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 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 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.

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 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.