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

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 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.

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 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 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%.

The invention discloses a triple fluorescence PCR primer set for detecting pepper transgenic ingredients. The triple fluorescence PCR primer set comprises a triple primer set A for CaATL1, CaMV35S and NPT II genes and a triple primer set B for NOS, CMV and TMV genes and is specifically shown as Seq. ID No.1 to Seq. ID No.12. The invention belongs to the technical field of genetic engineering detection; all the primers and probes cannot mutually interfere; a specific target sequence is amplified and excites a fluorescence signal, the non-target sequence is not amplified and generates no fluorescence signal and accurate detection for the pepper transgenic ingredients can be realized; the triple fluorescence PCR primer set has the advantages of high specificity, low standard error, short detection time, capability of saving reagent cost and the like, and is suitable for the detection for the transgenic ingredients of the pepper product.