84results about How to "Increase the Tm value" patented technology

This invention relates to one polymer enzyme linkage reaction fluorescence test method to dialogue dangerous human body nipple shape virus and to isolate DNA sample HPV gene type to test one set of dangerous HPV infection from patient by the method, wherein, it belongs to life science and biological technique. This invention agent case comprises one fluorescence meter PCR technique as base of multi-layer polymer enzyme reaction composed of multiple HPV positive lead object, reaction lead object and fluorescence detector.

The invention discloses a primer pair for specific amplification of EGFR gene 20 exon T790M and C797S loci. The sequence of the primer pair is shown in SEQ ID No:1-2. The invention further discloses a probe set for specific detection of the T790M and C797S loci. The probe set comprises a wild type probe and a mutant type probe, and the sequences are shown in SEQ ID No:3-7. The invention further discloses a reagent kit comprising the primer pair and the probe set, and a method for adopting the primer pair and the detection set for detecting EGFR gene 20 exon T790M and C797S mutation. By designing the specific primers and probes, the 3' end of the probes is connected with a minor groove binder and a non-fluorescent quenching group, the interference of background signals is lowered, hybridization of the probes and a template is stabilized, a Tm value of the probes is increased, and therefore the mutation detection sensitivity is improved.

The new quantitative PCR detection process of micro RNAs (microRNAs) and small interference RNAs (siRNAs) includes: complementing the bases near the 5' end and the 3' end of retroprimer to form a stem-loop structure, combining the 3' end of retroprimer and target nucleic acid complementarily, and performing reverse transcription reaction with reverse transcriptase; amplifying cDNA obtained through the reverse transcription reaction with partial sequence of the retroprimer as one of the PCR primers; and real-time monitoring quantitative PCR with fluorescence labeled molecular beacon probe.

The invention relates to the technical field of molecular biology detection, in particular to a TaqMan-MGB fluorescent quantitative polymerase chain reaction (PCR) detection specific primer (as shown in SEQ ID No. 1 and SEQ ID No. 2), a TaqMan-MGB fluorescent quantitative PCR detection probe as shown in SEQ ID No. 3) and a TaqMan-MGB fluorescent quantitative PCR detection method for seneca valley virus (SVV). The method comprises the steps of drawing a standard curve; extracting ribonucleic acid (RNA) of a sample virus; carrying out reverse transcription on the RNA of the sample virus; enabling the product of the reverse transcription to have a TaqMan-MGB fluorescent quantitative PCR, and reading the result. The primer provided by the invention has better specificity and sensitivity; the MGB probe provided by the invention is shorter and is beneficial to probe design, and the Tm value difference between a paired template and a non-paired template is improved, so that the experimental result is more stable and accurate; the method provided by the invention has the advantages of being simple and rapid, easy to operate, visual in results, high in sensitivity, good in stability, real-time quantitative, and the like, and shortens the reaction time.

The invention provides a detection probe for an SNP (Single Nucleotide Polymorphism) of a human CYP2C19 gene. A detection primer consists of a specific upstream and downstream primer pair of the gene and a specific Taqman double fluorescent probe which are respectively used for correspondingly detecting SNPs at CYP2C19*2, CYP2C19*3 and CYP2C19*17 sites. The detection probe has the characteristics of quick detection and high specificity; furthermore, the detection method is simple; positive contrast and negative contrast which are necessary to conventional fluorescent quantitation PCR are eliminated, so that the operation steps and the experimental cost are reduced; the subsequent clinical treatment strategy can be guided more quickly and better.

The invention discloses an identification method for dalbergia odorifera chen in rosewood. According to the method, a primer, a probe and a molecular level PCR (polymerase chain reaction) detecting method are used, and the primer and the probe have exclusive specificity of the dalbergia odorifera chen. According to the method, detection sample capacity is low, proper DNA (deoxyribonucleic acid) information of wood serves as a basis, a species identification result of the dalbergia odorifera chen can be obtained quickly, accurately and objectively, and the shortcoming that the traditional identification method is high in subjectivity and is necessarily established on the basis that detected wood has a complete morphological structure which is easy to recognize is overcome. Therefore, the identification method can be used for performing enforcement detection for relevant departments such as an agriculture and forestry department, an industry and commerce department and an inspection and quarantine department, can also be used for consumer rights protection identification of a third-party detecting organization, and has an important role in standardizing markets, protecting consumer rights and interests and promoting protection and utilization of species resources.

