36 results about "Single-base extension" patented technology

The invention discloses a method for rapidly detecting common candida of urinary tract infection based on a nucleic acid mass spectrometry technology. The method comprises the following steps: extracting total nucleic acid from a urine sample of a patient; amplifying the extracted total nucleic acid by adopting a PCR (Polymerase Chain Reaction) technology to obtain an amplification product containing a target sequence; analyzing a characteristic peak map of the amplification product containing the target sequence by using a single-base extension primer and a matrix-assisted laser desorption / ionization time flight mass spectrometry technology; and determining whether specific candida exists in the sample according to the characteristic peak map. According to the invention, a set of detection system suitable for common candida is obtained through sequence screening and optimization of the amplification primers and the single-base extension primers, five common candida of UTI are detected based on the system, and a nucleic acid mass spectrometry technology is combined, so that the result can be clearly distinguished, a complex traditional detection means is avoided, and the detection efficiency is improved.

This invention discloses primer compositions, detection products, and applications for detecting 17 pathogens, relating to the field of biotechnology. A primer composition for detecting 17 pathogens includes 17 pairs of amplification primers and 17 extension probes, as shown in SEQ ID NO: 1-51, respectively. This invention also provides detection products comprising the above primer compositions and their application in simultaneously detecting 17 pathogens for non-disease diagnostic purposes. The beneficial effects of this invention are that it designs 17 primer sets to simultaneously amplify DNA fragments containing specific target sequences of 17 pathogens in multiplex PCR, and performs single-base extension of these products using highly efficient single-base primers. Simultaneous detection and identification of 17 pathogens can be achieved using MALDI-TOF MS mass spectrometry, with accurate and highly specific detection results.

The invention discloses a joint detection kit for genetic risk genes and medication genes of Alzheimer's disease and a multivariable risk assessment model. The kit comprises a detection reagent for detecting 25 mutation sites of 15 genes, wherein the 25 mutation sites of the 15 genes comprise 17 mutation sites of 10 risk genes and 10 mutation sites of 6 medication genes. Amplification primer pairs and single-base extension primers of each site are designed for 25 mutation sites of 15 genes, multiple PCR amplification and single-base extension reactions are carried out, and the genotype of each site of a product is analyzed by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. A multivariable risk assessment model for the Alzheimer's disease is constructed by taking a genetic risk score GRS, age, gender and plasma p-tau217 concentration of a risk gene mutation site as markers, one-stop detection of'early screening and medication guidance 'can be realized in combination with a medication gene detection result, and the application prospect and demand are broad.

This invention discloses a method and application for detecting TA repetitive sequence polymorphisms in the UGT1A1 gene based on nucleic acid mass spectrometry, belonging to the field of biotechnology. The method uses primer sets with sequences as shown in SEQ ID NO. 1-24, 26-36, and 38, where SEQ ID NO. 1-24 are amplification primer sequences for each mutation site, and the remaining primers are single-base extension primers. This invention enables multiplex detection of irinotecan-related gene polymorphic sites based on a nucleic acid mass spectrometry platform, achieving the detection of 12 polymorphic sites in one well, with low detection cost and small sample volume. Furthermore, nucleic acid mass spectrometry technology has advantages such as ease of operation, high sensitivity, high specificity, and high accuracy, while also overcoming the shortcomings of insufficient throughput in qPCR and high cost in sequencing technology.

The invention discloses a method for high-throughput detection of target DNA in adeno-associated viruses. The method provided by the invention comprises the following steps: A1) digesting the shell of a recombinant adeno-associated virus to be detected by using protease to obtain a treated recombinant adeno-associated virus sample; the method comprises the following steps: designing and synthesizing single-base extension primers according to target DNA to be detected, and forming a single-base extension primer group by aiming at the single-base extension primers of all the target DNA to be detected; a2) extending the treated recombinant adeno-associated virus sample under the action of the single-base extension primer group to obtain a single-base extension product; and A3) MALDI-TOF nucleic acid mass spectrometry detection: determining whether the recombinant adeno-associated virus contains target DNA to be detected according to the size of a single base extension product.According to the method for high-throughput identification of the adeno-associated virus insertion gene, the process is extremely simplified, the detection efficiency is greatly improved, the cost is remarkably reduced, the high-throughput capacity is outstanding, and the industrialization requirement is met.

