high-throughput method for rapid detection of virus pathogens comprises the following steps of: (1) designing probes and spotting on a chip to obtain an oligonucleotide chip; (2) designing and synthesizing primers to obtain primers corresponding to a detected pathogen; (3) performing synchronous asymmetric PCR amplification to a detected sample with the primers corresponding to the detected pathogen to obtain a single-stranded DNA product specifically complementary with the oligonucleotide; (4) labeling a fluorescent substance on the single-stranded DNA product to obtain a fluorescent-labeled single-stranded DNA product; (5) hybridizing with the oligonucleotide chip, and cleaning the chip; and (6) scanning with a fluorescent scanner to obtain virus detection and analysis results. The invention solves the time-consuming, labor-consuming, low sensibility and specificity and inaccurate detection result problems in the detection method of virus causing porcine epidemic diseases in the prior art. The invention can be used for rapid, accurate and efficient detection of multiple genes.

The invention discloses a nucleotide composition, a kit containing nucleotide and a detection method. When a sample to be detected contains target molecules to be detected, a large amount of single-chain products can be obtained through subjecting a composition II and a primer pair, which are designed in the invention, to an asymmetric PCR, these single-chain products can be hybridized with a silver nanocluster probe prepared by using a composition I designed in the invention and then can emit fluorescence, and thus, the visible detection on the target molecules to be detected is achieve; thedisadvantage that the traditional methods are dependent on instrument discrimination is overcome, a visualized qualitative quantitative novel detection method which is independent of complicated instruments is established, the existing detection techniques and methods are enriched, and thus, a way of think and a technical base are provided for applying silver nano clusters in the aspect of transgenosis in the future. In a specific embodiment, the compositions and the method, disclosed by the invention, have the sensitivity of 0.5ng per microliter and are good in repeatability and high in specificity.

The invention discloses a reagent kit used for detecting CYP2C19 genotypes. Allelic genotypes of CYP2C19*2(681G / A) and CYP2C19*3(636G / A) of a CYP2C19 gene are detected by designing a specific primer pair and a fluorescent probe. Asymmetric PCR and the fluorescent probe melting curve analysis technology are combined, different genotypes are judged through melting peaks of specific temperatures, detection of six genotypes related to CYP2C19*2 and CYP2C19*3 can be achieved in one-tube PCR, subsequent processing is not needed, the possibility of reactant pollution is avoided to the largest extent, operation is easy, consumed time is short, specificity is good, accuracy is high, and results are easy to interpret.

The invention discloses a primer and probe composition for detecting polymorphism of human CYP2C9 and VKORC1 genes, a kit and application. The primer and probe composition comprises three pairs of specific primers for amplifying CYP2C9*2, CYP2C9*3 and VKORC1 sites, and three specific fluorescent probes. The primers and the probes, disclosed by the invention, have high sensitivity, strong specificity and strong anti-interference capability; a manner combining an asymmetric PCR (Polymerase Chain Reaction) amplification and fluorescent probe melting curve analysis technologies is used for detecting the gene polymorphism; different gene types can be effectively distinguished according to the quantity and Tm value of a melting peak; a result is convenient, clear and objective to judge and read.Single-tube sampling can be used for simultaneously detecting 6 mutation types of 3 gene sites; the primer and probe composition is simple and convenient to operate and the detection efficiency is improved; a large batch of samples can be detected and clinical operation is facilitated.

The invention provides a primer pair, specific oligonucleotide fluorescence probe and kit for detecting the nucleotide polymorphism of a C677T locus of an MTHFR gene. The new specific primer pair and the corresponding fluorescence probe are designed, PCR amplification is performed by adopting an asymmetric PCR technology, melting curve analysis is performed after PCR amplification is finished, and a genetype is judged through the melting peak at specific temperature. According to the kit, the MTHFR gene can be amplified in a highly specific mode, detection of three kinds of genetypes of the C677T locus in a single-pipe PCR system can be completed, the detection specificity is high, the result is easy to judge and read, the operating steps are simple, the detection cost is low, the cycle is short, and the efficiency is high.