The invention discloses a creatinase mutant with increased thermal stability, and belongs to the field of enzyme engineering. The mutant is obtained by mutating a 368th amino acid of a creatinase amino acid sequence. Creatinase roots in escherichia coli. The mutant is a mutant V368M obtained by mutating a 368th valine into methionine. An amino acid in creatinase protein rooting in the escherichia coli is subjected to site-specific mutagenesis, so that thermal stability of the creatinase is improved. Compared with the existing mutant V368M, the creatinase mutant with increased thermal stability has the advantages that half-life is prolonged for 5 times, and Tm value is increased by 3 DEG C.

The invention discloses a kit for detecting CYP3A4 and CYP3A5 polymorphic sites and a method thereof. The kit comprises specificity upstream and downstream primer sequences of CYP3A4*1G shown in sequences 1-4 in a sequence table, specificity TaqMan-MGB bi-fluorescence probe sequences of CYP3A4*1G shown in sequences 5-8, specificity upstream and downstream primer sequences of CYP3A5*3 shown in sequences 9-12 and specificity TaqMan-MGB bi-fluorescence probe sequences of CYP3A5*3 shown in sequences 13-16. The kit has the advantages that the specificity is high, pollution is prevented, the sensitivity is high, the detection speed is high, result analysis is simple, and the accuracy is high; the kit can judge the metabolism capacity of to-be-detected objects to fentanyl medicine by detecting polymorphic sites of CYP3A4 and CYP3A5 genes, the using amount of medicine such as fentanyl can be more accurately and effectively guided accordingly, and the kit has the actual clinical application value. The kit is applied to clinical diagnosis, the fit degree between the kit and the prior art is higher, the cost is low, and repeatability is high.

The invention discloses a mannose PMan5A mutant with improved heat resistance and a gene and application of the mannose PMan5A mutant. The mannose PMan5A mutant is used for mutating histidine (H), phenylalanine (F), leucine (L) and alanine (A) at sites of 93, 94, 356 and 389 in a female parent into tyrosine (Y), tyrosine (Y), histidine (H) and proline (P) by means of molecular biology, and mannosePMan5A of a GH5 family derived from Penicillium sp.WN1 is adopted as the female parent. Results show that the heat resistance of single point mutants H93Y, L356H and A389P is greatly improved when being compared with that of a wild enzyme PMan5A, in addition, the heat resistance of combined mutants H93Y / F94Y, H93Y / L356H, H93Y / A389P, L356H / A389P, H93Y / F94Y / L356H, H93Y / L356H / A389P and H93Y / F94Y / L356H / A389P shows a single point mutation overlapping effect, which means that the sites 93, 94, 356 and 389 in PMan5A play significant roles in heat resistance of mannose in the GH5 family, and the research has significant instruction meanings in studying heat resistance mechanisms of other (alpha / beta) 8 tubbiness enzymes of the GH5 families.

The invention relates to a marking method of oligonucleotide and discloses a co-modified nucleic acid segment of a locked nucleic acid (LNA) and a minor groove binding agent (MGB), namely, both the LNA and the MGB are marked on the same oligonucleotide chain. The invention enables that the capacity of identifying nucleotide sequence mutation and SNP of the oligonucleotide is enhanced by applying the combined modification of the MGB and the LNA.

