59results about How to "Inhibition of nonspecific amplification" patented technology

The invention relates to the field of molecular biology and aims to provide an ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and a detection method. The kit comprises a qPCR hybrid reaction solution, a locked nucleic acid retardant probe, a reference primer, an ARMS primer and a positive control sample, wherein the qPCR hybrid reaction solution comprises a PCR buffer solution, dNTPs (Deoxynucleotide Triphosphates), MgCl2, GoldStarbest Taq enzyme, a universal PCR reverse primer and a universal TaqMan probe. The kit provided by the invention can be used for rapidly and accurately detecting specific locus mutation of KRAS genes in various cancer tissues with high sensitivity, has high sensitivity, and can be used for detecting genome DNA with various tissue origins, specially free DNA segments adopting cell-free systems, such as blood serum and blood plasma, orother body fluid origins, wherein the genome DNA is derived from cell systems. Compared with direct sequencing and other mutation detection technologies, the kit and the detection method thereof havethe advantages of strong specificity, high sensitivity, simplicity and rapidness in operation, high throughput, safety, definiteness and objectivity in result identification and the like for detecting the KRAS gene mutation.

The invention discloses a method for detecting thymine DNA glycosylase activity based on a cyclophorase-remediation mediated double-signal amplification strategy. A fluorescent method for detecting the thymine DNA glycosylase activity in real time can be achieved through circular rolling circle index amplification triggered by thymine DNA glycosylase and assisted by enzyme remediation and circular cutting catalyzed and mediated by endonuclease IV. As efficiency of circular rolling circle amplification assisted by uracil excision and efficiency of circular cutting based on endonuclease IV are both high and index amplification mediated by uracil can greatly inhibit non-specific amplification, the method has very high detection flexibility; detection shows that lower detection limit of the method to thymine DNA glycosylase is 5.6*10<-7>U / mu L; meanwhile, the method disclosed by the invention utilizes self repair characteristics of the thymine DNA glycosylase and uracil DNA glycosylase to enable the whole repairing reaction to be performed strictly according to a natural repairing mechanism, so that the method disclosed by the scheme of the invention has very high specificity.

The invention relates to the technical field of biology and particularly relates to a next generation sequencing library building technology. A library is built through utilizing two rounds of multiplex PCR (Polymerase Chain Reaction). Aiming at each target region or target site, two specific primers are designed; and an upstream primer OP is used for the first round of multiplex PCR and a downstream primer IP is used for second round of multiplex PCR. After a product of the second round of PCR is purified, Q-PCR quantification is carried out; and then the product is diluted to a proper concentration and is used for next generation sequencing. According to the next generation sequencing library building technology, the problems of an Ampliseq kit can be effectively reduced, and a customization-oriented panel design production period only needs two weeks; the cost of building a library by a single sample is reduced; and the technology is general for Ion Torrent and Illumina sequencing platforms.

The invention relates to a warm starting TaqDNA polymerase and its preparation method and an application. The warm starting TaqDNA polymerase is prepared by an antibody modification method, and includes steps of (1), structuring and transformation of a recombination expression carrier; (2), inductive expression of recombinant bacteria; (3), preparation and purification of crude enzyme; (4), antibody modification. The warm starting TaqDNA polymerase is high in specificity, high in sensitivity, and convenient to operate; the warm starting TaqDNA polymerase can be applied to regular PCR amplification, real-time fluorogenic quantitative PCR, multiplex PCR, and digital PCR and TA cloning.

The invention discloses a primer, a probe and a kit for detecting a mutation of a human JAK2 gene V617F. The primer comprises a mutation forward primer JW1-F and a mutation reverse ARMS primer JM1-R, and the probe comprises a detection probe JW1-P and a blocking probe JW1-B, wherein the mutation forward primer JW1-F is combined with a JAK2 gene conserved sequence; the mutation reverse ARMS primer JM1-R is specifically combined with a V617F (1849G>T) site mutation sequence to selectively amplify the mutation sequence; a 5minute end of the detection probe JW1-P is marked with an FAM (carboxy fluorescein) signal; a 3minute end of the detection probe is marked with an MGB (Minor Groove Binder); the detection probe JW1-P can be combined with an amplified fragment; a 5minute end of the blocking probe JW1-B is double-deoxidized and modified; a 3minute end of the blocking probe JW1-B is marked with the MGB; the blocking probe JW1-B can be specifically combined with a V617 site wild-type sequence to inhibit wild-type nonspecific amplification. The primer and the probe which are disclosed by the invention are high in specificity and good in sensitivity, and have high detectability of 1 percent. The kit prepared from the primer and the probe is accurate in detection result and easy in reading of the detection result, is simple and rapid to operate, and is wide in application range.