The invention discloses a nested asymmetric PCR reagent kit for detecting alpha 2 globin gene point mutation. The reagent kit comprises a nested asymmetric PCR amplification primer, a fluorescent probe, a quenching probe, an LADNA polymerase and the like. The alpha-thalassemia point mutation genotype of a detected sample is detected by virtue of specific amplification of five point mutation destination fragments WS, QS, CS, CD30 and CD31 of the human alpha 2 globin gene and molecular hybridization and unwinding analysis of a specific fluorescent probe. The reagent kit has good sensitivity and accuracy to wild type and mutation type of alpha-thalassemia five point mutation positions WS, QS, CS, CD30 and CD31, has good repeatability and stability, and is totally suitable for clinical detection of alpha-thalassemia point mutation.

The invention relates to the field of nucleic acid molecule detection and particularly discloses a multiplex ligation-dependent probe amplification (MLPA) long probe preparation method, a transgene corn MLPA long probe, a transgene corn MLPA short probe and a transgene corn MLPA detection method. The MLPA long probe preparation method can be carried out by adopting conventional laboratory technology of asymmetric polymerase chain reaction (PCR) amplification, enzyme restriction, gel electrophoresis, cut gel recovery and the like. Compared with the existing preparation method, the method has the advantages that the operation is simple, the cost is low, and the operation can be realized in ordinary laboratories. The problem of limited application of the MLPA detection method caused by difficult long probe preparation and high cost is solved. The transgene corn MLPA long probe and the transgene corn MLPA short probe can be used for detecting transgene corn, and the basis is provided for the high-flux MLPA detection of the transgene corn. The transgene corn MLPA detection method carried out on the basis has good expansion performance and high specificity, a simple, convenient, effective and reliable high-flux detection method is provided for the transgene corn detection, and the method is particularly suitable for being used for departments such as inspection and quarantine departments and the like.

The invention discloses an electrochemical luminescence nucleic acid detection method based on branched a DNA (Deoxyribonucleic Acid) amplification signal. The electrochemical luminescence nucleic acid detection method comprises the following steps: acquiring single-chain target DNA by using asymmetric PCR (Polymerase Chain Reaction) amplification target genes, designing an electrochemical luminescence nucleic acid detection primer group for target DNA specificity sequences according to branched DNA amplification signal principles, performing hybridization on the single-chain target DNA and the electrochemical luminescence nucleic acid detection primer group, performing electrochemical luminescence nucleic acid detection on a hybridization product, and performing qualitative and quantitative analysis on target DNA according to existence and intensity of an electrochemical luminescence signal, thereby finally achieving detection on a target gene. The invention further provides a kit for nucleic acid detection by using the method. The electrochemical luminescence nucleic acid detection method has the advantages of being sensitive in reaction, high in detection speed and accurate in result.

The invention discloses primers, probes and a method for detecting plasmodium, belonging to the technical fields of biological detection and biological chemistry. The adopted technical scheme is as follows: the invention provides a method for detecting plasmodium, universal primers SEQ ID No: 1-2 for PCR (Polymerase Chain Reaction) amplification of gene sequences of plasmodium in the method, and probes SEQ ID No: 3-6 for detecting plasmodium. The primers, probes and method have the advantages that (1) the universal primers for PCR amplification of the gene sequences of plasmodium and the specific nucleic acid probes are high in sensitivity and good in accuracy; (2) due to the adoption of an asymmetric PCR method and liquid chip combined technology, the yield of a single strand of a biological marker is increased so that the hybridization and combination efficiencies of PCR products and microspheres coupled with the specific nucleic acid probes are increased; the method has the advantages of high-flux test, high specificity and sensitivity, stable result, good repeatability, simplicity in operation, high detection speed and capability of rapidly detecting plasmodium.

The invention relates to a kit for quickly detecting a drug resistance gene of pneumophila pathogenic bacteria, which comprises a gene chip capable of detecting 24 main drug resistance genes of pneumophila pathogenic bacteria and a reaction system for multiple asymmetric PCR reactions. The 24 drug resistance genes are derived from aminoglycosides, quinolones, extended spectrum beta-lactamases (ESBLs), cephalosporins (AmpC), carbapenems and drug resistance genes with vancomycin resistance and methicillin resistance causing membrane permeability transition. The kit can be used for directly detecting drug resistance genes of pneumophila pathogenic bacteria in clinical samples, is simple and quick to operate, does not need culture or drug sensitive tests, gains valuable time for reasonable application of antibiotics to a great extent, and is worthy of clinical popularization and application.