The present invention provides a Candida rugosa lipase mutant, wherein Candida rugosa (ATCC NO:14830) lipase LIP1 (CRL1) is subjected to thermal stability modification by using a site-directed mutation technology so as to optimize the practicality in the high temperature industrial environment. According to the present invention, the CRL1 gene is used as the template, the protein rational design technology is used, and the amino acid Asp at 4578 site of the CRL1 lipase gene sequence is substituted with Phe so as to obtain the lipase mutant with the improved thermal stability, wherein the application range and the catalytic efficiency of the enzyme are improved; and the CRL1 is subjected to site-directed mutation by using the molecular biology method to obtain the lipase mutant, wherein theTm value of the lipase mutant is increased by 9.4 DEG C, the optimum temperature is increased by 10 DEG C, and the T1 / 2 (50 DEG C) is 6.5 times the T1 / 2 (50 DEG C) of the parent lipase.

The invention discloses an Allglo probe-based detection method of an anopheles sinensis knockdown resistance gene mutation site. A special primer provided by the invention consists of a primer 1 and a primer 2 shown in a SEQ ID No.1 and a SEQ ID No.2; a reagent consists of the special primer, a probe 1 (sequence SEQ ID No.3), a probe 2 (sequence SEQ ID No.4) and a probe 3 (sequence SEQ ID No.4); a reaction reagent consists of a reagent and an Allglo reaction buffer liquid; and a kit comprises the reaction reagent. The invention provides an efficient, rapid, simple and convenient anopheles sinensis kdr gene mutation site detecting method which is high in specificity and sensitivity, and according to the method, the anopheles sinensis kdr gene mutation type can be rapidly and accurately detected.

A hepatitis B virus gene grouping method by retroactive point hybridizing technology includes: 1) taking X zone sequence of HBV gene group as main designing grouping primmer and probe; 2) fixing active amino-marked probe on nylon membrane to obtain detecting membranous strip; 3) HBV DNA amplifying marked biotin of primmer; 4) amplified product hybridizing with detecting membranous strip, and color developing POD and TMB; 5) discriminating gene grouping end. It achieves low cost and simple operation.

The invention discloses a real-time fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detection of foot and mouth disease virus and seneca valley virus and application. The kit comprises primers and probes for detecting the foot and mouth disease virus and the seneca valley virus and preferably further comprises nucleic acid extract liquid, 2*Direct qRT-PCRMix, enzyme mixed liquid, negative control and positive control. By adoption of the kit for detecting the foot and mouth disease virus and the seneca valley virus, high specificity, high sensitivity,high stability, simplicity and convenience in operation and the like are achieved. Without extra extraction of virus RNA and reverse transcription, a user only needs to add a to-be-tested sample intoa reaction tube, then performs quantitative analysis on a start template according to fluorescence signal changes and a Ct value and standard curve relation and finally calculates a copy number of the to-be-tested sample. The kit is not only applicable to quantitative analysis in research and development institutions but also suitable for pathogen detection and analysis in all levels of prevention and control institutions, basic veterinary stations, large and medium sized farms and the like, thereby having a promising application prospect.

The invention belongs to the field of biotechnology, and in particular relates to a kit and method for detecting the A746G mutation of a sheep BMPR-IB gene by using a Taqman-MGB probe. The kit mainly comprises three tubes of positive control samples, a DNA extraction solution I, a DNA extraction solution II, a 2 * Light Cycler 480 probes master reaction buffer solution, BMF and BMR primers, BPA and BPG probes and ddH2O. The Method includes the steps of rapidly extracting genomic DNA, conducting real-time fluorescent quantitative PCR amplification on the genomic DNA by using the primers, and performing gene interpretation. The kit and method for detecting the A746G mutation of the sheep BMPR-IB gene by using the Taqman-MGB probe has the advantages of being simple to operate, fast in detection and high in accuracy, and can judge types without subsequent analysis of PCR products; besides, the whole reaction process is performed in a closed tube, so that cross-contamination is reduced.