The invention discloses a primer probe composition, a kit and a method for detecting various cattle and sheep fever and respiratory tract pathogens based on nucleic acid mass spectrometry, and relates to the technical field of microbiological detection, a multiple PCR primer composition and a single-base extension probe group are innovatively designed, conservative stable regions in various pathogen genomes can be targeted, and the detection sensitivity is high. Single-tube multi-target synchronous amplification is realized, and further extension amplification is carried out, so that an extension product suitable for MALDI-TOF MS detection is obtained. The method realizes multi-target, high-sensitivity and automatic synchronous detection of fever pathogens of cattle and sheep, provides a basis for rapid detection of nucleic acids of multiple pathogens, and has a good application prospect in quarantine and prevention and control.

This invention provides a method for detecting the TLR10rs11096957 genotype and its application in assessing transplanted liver function. The method includes the following steps: extracting genomic DNA from whole blood of liver transplant patients, adjusting the concentration, and performing PCR amplification; treating the amplified PCR product with SAP and then performing a single-base extension reaction; purifying the single-base extension reaction product, detecting it using time-of-flight mass spectrometry, and performing genotyping. This invention discovers that TLR10rs11096957 can be used to predict the function of transplanted livers in liver transplant patients, thereby guiding clinical treatment to improve transplant survival rates. Therefore, the method for detecting the TLR10rs11096957 genotype provided by this invention can improve the sensitivity of predicting transplanted liver function in liver transplant patients, providing a strategy for preparing corresponding predictive or assessment products, and has significant social and economic benefits.

This invention discloses a semen-specific mRNA-cSNP primer composition, kit, and method based on SNaPshot technology, belonging to the field of molecular identification technology. The primer composition provided by this invention includes amplification primers with nucleotide sequences as shown in SEQ ID NO. 1-34 and single-base extension primers as shown in SEQ ID NO. 35-52. This invention further provides a method for identifying human semen samples, including extracting sample mRNA, reverse transcription to obtain cDNA, performing an amplification reaction using amplification primers, and then performing a single-base extension reaction using single-base extension primers. Experimental results show that the identification method of this invention has advantages such as high specificity, high sensitivity, and good reproducibility, and can accurately identify individual semen donors, which is of great significance for achieving accurate semen-related criminal investigations and forensic identification.

The present invention relates to the field of pathogenic microorganism detection, and in particular to a MALDI-TOF mass spectrometry detection method for bovine nodular skin disease virus, goat pox virus and sheep pox virus, and a primer combination used in the method. The present invention combines multiplex PCR amplification technology, a dual-gene target strategy, a single base extension reaction, and the high-precision analysis capability of MALDI-TOF MS to provide an efficient method for simultaneously performing multi-target accurate detection of three sheep pox viruses. The present invention adopts 2 pairs of specific amplification primers to target and amplify target gene fragments, and is supplemented by 2 extension primers for subsequent single base extension reactions, and then accurately measures the molecular weight difference generated by a single base in the extension product on MALDI-TOF MS. It can be achieved in a single-tube reaction system that not only efficiently identifies whether there are three sheep pox viruses in the sample, but also can further subdivide and clearly report whether the specific infection is one, several or all of the three viruses, thereby greatly improving the comprehensiveness, specificity and accuracy of the detection.

The invention discloses a primer group for detecting hereditary hearing loss gene locus polymorphism, a kit and a detection method thereof. The primer group comprises a multiple PCR amplification primer and a UEP primer used for a single base extension reaction, multiple PCR amplification is firstly carried out, SAP purification is then carried out, the extension reaction is then carried out, nucleic acid mass spectrometry detection is carried out on an extension product, and related genotype information is obtained. The method has the advantages of high accuracy, high flux, low cost and short period, 96 samples or 384 samples can be detected in one batch, only 8-10 hours are needed, the time is short, and the price is low. 27 hereditary deafness gene variation sites can be detected, 9 genes are involved, the coverage range is wider, and the misdiagnosis risk caused by incomplete detection sites is reduced. The method has high accuracy, sensitivity and repeatability, and the detection result has important guidance and research value for auxiliary diagnosis and treatment of hereditary hearing loss.