The invention discloses a method for detecting the activity of DNA methyltransferase based on single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification. The method comprises the following steps: (1) adding a Dam MTase acted hairpin substrate to a resection reaction buffer solution, and carrying out a cleavage reaction to produce a 24-nt product; and (2) adding the cleavage product to an amplification reaction buffer solution, carrying out a cyclic linkage-dependent strand displacement amplification reaction to cyclically generate an enhanced fluorescent signal, anddetecting the intensity of the fluorescent signal to determine the activity of the Dam MTase. The method can effectively inhibit nonspecific amplification independent of a target and a template / primer, so the background signal is greatly reduced, and the index amplification of the signal is finally achieved. The method has a high sensitivity and a high specificity, and the lower detection limit can reach 4.8 * 10<-6> U / mL.

The invention relates to an antisense interference oligonucleotide used for inhibiting primer non-specific amplification. Through combining a primer 3' end antisense oligonucleotide, the antisense interference oligonucleotide interferes PCR primer 3' end non-specific amplification. The antisense oligonucleotide is a section of oligonucleotide with 4-14 base composed of antisense nucleotide. The basic characteristics are that the antisense base oligonucleotide maintains a Watson base pairing hybrid property, but loses the functions as PCR amplification template and primers. Antisense oligonucleotide is immobilized by using nanospheres, such that primers are indirectly immobilized to a PCR system, and hot start is sustained-released. Mineral oil is used for sealing PCR, such that aerosol cross-contamination is avoided, and PCR reaction is prevented from non-specific amplification. The antisense oligonucleotide is suitable for various PCR technologies with primer annealing and extension, including array amplification technology.

The invention discloses primers and probes and a kit for detecting seven mutation types of a human RET gene. According to each group of the seven groups (RM1-RM7) of the primers and probes, the mutation upstream ARMS primer can be specially combined with a corresponding mutation sequence of the mutation upstream ARMS primer to amplify the mutation sequence, the mutation downstream primer can be combined with a conserved sequence of the RET gene, the detection probe can be combined with an amplified fragment, and the blocking probe can be specially combined with a wild type sequence corresponding to a mutation locus to inhibit wild type nonspecific amplification. Accordingly, by adopting a specific mutation primer and probe blocking technology, the seven mutation types of the RET gene can be accurately detected; through the established kit of a real-time fluorescence PCR amplification reaction system, rapid detection on the RET gene mutation is facilitated, operation is easy, and a result is easy to read; the detection method is high in sensitivity, and 50-copy mutation can be stably detected; the specificity is good, no nonspecific amplification is generated in 20-ng wild type genome DNA, and the detectability reaches up to 1%.

The invention discloses a probe, a primer and a kit for detecting seven kinds of mutations of human NRAS genes, wherein in seven groups of primers and probes from NM1 to NM7, the mutation primer in each group can be combined with a conserved sequence of the NRAS genes; the mutation ARMS primers can realize the specific binding with the corresponding mutation sequences; the mutation sequences are amplified; the detection probe can be combined with an amplification segment; the specific binding of the probe with the wild sequence corresponding to the mutation site is blocked; and the wild nonspecific amplification is inhibited. The specific mutation primer and probe blocking technology is used; the seven kinds of mutation types of the NRAS genes can be accurately detected; the built kit for a real-time fluorescent PCR (Polymerase Chain Reaction) amplification reaction system can be used for conveniently and fast detecting the mutations of the NRAS genes; the operation is simple; the result can be easily read; the sensitivity of a detection method is high; 50 copy mutations can be stably detected; the specificity is good; the 20ng wild genome DNA (Deoxyribose Nucleic Acid) does not have specific amplification; and the detection capability is as high as 1 percent.