The invention relates to a fluorescent probe primer group and a kit for an African swine fever virus P72 gene, and application thereof. The primer comprises an upstream primer ASFV-F: 5 '-CCACGGGAGGAATACCAA-3', the downstream primer ASFV-R is 5 '-GCAGATGCCGATACCACA-3'; a fluorescent probe ASFV-P, containing a fragment 5 '-TCATTAACGTATCCAGAGCAAGA-3', the 5' end of the fluorescent probe ASFV-P is marked with a fluorescent reporter group, and the 3 ' end of the fluorescent probe ASFV-P is marked with a fluorescent quenching group. The kit comprises a fluorescent probe primer group, a positive control, a negative control and a PCR amplification solution. The application specifically comprises the following steps: 1) extracting total DNA of a sample to be detected; 2) taking the total DNA obtained in the step 1) as a template, preparing a fluorescence reaction system by using the kit, and carrying out fluorescence amplification; and (3) establishing a standard curve by utilizing positive control, analyzing a fluorescence amplification product, and judging whether the sample to be detected is positive or negative according to an amplification reaction result. Compared with the prior art, the primer group has the advantages that all current epidemic ASFV strains can be identified to the maximum extent, and the sensitivity of the primer is improved to 1.15 * 10 < 1 > copise / [mu]L.

The invention discloses a fluorescent quantitative PCR method for detecting vibrio parahaemolyticus causing acute hepatic pancreatic necrosis. The method specifically comprises the following steps of(1) taking the PirAVp gene of VpAHPND as a detection target gene and designing a specific primer and a TaqMan-MGB probe; (2) extracting the DNA of the VpAHPND, constructing a recombinant plasmid, preparing a standard substance, and storing at -20 DEG C for use; (3) performing a quantitative PCR, and establishing a standard curve and a standard equation between the logarithm of the initial templatecopy number and the threshold cycle number; (4) detecting the to-be-detected sample with the primer and the probe in the step (1),and based on the measured threshold cycle number, according to the standard curve, calculating the copy number of the VpAHPND in the to-be-detected sample. The Fluorescent quantitative PCR method for detecting the VpAHPND for non-diagnostic purposes established by theinvention has the advantages of high sensitivity, strong specificity, good reproducibility, rapid quantification and the like, and can be used for clinically detecting the VpAHPND of shrimp samples and water samples.

The invention discloses a breast cancer 21 gene detection kit and a detection method thereof. The kit comprises primers and probes for detecting genes ACTB, GAPDH, RPLPO, GUS, TFRC, CD68, BAG1, GSTM1,HER2, GRB7, MMP11, CTSL2, KI67, STK15, CCNB1, MYBL2, Survivin, ESR1, PGR, BCL2 and SCUBE2. Two pairs of primers and two specific probes are respectively designed for 21 genes in the kit, a primer andprobe liquid of each gene is placed in one reaction tube, a same buffer solution and a procedure are adopted for QPCR (Quantitative Polymerase Chain Reaction) amplification, the operation is simple and convenient to carry out, the detection time can be shortened, and clear and accurate results can be achieved; the kit is wide in application, and applicable to both scientific research and clinicaldetection and applicable to single or multiple samples, and rapid and accurate detection results can be achieved.

The invention discloses a deafness susceptible gene mitochondrion 12SrDNA 1555A>G and 1494C>T mutant ratio detection kit which comprises amplification reagents and a series of standard substances, wherein the amplification reagents comprise a reaction mixture of a PCR (polymerase chain reaction) buffer solution, MgCl2 and dNTPs (deoxyribonucleotide triphosphates), Taq enzyme, ultrapure water, and a high-specificity amplified mitochondrion 2SrDNA:1494C>T and 1555A>G primer mixture; and the series of standard substances comprise a 1494C>T mutant ratio standard substance and a 1555A>G mutant ratio standard substance. By using the 2 deafness susceptible gene sites as the detection objects, the deafness susceptible gene sites are subjected to amplification and fluorescence quantitative detection and are compared with the mutant ratio standard substances to screen out individuals containing the site mutants and determine various mutant ratios. The kit has important meanings for screening deafness susceptible genes and especially newborn deafness genes.