Provided are a method and a means for improving a single base extension reaction method using capillary electrophoresis. Specifically, the present invention relates to a method for detecting presence of a target nucleic acid in a sample and / or determining a base of the target nucleic acid, the method including: preparing a sample containing or suspected of containing a target nucleic acid; preparing a primer 200 containing a double-strand nucleic acid tag 204 with an interstrand crosslink 203 and a primer nucleic acid 205 which specifically binds to the target nucleic acid; performing a single base extension reaction with the primer using the target nucleic acid as a template; and subjecting the resulting reactant to capillary electrophoresis for analysis.

The invention discloses a composition for detecting spinal muscular atrophy typing based on nucleic acid mass spectrometry and application of the composition. The invention relates to the field of gene detection, and provides a composition for detecting spinal muscular atrophy typing based on nucleic acid mass spectrometry, which comprises a target gene primer pair, a reference gene primer pair and an IIS type restriction enzyme. The composition can be used for detecting spinal muscular atrophy typing based on nucleic acid mass spectrometry, and the traditional technical method of nucleic acid mass spectrometry is improved, so that nucleic acid mass spectrometry detection does not need the steps of SAP treatment and single base extension reaction, the reaction time is shortened, the result is accurate and reliable, the steps are simple, and the cost is greatly reduced. The invention is of great significance to preparation of screening products suitable for large-scale screening of spinal muscular atrophy.

The invention discloses a pseudomonas aeruginosa drug resistance gene detection primer group based on multiple PCR-time-of-flight mass spectrometry, a kit and a use method of the kit. The pseudomonas aeruginosa drug-resistant gene comprises armA, rmtB, rmtC, mexA, blaVIM, blaNDM, blaIMP, blaKPC, blaOXA and blaGES, and the pseudomonas aeruginosa drug-resistant gene is a drug-resistant gene of pseudomonas aeruginosa. The detection primer group comprises upstream and downstream amplification primers of each gene and a single-base extension primer; the nucleotide sequences of upstream and downstream amplification primers of each gene are respectively as shown in SEQ ID NO.1-NO.20; nucleotide sequences of single base extension primers of all the genes are shown as SEQ ID NO. 21 to NO. 30 respectively. By adopting the kit comprising the primer group, whether pseudomonas aeruginosa infection exists or not can be known within 40 minutes, and if the pseudomonas aeruginosa infection exists, the drug resistance condition of the pseudomonas aeruginosa can be known. The detection method has high sensitivity and specificity in the detection process, the sample detection accuracy rate can reach 92-97%, and the method can be used for rapid, accurate, high-sensitivity and high-throughput detection of the pseudomonas aeruginosa antibiotic-resistant strains and is beneficial to application and popularization in laboratories of hospitals and grass-roots departments.

The present disclosure provides a kit and its application, the kit comprising two sets of amplification primer groups, single base extension primers and processing reagents; the amplification primer groups are used for amplifying Mycobacterium tuberculosis flora identification sites and anti-tuberculosis drug pharmacogenetic polymorphism sites; each pair of primers corresponds to the upstream and downstream regions of a SNP site; the processing reagents include PCR reaction mixture, PCR enzyme mixture, SAP reaction mixture, SAP enzyme mixture, extension reaction mixture and extension enzyme mixture. By designing specific amplification primer groups and single base extension primers, the system and reagents are optimized, and the sensitivity is high. The kit combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry can complete the detection of all sites, and the detection cost is low and the efficiency is high.

The present invention relates to a method, a reagent, and a kit each for detecting a gene by a single nucleotide extension reaction. Specifically, the present invention relates to a method, a reagent, and a kit for detecting a gene while reducing noise coming from a template nucleic acid, wherein the template nucleic acid is treated with an enzyme having a terminal transfer activity to form a 3'-terminal-protruding terminal structure in the template nucleic acid before a single nucleotide extension reaction is performed.