The invention relates to a triple fluorescence PCR method for detecting bovine-derived, sheep-derived and porcine-derived ingredients. The method comprises four elements, namely a sample DNA, a pair of universal primers and three probes, fluorescence PCR premixed liquid as well as a positive control. The method comprises the following steps: designing primers and probes according to glyceraldehyde-3-phosphate dehydrogenase genes of cows, sheep and goats as well as pigs, amplifying a target sequence, and exciting and quenching a fluorescence signal, wherein a non-target sequence is not amplified and has no fluorescence signal; and preparing a kit according to a formula of a reagent used in fluorescence PCR amplification carried out on a to-be-detected sample DNA and amplification conditions. Concrete actions are as follows: a triple fluorescence PCR primer system used for detecting the bovine-derived, sheep-derived and porcine-derived ingredients is established; a triple fluorescence PCR kit used for detecting the bovine-derived, sheep-derived and porcine-derived ingredients is established; and a triple fluorescence PCR identification method used for detecting the bovine-derived, sheep-derived and porcine-derived ingredients is determined. The method provided by the invention has the advantages that the standard error is small, the detection time is short, multiple target genes can be detected at the same time, and the reagent cost is saved; and the method is applicable to true and false identification of an animal product.

The invention discloses a probe, a primer and a kit for detecting 7 mutations of a human CKIT gene. 7 groups of primers and probes CM1 to CM7 are provided; in each group, a mutation forward primer can be combined with a CKIT gene conserved sequence, and a mutation reverse ARMS primer can be specifically combined with a corresponding mutation sequence so as to amplify the mutation sequence; the detection probes can be combined with amplified fragments, and blocking probes can be specifically combined with a wild-type sequence corresponding to a mutation site so as to inhibit wild-type non-specific amplification. According to the probe, the primer and the kit which are disclosed by the invention, a specific mutation primer and probe blocking technology is adopted; 7 mutations of the CKIT gene can be accurately detected; the established real-time fluorescence PCR (Polymerase Chain Reaction) amplified reaction system kit is convenient to rapidly detect the mutations of the CKIT gene, is simple to operate, and is easy in reading of a result; a detection method is high in sensitivity, and 50-copy mutants can be stably detected out; specificity is good, 20ng wild-type genome DNA has no phenomenon of nonspecific amplification, and detectability reaches 1 percent.

The invention discloses primers and probes and a kit for detecting five mutation types of a human PIK3CA gene. According to each group of the five groups (PIM1-PIM5) of the primers and probes, the mutation primer can be combined with a conserved sequence of the PIK3CA gene, a mutation ARMS primer can be specially combined with a corresponding mutation sequence of the mutation ARMS primer to amplify the mutation sequence, the detection probe can be combined with an amplified fragment, and the blocking probe can be specially combined with a wild type sequence corresponding to a mutation locus to inhibit wild type nonspecific amplification. Accordingly, by adopting a specific mutation primer and probe blocking technology, the five mutation types of the PIK3CA gene can be accurately detected; through the established kit of a real-time fluorescence PCR amplification reaction system, rapid detection on the PIK3CA gene mutation is facilitated, operation is easy, and a result is easy to read; the detection method is high in sensitivity, and 50-copy mutation can be stably detected; the specificity is good, no nonspecific amplification is generated in 20-ng wild type genome DNA, and the detectability reaches up to 1%.

The invention discloses a kit capable of rapidly detecting staphylococcus aureus in meat and application and belongs to the technical field of food safety. The kit is composed of an LAMP amplified reaction system, negative control liquid and positive control liquid, and the LAMP amplified reaction system is composed of Bst 2.0WarmStart DNA polymerase and an SEQ IDNO1-4 primer group. The invention further provides the application of the kit capable of rapidly detecting the staphylococcus aureus in the food-grade fresh meat or meat products. The kit has the advantages of being sensitive, rapid, simple and convenient. The specificity is good, cold temperature condition is not needed to be created, the sensitivity is high, the sensitivity of the kit detection is 100 times the sensitivity of common PCR detection, and the kit is suitable for rapidly detecting the staphylococcus aureus in the food grade fresh meat or the meat products.