The invention discloses a lumpy skin disease virus (LSDV) Taqman-MGB (minor groove binder) probe real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, a kit and a detection method. According to the method, 2 pieces of the primers and a probe are designed according to one region of a target sequence, the kit includes 2* PremixExTaqTM buffer, a positive control, a negative control and sterilized deionized water. The method only needs two step amplification method and simple reaction conditions for fast, efficient, specific and highly-sensitive detection of an objective target sequence, is simple in operation, does not need expensive equipment and reagents, has no technical requirement on operators, and is low in detection cost and short in testing time.

The invention provides a method and oligonucleotide for highly-sensitive detection of a mutation site of CYP2C19*2 by utilizing a real-time fluorescent PCR (Polymerase Chain Reaction) technology. The oligonucleotide comprises a pair of amplification primers SEQNO1 and SEQNO2, and detection probes SEQNO3 and SEQNO4, wherein MGB groups are modified at the 3 end of each of two detection probes so as to enhance the specificity. The method and oligonucleotide provided by the invention have the advantages of short detection period, high specificity, high accuracy, high sensitivity, low pollution risk and the like, and nearly do not depend on conditions.

A method of testing colibacillus O157 with MGB probe multiline fluorescence gauging PCR, is disclosed, the recombinant plasmid is constructed, the synthesized primer and the Taqman-MGB probe are designed, diploid fluorescence gauges PCR, the segment of saving sequences of rfbE, vt2 of the colibacillus O157 are augmented by PCR to obtain the goal segment, the goal segment is connected with PMD 18-T Vector to obtain the recombinant plasmid, then the recombinant plasmid is converted to DH5a competence bacillus coli cell, enlarged the culture to obtain the recombinant plasmid with high concentration, then the recombinant plasmid with high concentration is diluted by 10 times ratio to be the standard sample, the standard sample and the sample to be measured are combined together to gauge the PCR by diploid fluorescence, the calibration curve is formed by the standard sample, the content of the colibacillus O157 can be calculated in the sample to be measured based on the calibration curve. The MGB probe is used in the invention that has higher sensibility and specificity, and detects the content of the colibacillus O157 in the sample quickly and precisely; two specificity virogenes of the colibacillus O157 can be detected at the same time, so it is a quick and precise method of testing colibacillus O157.

The invention relates to a method for detecting poliomyelitis viruses in food in real time through fluorescent reverse transcription-polymerase chain reactions (RT-PCR). Particularly, the invention discloses a method for detecting the poliomyelitis viruses. In the method, a polymerase chain reaction is performed in a polymerase reaction system which contains a specific primer pair for amplifying the poliomyelitis viruses and a specific probe of the poliomyelitis viruses. The invention also provides a corresponding kit. By using the method and the kit, the poliomyelitis viruses can be detectedand identified sensitively, simply and conveniently.

The invention relates to a kit for detecting a genotype of a mitochondrion locus 3243. The kit comprises a PCR primer and a probe Taqman-MGB for amplifying a locus 3243 of a mitochondrion gene ATP6. Afluorescent quantitative PCR method is one of detecting means most extensively applied in the field of molecular diagnosis and is high in sensitivity and simple and convenient in operation, and the kit is applicable to large-scale clinical application. A furrow conjugate on a probe MGB can increase a Tm value of the probe, the probe MGB is designed to be shorter than an ordinary probe Taqman, andthe kit is free of background fluorescence, has the capacity for being capable of distinguishing base difference and is very applicable to SNP typing detection.

A peptide nucleic acid chip for detecting the mutation sites of hepatitis B virus is composed of glass substrate or nylon film, peptide nucleic acid probes array and reference point-type coated layer. Its preparing process includes PCR amplification of hepatitis B virus DNA speciment, fluorescence labelling, and hybridization. The mutation sites can be determined according to the intensity of hybrid signals. Its advantages are high speed, high effect, and high correctness.