The invention discloses a seminal fluid specificity mRNA-cSNP primer composition based on an SNaPshot technology, a kit and a method, and belongs to the technical field of molecular identification. The primer composition provided by the invention comprises an amplification primer of which the nucleotide sequence is as shown in SEQ ID NO. 1 to 34 and a single-base extension primer of which the nucleotide sequence is as shown in SEQ ID NO. 35 to 52. The invention further provides a method for identifying the human semen sample. The method comprises the following steps: extracting mRNA of the sample, carrying out reverse transcription, obtaining cDNA, carrying out amplification reaction by using the amplification primer, and carrying out single-base extension reaction by using the single-base extension primer. Experimental results show that the identification method disclosed by the invention has the advantages of strong specificity, high sensitivity, good repeatability and the like, can be used for accurately identifying individual semen donors, and has important significance for realizing accurate semen-related criminal investigation and judicial expertise.

The invention discloses a nucleic acid composition for detecting an isoniazide drug gene based on a nucleic acid mass spectrometry technology, a kit and application thereof, and relates to the technical field of gene detection. The nucleic acid composition disclosed by the invention comprises a multiple amplification nucleotide composition and an extension nucleotide composition, wherein the multiple amplification nucleotide composition is used for carrying out multiple PCR (Polymerase Chain Reaction) amplification on sites of rs1801280, rs1799930, rs1799931, rs1799929, rs1041983 and rs1801279 of an isoniazide drug gene NAT2, and the extension nucleotide composition is used for carrying out a single base extension reaction. By using the nucleotide composition and the kit provided by the invention, the genotype of the 6-site of the isoniazide drug gene NAT2 can be detected by adopting a nucleic acid mass spectrometry technology, and the nucleotide composition and the kit have the characteristics of high sensitivity, good specificity, short detection period and low detection cost. The invention provides a more reliable and low-cost detection kit for detecting related genes of isoniazide medication, and a detection result provides a basis for accurate medication of isoniazide.

This application relates to the fields of biomedicine and molecular diagnostics, specifically disclosing a method and kit for simultaneously detecting four genes. The method includes the following steps: extracting genomic DNA from the sample to be tested; using the extracted genomic DNA as a template, performing multiplex PCR amplification using a specific amplification primer set to obtain amplification products; digesting the amplification products, using the digested products as template, performing a single-base extension reaction using a specific single-base extension primer set to obtain extension products; desalting and purifying the extension products, and then detecting them using a nucleic acid mass spectrometer; analyzing and determining the genotyping results of the four genes based on the nucleic acid mass spectrometry detection results. This application achieves rapid detection of four genes at low cost and with simple operation.

The invention discloses a primer composition for detecting 17 pathogens, a detection product and application, and relates to the technical field of biology. The primer composition for detecting the 17 pathogens comprises 17 pairs of amplification primers and 17 extension probes, and the amplification primers and the extension probes are respectively as shown in SEQ ID NO: 1-51; the invention further provides a detection product comprising the primer composition and application of the detection product to simultaneous detection of 17 pathogens with the purpose of non-disease diagnosis. The kit has the beneficial effects that 17 primer groups are designed, DNA fragment products containing specific target sequences of 17 pathogens are amplified simultaneously in multiple PCR, the products are subjected to single-base extension through efficient single-base primers, the 17 pathogens can be detected and identified simultaneously through MALDI-TOF MS mass spectrometry, the detection result is accurate, and the specificity is good.

The application provides a method for detecting 19 Neisseria gonorrhoeae drug resistance site mutations. The method uses multiplex PCR-mass spectrometry to detect 19 Neisseria gonorrhoeae drug resistance site mutations. The detection method comprises the following steps: 1) designing primers for different drug resistance sites; 2) multiplex PCR amplification reaction; 3) shrimp alkaline phosphatase treatment; 4) single base extension reaction; 5) resin desalting purification; and 6) mass spectrometry detection.

The application discloses a terminal transferase mutant, a recombinant protein, a nucleic acid molecule and a method for single-base extension of oligonucleotide, and relates to the technical field of oligonucleotide synthesis. The terminal transferase mutant has at least 80% identity with the amino acid sequence shown in SEQ ID NO:1, and has amino acid mutations in at least one of the following positions: R323, W427 and R438. Compared with a wild-type terminal transferase, the catalytic domain amino acid of the terminal transferase mutant is changed, and the catalytic capacity for a 3'-O-closed group triphosphate nucleoside is significantly improved, so that the 3'-O-closed group triphosphate nucleoside is efficiently coupled to a single-stranded oligonucleotide chain. On this basis, the application also develops a method for single-base extension of oligonucleotide. The method has technical advantages such as no need to control humidity, less use of chemical reagents, higher product purity, less chemical synthesis byproducts and the like.

