209results about How to "Increased fluorescence signal value" patented technology

The invention discloses a specific primer, a liquid-phase chip and a method for SNP detection of CYP2C9 and VKORC1 genes. The liquid-phase chip comprises wild-type and mutable-type ASPE primer pairs and microspheres coated by a specific anti-tag sequence respectively, which are designed respectively aiming at each type of mutable loci, primers used for amplifying a CYP2C9 gene target sequence having CYP2C9*2, CYP2C9*3, CYP2C9*5 and CYP2C9*6SNP loci, and / or primers used for amplifying a VKORC1 gene target sequence having G1639A, G1173T and G3730A SNP loci. The liquid phase chip of the invention has a quite good signal-noise ratio, and the cross reaction does not happen between a designed probe and the anti-tag sequence basically; the ASPE primer designed by the invention has quite good specificity, and can accurately differentiate various types of mutable loci; and the detection method has the advantages that: a few steps are adopted, 7 types of SNP loci can be detected in one step, the operation is convenient, a lot of uncertain factors existing in a process of repeated operations can be avoided, and the detection accuracy is greatly improved.

The invention discloses a liquid phase chip, a specific prime and a method for the SNP detection of TPMT gene, the liquid phase chip comprises wild-type and mutant-type specific ASPE primer pairs designed respectively in accordance with mutant sites of each type, microspheres respectively enveloped with specific anti-tag sequences, and primers for amplifying target sequences having SNP sites of TMPT gene G238C, G460A and / or A719G. The prepared liquid phase chip for the SNP detection of TPMT gene has quite excellent signal-to-noise ratio and, basically, no cross reaction is present between thedesigned probe and the anti-tag sequence. The ASPE primers designed according to the invention has quite excellent specificity and can accurate distinguish the mutant sites of each type. The detectionmethod has simple steps and convenient operation, can complete the detection of 3 types of mutant sites in one step, and can avoid plenty of uncertain factors existing in the process of repeated operation, therefore, the accuracy of the detection can be enhanced enormously.

The invention discloses a PCR (Polymerase Chain Reaction) primer, a kit and a liquid chip for detecting an RET fusion gene. The liquid chip comprises a PCR amplification primer, an ASPE (Allele Specific Primer Extension) primer which consists of tag sequences and specific primers, and microspheres, wherein the specific primers comprise the following sequences: SEQ ID NO.23 for K15;R12, SEQ ID NO.24 for K16;R12, SEQ ID NO.25 for K22;R12, SEQ ID NO.26 for K23;R12, SEQ ID NO.27 for K24;R8, SEQ ID NO.28 for K24;R11 and / or SEQ ID NO.29 for C1;R12. The liquid chip disclosed by the invention has a quite good signal to noise ratio and can be used for implementing amplification of seven fusion subtypes through one step, and the specific primers have quite good specificity.

The invention discloses an SNP (Single Nucleotide Polymorphism) detection specific primer, a liquid-phase chip and a detection method of an RYR1 (Ryanodine Receptors 1) gene. The liquid-phase chip comprises wild type and mutant type ASPE (Allele Specific Primer Extension) primer pairs respectively designed aiming at the mutational sites of each type, microballoons respectively coated with a specific anti-tag sequence and primers used for respectively amplifying target RYR1 gene sequences with G341R, R614C, T2206M and / or G2434R sites. The SNP detection liquid-phase chip of the RYR1 gene has very good signal-to-noise ratio, and a designed probe and the anti-tag sequences have no cross reactions basically. The designed ASPE primers have very good specificity and can accurately distinguish various types of mutational sites. The detection method has simple steps, and four SNP sites can finish being detected in one step, thus the operation is convenient; and moreover, various uncertain factors existing in the processes of multiple operations are avoided, thus the detection precision is greatly improved.