The invention discloses a quantitative polymerase chain reaction (PCR) method capable of improving sensitivity and specificity. Fluorescent dye GelgreenI and hot start hTaq enzyme are added into a PCR system, and the fluorescent dye GelgreenI can be specifically combined with DNA double chains to send out a fluorescent signal. The high-sensitivity and high-specificity quantitative PCR method provided by the invention adopts the hot start hTaq enzyme and proper magnesium ion concentration, so non-specific amplification can be inhibited well, false positive and high background results are avoided, and a solubility curve can obtain single peak; the fluorescent dye GelgreenI is low in price, convenient to use, good in universality, high in sensitivity and applicable to a qPCR technology; the invention provides a new quantitative PCR method for molecular biology research.

The present invention discloses a method for detecting Dam methyltransferase activity based on base excision repair-induced strand displacement amplification and exponential isothermal amplification reaction fluorescence. The method has simple operation steps, good selectivity and high sensitivity. Experimental results show that the linear range of determination of Dam methyltransferase is 0.02-10U / mL, and the detection limit is 0.014 U / mL. The method can also be used for detection of endogenous Dam methyltransferase in E. coli cells. The detection limit of Dam methyltransferase in E. coli is0.61*10<-6> mg / mL.

The invention discloses probes, primers and reagent kits for detecting five mutations of a human gene TP53. According to five primers and probes for TM1-TM5, each mutation primer can be combined with a conserved sequence of the gene TP53, the primer for the mutation ARMS can be specifically combined with the corresponding mutation sequence, and the mutation sequence is amplified. The detection probe can be combined with amplified fragments, the blocking probe can be specifically combined with a wild type sequence corresponding to a mutation site, and wild type non-specificity amplification is restrained. The specificity mutation primers and the probe blocking technology are adopted, and the five mutation types of the gene TP53 can be accurately detected; mutations of the gene TP53 are rapidly detected conveniently through the built reagent kits of a real-time fluorescence PCR amplified reaction system, operation is easy, and the result is easy to read. The detection method is high in sensitivity, and 50-copy mutation can be stably detected; the specificity is good, 20 ng wild type genomic DNA non-specificity amplification is achieved, and the detectability reaches up to 1%.

The invention discloses transgenic tobacco multi-fluorescence PCR gene loci and primers and a detecting method thereof. The transgenic tobacco multi-fluorescence PCR gene loci and primers comprise loci, primers and probes of genes of Nr, Btc, CpTI, EPSPS, CMV-cp, PVY-cp, TMV-cp, CMV-sat, PVY-nib and TMV-54D. According to the transgenic tobacco multi-fluorescence PCR gene loci and primers, endogenous and specific exogenous genes of transgenic tobacco lines are simultaneously applied to design of specific primer-probe groups, and the primers and the probes can avoid mutual interference and can only perform amplification and fluorescence signals on specific target sequences but have no amplification or fluorescence signals on non-targeted sequences, so that high specificity and detecting sensitivity can be achieved.

The invention relates to a gene mutation detection product, in particular to a primer probe combination for KRAS gene mutation detection and application thereof. The primer probe combination comprisesdetection primer probes, wherein the detection primer probes include a KRAS gene mutation detection-specific primer pair, a KRAS gene-specific probe, an amplification blocking probe and an internal reference system, wherein KRAS gene mutation detection sites are 12 and 13 codons of an exon 1, particularly G12S, G12C, G12R, G12D, G12A, G12V and G13D mutation sites. The primer probe combination hasthe advantages of strong specificity, high sensitivity, simple and rapid operation and the like for KRAS gene mutation, a detection result has relatively good accuracy and repeatability, and the primer probe combination can detect a tumor tissue sample, can assist clinical treatment, and has an important value.

This invention relates to a method for preparing host-starting heat-resistant DNA polymerase. The activity of the host-starting heat-resistant DNA polymerase is reversibly blocked through chemical modification, and can be recovered after treatment at 94 deg.C or higher and pH = 8-9. The host-starting heat-resistant DNA polymerase has low 3'-5' elongation activity at low temperatures, and has suchadvantages as high specificity, high reliability, high uniformity and high sensitivity. The host-starting heat-resistant DNA polymerase can be used in PCR, especially human STR-PCR DNA composite proliferation gene separation.