The present disclosure provides methods, devices and systems for detecting a presence of a nucleic acid molecule having a nucleic acid sequence. Detection of cyclic single base extension can be used to detect a nucleic acid molecule hybridized to a probe and detect a presence of a nucleic acid. The methods disclosed herein can detect a nucleic acid molecule present in a nucleic acid sample at low concentrations and in the presence of background nucleic acids having high sequence similarity.

The invention belongs to the technical field of gene detection, discloses a kit for detecting thrombophiliosis related gene loci through an MALDI-TOF mass spectrum platform, and provides a group of multiple PCR amplification primers and single base extension primers for detecting the thrombophiliosis related gene loci through the MALDI-TOF mass spectrum platform and application of the multiple PCR amplification primers and the single base extension primers in the kit. The detected object comprises the genotypes of seven gene loci related to thrombophilia. The established detection kit has both flexibility and expandability, is simple to operate, high in throughput, automatic, high in throughput and low in cost, can effectively screen the problems of thrombophiliosis susceptibility and the like caused by genetic polymorphism in advance, and can help doctors to adjust a medication scheme so as to obtain an efficient, safe and economic optimal treatment effect.

The invention provides a primer composition for detecting hereditary thrombophilia related gene mutation and application of the primer composition, and relates to the technical field of gene detection. The primer composition comprises a PCR (Polymerase Chain Reaction) amplification primer and an extension primer, wherein the PCR amplification primer is used for carrying out specific amplification on 24 single nucleotide polymorphic sites of 22 genes, and the extension primer is used for carrying out single base extension on the 24 single nucleotide polymorphic sites. According to the primer composition, by selecting twenty-four specific loci of twenty-two genes covering key systems such as coagulation, anticoagulation and the like, a detection system which is adaptive to genetic characteristics of specific people and takes pathopoiesis and risk factors into account is constructed, so that the limitation of race heterogeneity of conventional indexes is effectively overcome; and major mutation and a multi-gene minor accumulation effect can be captured at the same time, so that individual genetic susceptibility is comprehensively analyzed, and a systematic and accurate basis is provided for accurate screening and diagnosis and treatment of hereditary thrombophilia.

The present invention relates to a gene analysis method which includes: conducting a single base extension reaction with use of a single base extension primer for detecting a target base sequence, and a fluorescent dye-labeled substrate for single base extension; subjecting a reaction product of the single base extension reaction to electrophoresis; and measuring mobility in the electrophoresis and fluorescence intensity of the fluorescent dye, and quantifying a content ratio of a plurality of target base sequences from a magnitude of the fluorescence intensity, wherein in the single base extension reaction, a fluorescent dye-free substrate for single base extension is mixed.

The invention discloses a primer group and a kit for simultaneously detecting five pathogens of TORCH and a use method of the kit, and aims at genomes of five pathogens including herpes simplex virus I / II (HSVI / II), rubella virus (RV), toxoplasma gondii virus (TOX) and cytomegalovirus (CMV) based on multiple PCR (Polymerase Chain Reaction) in combination with time-of-flight mass spectrometry. A primer group capable of detecting the pathogen in the same reaction system is provided, and the primer group comprises a multiple PCR amplification primer group and a single base extension primer group; the invention also provides a TORCH detection kit containing the primer group, the detection cost is low, the problem of high false positive rate can be avoided, the sensitivity is high, the specificity is good, the detection rate is high, and batch instant detection of samples can be realized.

The invention discloses a method for enriching mutation polymorphic site sequences, which comprises the following steps: by taking a 5 '-terminal phosphorylated selector as a primer, carrying out single base extension by taking a nucleic acid sample to be detected as a template under the condition of a mixture formed by DNA (deoxyribonucleic acid) synthetase and a buffer solution single dNTP (deoxyribonucleoside triphosphate) or reversible termination dNTP corresponding to the DNA synthetase; adding a T4DNA ligase buffer solution, heating at 90 DEG C for denaturation for 1 minute, and placing on ice for 2 minutes for intramolecular renaturation to form nucleic acid molecules with open-loop structures; adding T4DNA ligase, and connecting to form a ring when the information of the extended basic group is consistent with that of the basic group on the selector; exonuclease is added, and DNA which is not connected into a ring is digested. According to the method, a library does not need to be built in advance, and mutation information can be enriched by dozens of times through multiple rounds of single primer extension in the enrichment process; according to the invention, the extension of the single-ended primer can be completed in several seconds, multiple enrichment can be carried out by increasing the cycle number, and the later extension or ligation reaction can be completed in several minutes.