The invention discloses a PDGFRA gene mutation detection liquid-phase chip, which comprises tag sequence at 5' terminal and ASPE (Allele Specific Primer Extension) primers specific to mutational sites at 3' terminal. The ASPE primers consist of sequences shown in SEQ ID No.8-SEQ ID No.9 specific to V561D sites, sequences shown in SEQ ID No.10-SEQ ID No.12 specific to DIMH 842-845 and / or IMHD 843-846 deletion mutational sites, and / or sequences shown in SEQ ID No.13-SEQ ID No.14 specific to D842V site, the tag sequence which is selected from sequences shown in SEQ ID No.1- SEQ ID No.7, color coding microballoons which are respectively enveloped with a specific anti-tag sequence and amplification primers. The coincidence ratio with a sequencing method of the PDGFRA gene mutation detection liquid-phase chip reaches up to 100 percent. The PDGFRA gene mutation detection liquid-phase chip can simultaneously detect aiming at a plurality of deletion mutational sites with excellent signal to noise ratio.

The invention discloses a method for fast and accurate quantitative determination of circulating DNA in blood. The method comprises the following specific steps that processing of plasma or serum is performed: peripheral venous blood is taken and arranged in an EDTA anticoagulant tube for standing at room temperature, or self-clotting whole blood is taken to undergo centrifugation under 1800-2200 g of centrifugal force for 4 to 6 minutes, upper-layer plasma is taken, the same volume of diluted reagent is added, even mixing is performed, stirring is performed at the temperature of 92-97 DEG C for 6 to 10 minutes, then centrifugation is performed again under 15000-17000 g of centrifugal force for 8 to 12 minutes, and supernate is taken as a template for PCR; a quantitative PCR reaction system is configured; quantitative PCR reaction is performed. The quantitative PCR reaction can be performed by directly using the processed plasma or serum, PCR reaction is not influenced, the amplification efficiency is consistent with the amplification efficiency of purified DNA, DNA extraction steps and experimental errors are decreased, a new fluorescent dye SuperGreen which is higher in concentration, high in fluorescent signal value and response sensitivity and capable of accurately detecting plasma free DNA as low as the nanogram level can be adopted, the reagent price is low, the using cost of a DNA extraction kit is reduced, and operation is easy and convenient.

The invention discloses a method for quantitatively determining blood circulation DNA (Deoxyribonucleic Acid). The method comprises the following steps of: obtaining 1ml of peripheral venous blood of the same tumor patient and placing the peripheral venous blood into an EDTA (Ethylene Diamine Tetraacetic Acid) anti-freezing pipe; standing for 4 hours at 10-20 DEG C; centrifuging 2000g of peripheral venous blood for 5 minutes; taking 100-200 microliters of upper-layer blood plasma; adding a buffering solution with the same volume and sufficiently and uniformly mixing; standing at 95 DEG C for 5-10 minutes; centrifuging 16000g of peripheral venous blood for 10 minutes; and taking a liquid supernatant as a template to be directly used for quantitative PCR (Polymerase Chain Reaction) amplification. The method disclosed by the invention has the beneficial effects that 1. blood plasma or blood serum can be directly used for a quantitative PCR reaction through special technical process treatment and the PCR reaction is not influenced; ratios of amplification efficiencies to the purified DNA are consistent, and extraction steps and experimental errors of the DNA are reduced; and 2. an adopted novel fluorescent dye Super Green has slight inhibition effect in a PCR amplification process and high concentration can be used; and a fluorescence signal value is high and the reaction sensitivity is high, and blood plasma free DNA which is at a nanogram level can be accurately detected.

The invention discloses an HRAS gene mutation detection specificity primer and a liquid chip thereof. The liquid chip mainly comprises: an ASPE primer composed of a 5'-terminal tag sequence and 3'-terminal specificity primer sequences focused on target gene mutation sites, wherein the specificity primer sequences comprise SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 and / or SEQ ID NO.21 focused on a Codon12 site, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and / or SEQ ID NO.25 focused on a Codon13 site, and / or SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 and / or SEQ ID NO.30 focused on a Codon61 site; a microsphere coated by an anti-tag sequence; and an amplimer. The consistency between the detection result of the detection liquid chip provided by the invention and the detection result of a sequencing method is high to 100%, and the wild-type and mutant parallel detection of a plurality of mutation sites is realized.