The invention relates to a preparation method?of a hot start DNA polymerase,?comprising the following steps:?obtaining a?single stranded DNA binding protein, a Taq DNA polymerasemonoclonal antibody?and?a Taq DNA polymerase; and mixing the?single stranded DNA binding protein, the Taq DNA polymerase?monoclonal antibody?and the Taq DNA polymerase in accordance with?aproportion. The contents of the?single stranded DNA binding protein, the Taq DNA polymerasemonoclonal antibody?and the Taq DNA polymerase are all 20-40%?respectively. The invention also relates to the hot start DNA polymerase prepared by the above method and an application thereof. The hot start DNA polymerase provided by the invention can also be used to solve problems which appear easily in hot start PCR technology such as DNA damage, product contamination?and bad effect of hot start caused by incomplete sealing.

The invention discloses a nano PCR method for detecting fowl adenovirus type 4 and a kit, Ag / Au / Fe3O4 nanoparticles and primers shown as SEQ ID No.1-2 are added into a reaction system of the nano PCR,and the reaction system and reaction conditions are optimized. By adopting the Ag / Au / Fe3O4 nano composite material, the amplification efficiency of PCR is improved, the sensitivity is improved by 10times, non-specific amplification is effectively inhibited, and the defect of insufficient primer specificity is overcome.

The invention discloses primers and probes and a kit for detecting human PDGFRA gene D842V mutation. The primers and probes comprise the mutation upstream ARMS primer PM1-F which is specially combined with a D842V (2525A>T) locus mutation sequence to selectively amplify the mutation sequence, the mutation downstream primer PW1-R which is combined with a conserved sequence of the PDGFRA gene, the detection probe PW1-P of which the 5' end is marked with an FAM signal and the 3' end is marked with MGB and the blocking probe PW1-B of which the 5' end is modified through double deoxidation and the 3' end is marked with the MGB, wherein the detection probe PW1-P can be combined with an amplified fragment, and the blocking probe PW1-B can be specially combined with a D842V locus wild type sequence to inhibit wild type nonspecific amplification. The primers and probes are high in specificity and good in sensitivity, and the detectability reaches up to 1%; the kit composed of the primers and probes is accurate and readable in detection result, easy and rapid to operate and wide in application range.

The invention discloses a detection method of multi-drug-resistant microorganisms, the detection method comprises the following steps: capturing the multi-drug-resistant microorganisms by using nucleic acid aptamer magnetic beads, releasing a blocking sequence complementary with an aptamer, converting a protein signal into a nucleic acid signal, amplifying the blocking sequence through an index amplification reaction, and measuring the amplified blocking sequence. According to the invention, a molecular enhancer is added into an index amplification reaction system to inhibit non-specific amplification reaction, and a nanoscale transition metal dihalogen compound is added as a result reading carrier in the determination process, so that the intensity of a background signal is further reduced. The method is simple, convenient and efficient to operate, can achieve the purpose of background-free, rapid and hypersensitive detection, and can solve the key technical problems that a traditional detection technology is long in detection period and low in sensitivity, a background signal exists in a detection result, result interpretation is inaccurate due to the influence of human subjective factors, and severe non-specific amplification exists in isothermal amplification reaction. The method is suitable for detecting the drug resistance of the microorganisms.

The invention discloses a method for improving nucleic acid (nucleic acid) amplification reaction efficiency, and belongs to the technical field of biology. According to the method provided by the invention, carbon quantum dots are added to a reaction system, and nucleic acid in vitro amplification is performed. The method can effectively improve the sensitivity of a nucleic acid in vitro amplification reaction, can restrain the appearance of a false positive result, and can reduce non-specific amplification, so that the detection efficiency is improved. The method can be applied to the fieldof nucleic acid in vitro amplification of PCR amplification, loop-mediated isothermal amplification and the like, and has wide application prospects.