The invention relates to a method for preparing a visual classical swine fever virus (CSFV) gene detection reagent based on G4DNAzyme coloration. The method comprises the following steps: selecting cells infected with viruses, after freeze thawing and lysing, adopting a gene extraction kit for extracting a target gene and obtaining cDNA after carrying out reverse transcription and inactivation onMoloney murine leukemia virus (MMLV) reverse transcriptase; adding the cDNA to an asymmetric polymerase chain reaction (PCR) system and obtaining an asymmetric PCR product through degeneration, annealing and extension amplification for 50-100 cycles; and adding upstream and downstream probes and the asymmetric PCR product to G4DNAzyme coloration reaction buffer, and after degeneration and annealing, adding Hemin, ATBS and H2O2 to carry out coloration reaction and observing whether macroscopic green color appears, thus judging whether CSFV infection exists. The method has the beneficial effects of high detection speed, accuracy, stability, good repeatability, simple and easy-to-operate detection steps, capability of directly observing the coloration reaction with naked eyes, intuitionisticresults and low cost.

The invention discloses a method for quickly identifying a food pathogenic bacteria subtype based on an asymmetric polymerase chain reaction (PCR) combined test strip platform and a kit, and belongs to the technical field of biological detection. The method comprises the main steps of: (1) extracting food pathogenic bacteria subtype DNA; (2) designing an amplification primer and a nucleic acid probe sequence; (3) performing amplification reaction based on asymmetric PCR; (4) preparing a nanogold probe; (5) preparing a colloidal gold nucleic acid test strip; (6) detecting a sample. According to the method, food pathogenic bacteria in food samples can be quickly, specially, sensitively, qualitatively and quantitatively detected and the special subtype of the pathogenic bacteria is determined; the probe is simply designed, the operation steps are simple and short, the product is a single chain, and the method is easy to detect and convenient to popularize.

The invention provides a method and a kit for genotyping point mutation. The kit uses the fluorescence probe melting curve of fluorescent quantitative PCR to directly genotype the point mutation; and the kit includes a pair of upstream and downstream primers for carrying out asymmetric PCR amplification on every point mutation point of a sample to be detected, a Taqman probe designed for every point mutation point of the sample to be detected, and a positive quality control substance for every point mutation point of the sample to be detected. The method and the kit have the advantages of convenience in use for the point mutation genotyping, and convenience in promotion and use.

The invention provides a method for identifying yak meat and cattle meat by adopting an HRM method. The method comprises the following steps: (1) selecting different loci of the mitochondrial 12S rRNA gene of the yak and the mitochondrial 12S rRNA gene of the cattle, and designing a marking primer and a marking probe according to the selected different loci; (2) extracting the whole genome DNA of a to-be-detected sample; (3) by taking the whole genome DNA of the to-be-detected sample obtained in the step (2) as a template, adding with the marking primer and the marking probe in the step (1), and carrying out asymmetrical PCR amplification, thus obtaining a PCR amplification product; and (4) detecting the melting temperature of the PCR amplification product obtained in the step (3) by adopting an HRM method, and drawing a melting curve, wherein if the melting peak is within 55-60 DEG C, the to-be-detected sample is detected to be the yak meat, and if the melting peak is within 65-70 DEG C, the to-be-detected sample is detected to be the cattle meat.

The invention relates to a rape transgenosis detecting kit in the field of detecting articles. The rapeseed transgenosis detecting kit comprises a primer combination and a membrane chip, and is characterized in that the primer combination is a 11-double-starting oligonucleotide primer combination, the membrane chip refers to 11 groups of probe sequences and one group of positive control probe sequences, which are fixed on the surface of a supporting film, and the 5' end of each probe is provided with an amino marker; asymmetric PCR (polymerase chain reaction) amplification primers, which are Tag primers with biotin marks at 5'ends, are included in the primer combination. The rapeseed transgenosis detecting kit provided by the invention overcomes the defects of time and energy waste and high cost in the prior art, the rape transgenosis detecting kit provided is simple to operate, is quick and sensitive, is high in detection flux, has visual detection results and is low in cost.