The invention discloses a method for detecting mutation on the locus deltaF508 of a CFTR (Cystic Fibrosis Transmembrane Regulator) gene and an oligonucleotide, relating to a pair of specific amplification primers SEQ NO 1 and SEQ NO 2 and a pair of specific detection probes SEQ NO 3 and SEQ NO4. The method has the advantages of short detection period, high specificity, high accuracy, high sensitivity, low conditional dependency, low pollution risk and the like, and can be used for assisting in accurately and rapidly diagnosing cystic fibrosis and male infertility.

The invention relates to single nucleotide polymorphism, in particular to an rs 3909184 detection genotyping kit based on an AllGlo probe and a genotyping method thereof. The kit comprises a real-time fluorescence quantification PCR (polymerase chain reaction) reagent, positive control and negative control, and rs 3909184 is an SNP (single nucleotide polymorphism) locus serial number provided by NCBI(National Center of Biotechnology Information). The detection genotyping method includes the steps that DNA in an EDTA anticoagulant whole blood sample is extracted by adopting a conventional method; the real-time fluorescence quantification PCR reagent provided by the rs 3909184 detection genotyping kit based on the AllGlo probe is used for conducting real-time fluorescence quantification PCR amplification on DNA; the rs 3909184 SNP locus is genotyped according to detected fluorescence signals. On the premise of keeping high specificity and sensibility of the AllGlo probe, the detection price is lower than that of a direct sequencing method, the process is simpler, and consumed time is shorter.

The invention belongs to the technical field of molecular diagnosis, and particularly relates to a method and detection kit for detecting ERBB2 gene amplification based on a digital PCR technology. The kit comprises primer and probe premix liquid, wherein the premix liquid comprises upstream and downstream primers for detecting an ERBB2 gene, a fluorescent probe for detecting the ERBB2 gene, upstream and downstream primers for detecting a human ATCB reference gene, and a fluorescent probe for detecting the human ATCB reference gene. The method comprises the following steps of S1, providing a detection sample of a testee, wherein the detection sample is a plasma sample; S2, extracting ctDNA of a to-be-detected sample from the plasma sample in the step S1; S3, performing an amplification reaction on the sample extracted in the step S2 by using a digital PCR platform; and S4, analyzing and interpreting an amplification result. The ERBB2 gene amplification detection method provided by theinvention is based on a micro-droplet digital PCR detection system, reduces the interference of manual operation, has the advantages of stable result, high accuracy and good data amplification effect,and can be more conveniently used for auxiliary diagnosis and clinical treatment guidance.

The invention relates to a primer, probes, a PCR reaction liquid and a kit for human EGFR (epidermal growth factor receptor) gene T790M site detection and belongs to the field of molecular biology. The primer has the nucleotide sequences shown as SEQ ID NO:1 and SEQ ID NO:2. The probes comprise a mutant probe and a wild probe for a human EGFR gene T790M site, the mutant probe has the nucleotide sequence shown as SEQ ID NO:3, and the wild probe has the nucleotide sequence shown as SEQ ID NO:4; different fluorescent report genes are marked at 5' terminals of the mutant probe and the wild probe,a non-fluorescent quencher is marked at 3' terminals, and the 3' terminals are connected with MGB modification genes; at least one basic group of the mutant probe and the wild probe is modified with locked nucleic acid. The amplification specificity and the sensitivity of the primer are higher. The probe is an LNA modified TaqMan MGB (minor groove binder) probe, and an MGB can increase the Tm value of the probe by about 10 DEG C; by means of an LNA modified probe, the stability and the affinity of DNA molecules in a PCR (polymerase chain reaction) are improved, and the specificity and the sensitivity are increased.

The invention discloses a method and oligonucleotide for detecting FGFR3 gene G380R site mutation, and relates to a pair of specific amplification primers SEQ NO 1 and SEQ NO 2, and a pair of specific detection probes SEQ NO 3 and SEQ NO 4. By adopting the method and oligonucleotide, congenital achondroplasia can be rapidly diagnosed and identified, and the method and oligonucleotide have the advantages of being short in detection cycle, good in specificity, high in accuracy, high in sensitivity, less in condition dependence, low in pollution risk and the like.