The invention provides a method of establishing a DNA molecular label of a Yunrui-series maize cultivar. The method is applied to cultivar molecular identification and cultivar management. The method is characterized by screening and differentiating 4 pairs of core primers of the Yunrui-series maize cultivar and optimizing a PCR reaction system, carrying out digital coding assignment of cultivar DNA information, forming a two-dimensional code representation, and marking the same on a commodity seed package for using for anti-counterfeiting and tracing of seeds of the fine maize cultivar.

The invention relates to a DNA in vitro amplification technology characterized by simplicity in operation and high specificity, namely a suppression polymerase chain reaction (S-PCR for short) kit. The process of the S-PCR comprises the following steps of: synthesizing a pair of special inhibition primers according to a target DNA sequence to be subjected to amplification, wherein about 15-18 basic groups at the 3' end of each primer are complementary with a template DNA sequence, and 11-14 basic groups at the 5' end of each primer are reversely complementary with the sequence of the primer; dissolving the primers to obtain a partial double-chain structure; and adding the inhibition primers into a PCR amplification buffer reaction system comprising heat-resistant DNA polymerase, dNTPs, template DNA, magnesium ions, PCR buffer, ultrapure water and the like, and performing high temperature, low temperature and intermediate temperature repeated thermal cycle by using a thermocycler. In the amplification process, when the temperature is reduced to annealing temperature, the inhibition primers are specifically combined with a template, and massive inhibition primers renature into double-stranded primers; and because of the priority of intra-strand renaturation, two ends of most non-specifically amplified single strands renature to form hairpin structures, the activity of becoming effective PCR templates is lost, and the amplification specificity is improved.

The invention relates to a B-raf gene mutation detection kit. The B-raf gene mutation detection kit comprises quality-control primer probe internal-standard mixed liquor and detection primer probe internal-standard mixed liquor, wherein the quality-control primer probe internal-standard mixed liquor comprises a quality-control primer pair, a B-raf gene specific probe, an internal-standard primer pair, an internal-standard specific probe and an internal-standard template, and the detection primer probe internal-standard mixed liquor comprises a B-raf gene V600E mutation detection specific primer pair, a B-raf gene specific probe, an amplification blocking nucleic acid sequence, an internal-standard primer pair, an internal-standard specific probe and an internal-standard template. The B-raf gene mutation detection kit has the advantages that an amplification refractory mutation system is combined with a wild type amplification blocking nucleic acid sequence with a phosphorylated terminal, deoxyinosine is introduced into detection of a B-raf gene V600E mutation detection specific ARMS (amplification refractory mutation system) forward primer to enable quality-control PCR (polymerase chain reaction) and detection PCR to be performed for detection of samples parallelly, and each reaction system can have an internal-standard system capable of effectively avoiding false negative and intra-assay variation; the B-raf gene mutation detection kit is low in cost, high in sensitivity and more capable of controlling intra-assay and inter-assay variation.

The invention relates to multiplex, asymmetric amplification of nucleic acid molecules. Particularly, the invention provides a method for simultaneously and asymmetrically amplifying one or more target nucleic acids in a sample. The method can be used for simultaneously and asymmetrically amplifying multiple target nucleic acids in the sample and simultaneously generating a large number of single-chain products.

The invention discloses probes, primers and kits for detecting seven kinds of mutations in human NRAS genes, wherein there are seven sets of primers and probes from NM1 to NM7, and the mutation primers in each group can combine with the conserved sequence of the NRAS gene, and the mutation ARMS primers It can specifically bind to the corresponding mutant sequence and amplify the mutant sequence; the detection probe can bind to the amplified fragment, and the blocking probe can specifically bind to the wild-type sequence corresponding to the mutation site, inhibiting the non-specific amplification of the wild-type . The present invention adopts specific mutation primer and probe blocking technology, can accurately detect 7 kinds of mutation types of NRAS gene; the kit of the real-time fluorescent PCR amplification reaction system established is convenient for rapid detection of NRAS gene mutation, and the operation is simple and the result is easy Read; the detection method has high sensitivity, and 50 copies of mutations can be detected stably; the specificity is good, 20ng of wild-type genomic DNA has no non-specific amplification, and the detection ability is as high as 1%.