The invention discloses a kit for detecting genetically modified soybean in the field of test items. The kit for detecting genetically modified soybean comprises a primer combination and a membrane chip. The kit for detecting genetically modified soybean is characterized in that the primer combination is a seven polymerase chain reaction (PCR) primer combinations; each pair of primers comprise Pat, Bar, Cry1Ab, Cry105, EPSPS, P35S and a soybean endogenous gene sequence Lectin; the membrane chip is seven groups of probe sequences, which are orderly fixed on the surface of a support membrane and a group of positive control probe sequences, the probe sequences are respectively a Pat probe, a Bar probe, a Cry1Ab probe, a Cry105 probe, an EPSPS probe, a P35S probe, a Lectin probe and a Positive probe; an asymmetric PCR amplification primer is formed in the primer combination, and is a Tag primer with a biotin marker (biotin) at the 5' end. By adopting the kit for detecting genetically modified soybean, the defects of time and labor waste, and high cost in the prior art are overcome; the provided kit for detecting genetically modified soybean is simple to operate, quick and sensitive, large in detection throughput, visual in detection result and low in cost.

The invention relates to a visual classical swine fever virus (CSFV) gene identification and detection reagent based on G4DNAzyme coloration and a preparation method thereof. The reagent is asymmetric (polymerase chain reaction) PCR reaction liquid. The 100mul reaction liquid comprises: 2mul of positive primerW3(+) (0.4-4mumol / L) and 2mul of negative primer W3(-) (20mumol / L), 10mul of PCR solution (10*buffer), 8mul of MgCl2 solution (2.5mmol / L), 2mul of dNTPs solution (10mmol / L), 2mul of TaDNA polymerase (2U) and balance of sterile double distilled water. A sequence of the positive primer W3(+) in the asymmetric PCR reaction liquid is: 5'-gcgcgggtaacccgggat-3', and the sequence of the gemeative primer W3(-) is 5'-GTCCAACTGTGGACGTCAGGAA-3'. The method has the beneficial effects of high detection speed, accuracy, stability, good repeatability, simple and easy-to-operate detection steps, capability of directly observing the coloration reaction with naked eyes, intuitionistic results and low cost.

The invention relates to a method for detecting gene chips of SS2 (streptococcus suis serotype 2) and an application method thereof, which can effectively solve the problems that the existing detection equipment and method are long in time consumption and large in investment. The technical scheme of the invention is as follows: a method for detecting gene chips of SS2 is implemented through designing and synthesizing a primer and a probe, dissolving the synthesized probe by using sterilized water, and carrying out contact sample application on an aldehydized substrate by using a gene chip sample application instrument according to a preset program. The application method comprises the following steps of: extracting the genomic DNA (deoxyribonucleic acid) of SS2 to be detected; preparing a PCR (polymerase chain reaction) product for hybridization by using an asymmetric PCR, and hybridizing the prepared PCR product with a prepared gene chip; and after the obtained product is washed and dried, placing the chip on a laser confocal scanner to scan the chip, and synthesizing results. The methods disclosed by the invention are simple and easy to operate; steps of carrying out hydration treatment and pre-hybridization on chips subjected to sample application are omitted, and the hybridization is implemented just through half an hour, thereby greatly saving the time; and the signal strength can be achieved by using a probe concentration of 5mu m / L, therefore, the detection cost is low, and the methods can be used for large-scale detection.