The invention relates to a primer group for detecting individualized medication gene locus polymorphism of hypoglycemic drugs, a kit containing the primer group and application, and belongs to the technical field of gene polymorphism detection. The primer group for the individualized medication gene locus polymorphism of the hypoglycemic drugs comprises at least one of 18 groups of primer pairs aiming at 18 SNP (Single Nucleotide Polymorphism) loci respectively, wherein the nucleotide sequences of reverse and forward primers of the 18 groups of primer pairs are SEQ ID NO: 1-SEQ ID NO: 36; the invention also comprises a UEP primer for the single base extension reaction, and the nucleotide sequence of the primer is selected from at least one of SEQ ID NO: 37-SEQ ID NO: 54. Based on the primer, the invention provides a kit containing the primer and an application method. The technical problems that the detection flux is limited or the biological information analysis and interpretation amount is large, and the detection cost is high are solved, the detection result can be obtained only through the trace genome DNA, the consumed time is short, the detection efficiency is high, the cost is remarkably reduced, and accurate medication of the hypoglycemic drug can be achieved.

The invention discloses an engineered terminal deoxynucleotidyl transferase mutant and application thereof. The engineering TdT enzyme variant containing single mutation and multi-site mutation is obtained by screening through a directed evolution technology on the basis of a DNA (Deoxyribonucleic Acid) sequence of wild type ZaTdT from a diphtheria sparrow. The engineered TdT enzyme variant designed by the invention has a unique function of specifically catalyzing an aminooxy modified nucleotide monomer to carry out single base extension, and high-precision controllable synthesis of a single-stranded DNA molecule can be realized under the condition of no template dependence. According to the method, the dependency of traditional enzymatic synthesis on a template chain is broken through, the single-stranded oligonucleotide synthesis efficiency and the sequence control precision are remarkably improved, and a novel tool platform is provided for innovative development in the fields of gene synthesis, molecular diagnosis, biological storage and the like.

The invention discloses a kit for detecting SNP (Single Nucleotide Polymorphism) of clopidogrel metabolism related genes and a use method of the kit. The kit comprises the following components: c.681Ggt; a, c, 636Ggt; the method comprises the following steps: A, c.-806Cgt; an upstream and downstream amplification primer mixture and a single-base extension primer mixture of three sites of T and T; the c.681Ggt is selected from the group consisting of The sequence of an upstream amplification primer of the site A is shown as SEQ ID NO.1, the sequence of a downstream amplification primer of the site A is shown as SEQ ID NO.2, and the sequence of a single-base extension primer of the site A is shown as SEQ ID NO.3; the c is 636Ggt; the sequence of an upstream amplification primer of the site A is shown as SEQ ID NO.4, the sequence of a downstream amplification primer of the site A is shown as SEQ ID NO.5, and the sequence of a single-base extension primer of the site A is shown as SEQ ID NO.6; the c is-806Cgt; the sequence of an upstream amplification primer of the T site is as shown in SEQ ID NO.7, the sequence of a downstream amplification primer of the T site is as shown in SEQ ID NO.8, and the sequence of a single-base extension primer is as shown in SEQ ID NO.9. The CYP2C19 * 2, CYP2C19 * 3 and CYP2C19 * 17 site genotypes of the drug metabolic enzyme CYP2C19 gene are mutually verified through two detection results, the drug metabolic capability difference of an individual can be rapidly and accurately determined, scientific guidance is provided for drug selection and drug dosage control of anti-platelet drugs, and the method has the advantages that the method is simple and convenient to operate, and the application prospect is wide. The method is beneficial to non-invasive, miniaturized, time-saving, low-cost design, specificity and rapid detection of gene detection products, and is simple to operate and convenient for clinical application.