The invention discloses a TERT gene mutation detection liquid chip and specific primers. The liquid chip mainly comprises the following parts: each ASPE primer contains a tag sequence at the 5' terminal and a specific primer sequence aiming at target gene mutation locus at the 3' terminal, and the specific primer sequences are as below: a SEQ ID NO.15 and a SEQ ID NO.16 aiming at a T174G locus, a SEQ ID NO.17 and a SEQ ID NO.18 aiming at a C98T locus, a SEQ ID NO.19 and a SEQ ID NO.20 aiming at a G162A locus, a SEQ ID NO.21 and a SEQ ID NO.22 aiming at a G168A locus, a SEQ ID NO.23 and a SEQ ID NO.24 aiming at a C209T locus, a SEQ ID NO.25 and a SEQ ID NO.26 aiming at a G151T locus, and / or a SEQ ID NO.27 and a SEQ ID NO.28 aiming at an A129T locus; microballoons coated with anti-tag sequences; and amplification primers. The detection results from the detection liquid chip provided by the invention are 100% identical with those from a sequencing method, so as to realize parallel detection on wild types and mutants of a plurality of mutation loci.

The invention discloses a specific primer and liquid chip for detecting polymorphism of a DPYD (dihydropyrimidine dehydrogenase) gene. The liquid chip mainly comprises ASPE (allele specific primer extension) primers, microspheres and amplification primers, wherein each ASPE primer is formed by tag sequences at the 5' terminal and specific primer sequences which are arranged at the 3' terminal and aim at the mutation site of the target gene; the specific primer sequences are SEQ ID NO.7 and SEQ ID NO.8 aiming at the G55A site, SEQ ID NO.9 and SEQ ID NO.10 aiming at the G141A site and / or SEQ ID NO.11 and SEQ ID NO.12 aiming at the G134T site; and the microspheres are enveloped by anti-tag sequences. The detecting liquid chip provided by the invention has the advantages that the consistency of the detection result and the sequencing method is as high as 100%, thus parallel detection of wild types and mutant types of a plurality of mutation sites is achieved.

The invention discloses an AGT1R (Angiotensin Type 1 Receptor) gene SNP (Single Nucleotide Polymorphism) detection specific primer and a liquid phase chip. The liquid phase chip mainly comprises an ASPE (Allele Specific Primer Extension) primer, a microsphere and an amplification primer, wherein the ASPE primer consists of tag sequences at an ends 5' and specific primer sequences specific to SNP sites at ends 3', the specific primer sequences are respectively selected from SEQ ID NO.5, SEQ ID NO.6, and / or SEQ ID NO.7 and SEQ ID NO.8, and the tag sequences are selected from SEQ ID NO.1-SEQ ID NO.4. The prepared AGT1R gene SNP detection liquid phase chip has excellent signal-noise ratio, and cross reaction does not exist between a designed probe and an anti-tag sequence basically. The ASPE primer designed by the invention has excellent specificity, and can accurately differentiate various kinds of mutant sites.

The invention discloses an FGFR1 gene mutation detection specific primer and a liquid chip. The liquid chip mainly comprises ASPE primers consisting of tag sequences at a 5' end and specific primer sequences aiming at target gene mutation sites at a 3' end, microspheres coated with anti-tag sequences and an amplification primer, wherein the specific primer sequences are as follows: SEQ ID NO.5 and SEQ ID NO.6 which aim at a C374T site, and / or SEQ ID NO.7 and SEQ ID NO.8 which aim at a C754A site. The detection liquid chip provided by the invention has the advantages that the matching rate between the detection result and a sequencing method is up to 100%, and the wild-type and mutant parallel detection of multiple mutant sites can be realized.

The invention discloses an RB1 gene mutation detection specific primer and a liquid chip. The liquid chip mainly comprises ASPE primers consisting of tag sequences at a 5' end and specific primer sequences aiming at target gene mutation sites at a 3' end, microspheres coated with anti-tag sequences and an amplification primer, wherein the specific primer sequences are as follows: SEQ ID NO.7 and SEQ ID NO.8 which aim at a C1654T site, SEQ ID NO.9 and SEQ ID NO.10 which aim at a C1666T site, and / or SEQ ID NO.11 and SEQ ID NO.12 which aim at a C1981T site. The detection liquid chip provided by the invention has the advantages that the matching rate between the detection result and a sequencing method is up to 100%, and the wild-type and mutant parallel detection of multiple mutant sites can be realized.