The present invention relates to a primer probe system, a method and a kit for detecting epidermal growth factor receptor exon 19 and 21 mutations. According to the method, an asymmetric PCR technology, a nucleic acid isothermal amplification technology and a fluorescence detection technology are organically integrated, wherein the asymmetric PCR amplification technology and special primers are adopted to obtain a lot of single-stranded DNA so as to overcome disadvantages of low efficiency linear amplification of 3WJ (an isothermal digestion nucleic acid detection technology), and specificity and simplicity of the 3WJ technology are adopted, such that a reaction temperature can be controlled at about 30 DEG C, enzyme activity reduction can not be generated, expensive and precise temperature control equipment is not required during a detection process, and discrimination can be achieved only through the simple fluorescence detection instrument with the special probe design. According to the present invention, advantages of rapidness, simpleness, specificity, sensitivity, and the like are provided.

The invention discloses an SARS-CoV-2 D614G mutation detection kit and an SARS-CoV-2 D614G mutation detection method. The sequences of a primer and a probe for SARS-CoV-2 D614G mutation nucleic acid detection are shown as SEQ ID NO: 1-4. According to the SARS-CoV-2 D614G mutation detection kit, asymmetric PCR amplification is adopted, a multicolor probe melting curve analysis technology is combined, typing detection of SARS-CoV-2 S-D614 and S-G614 strains can be achieved, and the kit has the advantages of being easy to operate, short in detection period, high in sensitivity and the like.

The present invention relates to compositions and methods for nucleic acid based diagnostic assays. In particular, the present invention provides probes and non-amplifiable controls for asymmetric PCR and other amplification modalities. In some embodiments, the present invention provides probe design criteria for probes for use in amplification / detection assays. Further embodiments of the present invention provide non-amplifiable controls for use in generating reference probe signal ratios in amplification detection assays.

The present invention provides a microarray for detecting a genotype at a polymorphic site in a plurality of nucleic acid samples, comprising a first set of nucleic acid fragments derived from the samples and a second set of nucleic acid fragments derived from a plurality of references immobilized thereon. The invention also provides a microarray comprising a set of nucleic acid fragments immobilized on the surface of the microarray, wherein the nucleic acid fragments are derived from the samples by amplifying a region in the sample containing the polymorphism through asymmetric PCR amplification. Methods of using and making the microarrays are also provided.

Provided herein is a method for detecting the presence of a COVID-19 virus in a human sample or an environmental sample having one or more viruses and bacterial pathogens. Samples are processed to obtain total nucleic acids. A combined reverse transcription and asymmetric PCR amplification reaction is performed to obtain fluorescent labeled COVID-19 virus specific amplicons. The amplicons are detected by microarray hybridization near the lowest limit of detection. Also provided is a method for detecting concurrently with COVID-19 virus, the presence of respiratory disease-causing pathogens including viruses, bacteria and fungus in a single assay using the above method.

The invention discloses a screening library of nucleic acid aptamers, belonging to the technical field of bioanalysis and bioengineering. The screening library comprises the following specific nucleotide sequences: 5'-ACCGACCGTGCTGGACTCT(N40)ACTATGAGCGAGCCTGGCG-3' as the library, 5'-ACCGACCGTGCTGGACTCT-3' as the forward primer, and 5'-CGCCAGGCTCGCTCATAGT-3' as the reverse primer. According to the invention, the library has short fixed sequences with the length being close to the minimum length and long random sequences, and the diversity of single-chain oligonucleotides in the library is fully guaranteed; the unique design of the fixed sequences (primer sequences) in the library can conduct PCR amplification at high annealing temperature, can effectively inhibit non-specific products and does not influence the amplification of target products, which is proved in symmetric PCR amplification, asymmetric PCR amplification and real-time fluorescent quantitative PCR amplification. The random single chain oligonucleotide library provided by the invention has excellent performance and provides guarantee for successfully screening nucleic acid aptamers of bio-target.

The invention discloses a micro array chip to check 45 gene locus of mitochondria diabetes, which comprises the following steps: choosing aldehyde slide as carrier; designing primer and probe; distributing and controlling chip dot matrix; distributing and controlling dot matrix of monitoring system and testing system; composing the monitoring system with negative contrast and parallel contrast of base point of discontinuity; setting the testing system as wild-type and saltant probe of mtDNA 45 site; setting each chip incorporates 1-3 0.04cm2 sampling point matrix, 90 trips probe of each matrix and 324 sites. The operation is simple, which can be used to check diabetes patient and high risk group.