7611 results about "Specific primers" patented technology

Methods for genotyping polymorphisms using a locus specific primer that is complementary to a region near a selected polymorphism are described. Methods for synthesizing pools of locus specific primers that incorporate some degenerate positions are also disclosed. A plurality of different sequence capture probes are synthesized simultaneously using degenerate oligonucleotide synthesis. The sequence of the locus specific regions of the capture probes are related in that they have some bases that are identical in each sequence in the plurality of sequences and positions that vary from one locus specific region to another. The sequences are selected based on proximity to a polymorphism of interest and because they conform to a similar sequence pattern.

Methods are provided for selective tagging of short nucleic acids comprising a short target nucleotide sequence over longer nucleic acids comprising the same target nucleotide sequence. The methods can involve performing one or two cycles of amplification of a sample comprising long nucleic acids and short nucleic acids, each comprising the same target nucleotide sequence with at least two target-specific primers or primer pairs under suitable annealing conditions, wherein the primer pairs comprise: an inner primer or primer pair that can amplify the target nucleotide sequence on long and short nucleic acids (wherein each inner primer comprises a 5′ nucleotide tag; and an outer primer or primer pair that amplifies the target nucleotide sequence on long nucleic acids, but not on short nucleic acids); whereby the amplification after a second cycle produces at least one tagged target nucleotide sequence that comprises two nucleotide tags, one from each inner primer, with the target nucleotide sequence located between the nucleotide tags.

The invention allows for the quantitative detection of a plurality of pathogens in a single sample. The method includes the amplification of a sample with a plurality of pathogen-specific primer pairs to generate amplicons of distinct sizes from each of the pathogen specific primer pairs. The method further includes the use of a plurality of competitor polynucleotide targets that correspond to each of the pathogen-specific primer pairs. The competitor polynucleotides are added to the reaction mixture at a known concentration to allow for the quantitation of the amount of pathogen in the sample. The method can be used for monitoring pathogen infection in an individual, preferably an immunocompromised individual.

The present invention provides methods, nucleic acids, compositions, and kits for detecting microRNA (miRNA) in samples. The methods comprise designing mRNA-specific primers, adding a polyA tail to the miRNA, and using reverse transcription and amplification to detect the miRNA. The nucleic acids, compositions, and kits typically comprise some or all of the components necessary to practice the methods of the invention.

Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type—pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional “bridge” amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5×10⁻¹² g (equivalent to 10²-10³ genomes) of a bacterial genomic DNA are disclosed.

The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and/or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates.

The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and/or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates.

The invention discloses a rapid fluorescence PCR detection kit for ASFV (African swine fever virus). The kit comprises specific primers ASFV-F and ASFV-R as well as a TaqMan probe ASFV-P, the 5' end of the probe labels fluorescence dye as FAM and the 3' end labels a fluorescence quenching group as BHQ-1. The kit can be used for detecting the ASFV in nasal swabs, blood, serum, plasma and tissue ofswine to realize rapid detection of the ASFV. In the whole ASFV detection process, only 40 min is taken from DNA extraction to obtaining of detection results, so that the detection time is greatly shortened, and the detection efficiency is improved.

The invention discloses a human leukocyte antigen HLA-A and HLA-B gene full-length sequencing method and an HLA gene sequencing and typing method. The HLA-A and HLA-B gene full-length sequencing method comprises the following steps of: a, performing PCR amplification on about 4kb full-length sequences of HLA-A and HLA-B genes by using a pair of primers respectively; and b, cloning the amplification products to a pGEM-Tea sy vector, sequencing the full-length sequences by using ten walking sequencing primers in positive and negative directions respectively, and totally obtaining 38 allele 4.3kb full-length sequences of the HLA-A and 30 allele 3.7kb full-length sequences of the HLA-B. The HLA-A and HLA-B sequencing and typing method comprises the following steps of: performing PCR amplification on typing target areas of the HLA-A and HLA-B by using two pairs of primers respectively; and performing two directional sequencing on products by using fourteen sequencing primers respectively, wherein the HLA-DRB1 and HLA-DQB1 sequencing and typing method comprises the following steps of: amplifying sequences of second and third exons of DRB1 and DQB1 by adopting group specificity primers respectively; performing two directional sequencing on the second and third exons of the DRB1 by adopting eight group specificity primers and three sequencing primers; and performing two directional sequencing on the second and third exons of the DQB1 by adopting four sequencing primers respectively.

The invention relates to a biological detection reagent capable of discriminating three types of ruminant animal varieties simultaneously, a preparation method and an application thereof. The invention selects the specificity of the cow, goat and sheep, and the conservative sequence segment of conservative mitochondrial gene as target, applies Primer Eexpress 3.0 software and Primer Select software in DNAStar, and designs and synthesizes a plurality of groups of primers and probes. After being synthesized and marked, the plurality of pairs of designed primers and the probes are carried out best pairing screening experiment so as to obtain the fittest combination of primers and probes. The reagent contains two pairs of specificity primers, and three Taq Man probes, wherein one pair of theprimer aims at cow source components, the other pair of primer aims at common primers of goats and sheep source component detection; the three probes are respective source components to the cows, thegoats and the sheep; and the amplified target fragment length to the source component detection of the cows, the goats and the sheep is respectively 92bp, 136bp and 136bp. The triple Taq Man fluorescence PCR detection method can simultaneously carry out accurate quantitative detection to the source components of the cows, the goats and the sheep, has simple and convenient operation, intuitive result, strong specificity, high sensitivity and good repeatability and can realize high throughput detection of various source components.