The invention discloses a specific primer and a liquid phase chip to test apo E gene and single nucleotide polymorphism (SNP) of the 9p21 segment of chromosome. The liquid phase chip mainly comprises a pair of allele specific primer extension (ASPE) primers. The specific primer has the SEQ ID No.17 and SEQ ID No.18 corresponding to the C609T SNP site, extension primers and / or SEQ ID No. 19 and SEQ ID No.20 corresponding to the T471C SNP site. The tag sequence is from SEQ ID No.1 to SEQ ID No.16. Micro particles which contain specific anti-tag sequence and have different color codes are respectively contained, and the anti-tap sequence is from SEQ ID No.33 to SEQ ID No.48. The invention also discloses a liquid phase chip to test the apo E gene and the SNP of the 9p21 segment of chromosome. The test result of the liquid phase chip fully matches that of a sequencing method. The prepared liquid phase chip to test the apo-E gene and the SNP of the 9p21 segment of chromosome has high signal to noise ratio.

The invention discloses a liquid phase chip for detecting mutation of GSTP1 (glutathione S transferase pl) genes, mainly comprising an ASPE primer consisting of a tag sequence at 5' end and a specific primer at 3' end and aiming at a mutant site, micro-balloons and an amplification primer; wherein the specific primer is SEQ ID NO.11 and SEQ ID NO.12 aiming at the A313G mutant site, and SEQ ID NO.13 and SEQ ID NO.14 aiming at the C341T mutant site; the tag sequence is selected from the group of SEQ ID NO.1 to SEQ ID NO.6; and the micro-balloons different color codes are respectively covered with the special anti-tag sequences. The liquid phase chip for detecting mutation of GSTM1(glutathione S transferase mu), GSTT1 (glutathione S transferase theta) and GSTP1 genes in the invention has a great signal-noise ratio; no cross reaction exists between the designed probes and the anti-tag sequences basically; the tag-tag sequence, the selection of the anti-tag tag sequence and the combinationof the tag-tag sequence with the specific ASPE primer can avoid the cross reaction, and realize parallel detection of a plurality of mutant sites.

The invention discloses PCR (Polymerase Chain Reaction) primers, a kit and a liquid-phase chip for detecting an ROS1 fused gene. The liquid-phase chip comprises PCR amplification primers, an ASPE primer and a micro-ball, wherein the ASPE primer is composed of a tag sequence and specific primers; the sequences of the specific primers are as follows: a probe pair C6; SEQ ID NO.34 of R32 and the probe pair C6; SEQ ID NO.35 of R34 and a probe pair C5; SEQ ID NO.36 of the R34 and a probe pair S2; SEQ ID NO.37 of the R32 and a probe pair S4; SEQ ID NO.38 of the R32 and a probe pair S4; SEQ ID NO.39 of the R34 and a probe pair SL13; SEQ ID NO.40 of the R32 and a probe pair SL4; SEQ ID NO.41 of the R34 and a probe pair E10; SEQ ID NO.42 of the R34 and a probe pair L16; SEQ ID NO.43 of R35 and / or a probe pair T5; SEQ ID NO.44 of the R35. The liquid-phase chip has a very good signal-noise ratio and can finish the amplification of 11 fused subtypes in one step; the specific primer has very good specificity.

The invention discloses specific primers and a liquid-phase chip for the single nucleotide polymorphism (SNP) detection of the kit ligand gene (KITLG). The liquid-phase chip mainly comprises allele specific primers (ASPE), each of which consists of a tag sequence at a 5' end and specific primers aiming at mutational sites of target genes at a 3' end, microspheres wrapped by an anti-tag sequence and amplification primers, wherein sequences of the specific primers are one or more from the following pairs of sequences: SEQ ID No. 19 and SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24, SEQ ID No. 25 and SEQ ID No. 26, SEQ ID No. 27 and SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30, SEQ ID No. 31 and SEQ ID No. 32, SEQ ID No. 33 and SEQ ID No. 34 and SEQ ID No.35 and SEQ ID No. 36. In the detection liquid-phase chip, the coincidence rate of detection results and a sequencing method is up to 100 percent, so the parallel detection of the wild type and mutanttype of polymorphic loci is realized.