The invention discloses a c-KIT gene mutation detection liquid-phase chip, which comprises tag sequence at 5' terminal and ASPE (Allele Specific Primer Extension) primers consisting of specific primers specific to mutational sites at 3' terminal. The specific primers consist of sequences shown in SEQ ID No.10 and SEQ ID No.11 specific to AY502-503dup mutational sites, sequences shown in SEQ ID No.12 and SEQ ID No.13 specific to V560G mutational site, sequences shown in SEQ ID No.14 and SEQ ID No.15 specific to T670I mutational site and/or sequences shown in SEQ ID No.16-SEQ ID No.18specific to D816H/V mutational site, the tag sequence which is selected from sequences shown in SEQ ID No.1-SEQ ID No.9, microballoons which have different color codings and are respectively enveloped with a specific anti-tag sequence and amplification primers. The coincidence ratio of the result of sequencing method with that of the c-KIT gene mutation detection liquid-phase chip reaches up to 100 percent. The c-KIT gene mutation detection liquid-phase chip can simultaneously detect a plurality of mutational sites with excellent signal to noise ratio.

The invention discloses a liquid phase chip for CYP19A1 gene SNP (Single Nucleotide Polymorphism) detection, which comprises wild type and mutant type specific ASPE primers designed targeting CYP19A1 gene SNPs of rs4646, rs10046C>T, rs700519C>T, rs1870050C>A, hCV1664178A>C, rs12900137G>C, rs730154G>A, rs936306T>C and rs1902586A>G, microballoon spheres respectively coated with specific anti-tag sequences and primers used for amplifying CYP19A1 gene SNPs with target sequences to be detected. Each ASPE primer comprises a tag sequence at 5' end and a specific primer sequence at 3' end, wherein the specific primers are selected from SEQ ID NO. 19-36 and the tag sequences are selected from SEQ ID NO.1-18; and the anti-tag sequences can be correspondingly in complementary pairing with the tag sequences. The invention also discloses a CYP19A1 gene SNP detection method. The coincidence rate of the detection method provided by the invention and a sequencing method is as high as 100%; and the prepared liquid phase chip for CYP19A1 gene SNP (Single Nucleotide Polymorphism) detection has excellent signal-to-noise rate, and basically no cross reaction exists between a design probe and the anti-tag sequences.

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) detection reagent, a kit and a detection method for an African swine fever virus (ASFV). The detection reagent is targeted to a conserved segment of an ASFV VP72 gene, and comprises a pair of specific primers, a specific probe and an internal labeling probe. The kit comprises a PCR mixed solution, Taq DNA polymerase, DEPC (diethylpyrocarbonate)-H2O, an ASFV positive quality product, an ASFV negative quality product, a quantification standard substance and a pseudovirus internal labeling solution, wherein the PCR mixed solution comprises a pair of specific primers, a specific probe and an internal labeling probe. The detection method comprises the steps of extraction of total DNAs, preparation of reaction components, making of a standard curve by diluting the standard substance, amplifying and result judgment. The reagent and the kit have the advantages of high specificity, high sensitivity, no pollution and high flux, and can be used for accurately detecting infection, inapparent infection or continuous carrying of a toxic host of low-content ASFV. The detection method is quick, specific and sensitive, and can be used for quantitatively detecting a large quantity of samples at the same time.

The invention relates to a reagent kit and a detecting method for using loop-mediated isothermal amplification (LAMP) technique to detect aeromonas caviae, which belongs to the pathogen diagnosis field. The main technical scheme of the invention comprises: utilizing the LAMP technique, designing four pairs of high specificity primers aiming at six zones of 16s and 23s ribosome RNA gene transcription spacer region, and achieving the purpose for detecting the aeromonas caviae with specificity. Compared with a traditional method, equipment and operational steps which are needed in the method are simple, the method has the advantages of rapidness and high specificity, and the detecting cost is lower, which is suitable for common clinics and field detections.

The invention discloses ethambutol (emb) B gene mutation detection specific primers and a liquid phase chip. The liquid phase chip comprises ASPE primers, microspheres and amplification primers, wherein the specific primers of the ASPE primers are selected from SEQ ID No.18 and SEQ ID No.19 at the codon 285th site, SEQ ID No.20 and more than one of sequences from SEQ ID No.21-25 at the codon 306th site, SEQ ID No.26 and SEQ ID No.27 at the codon 330th site, SEQ ID No.28 and more than one of sequences from SEQ ID No.29-32 at the codon 406th site, and/or SEQ ID No.33 and SEQ ID No.34 at the codon 497th site. The detection result of the emb B gene mutation detection liquid phase chip provided by the invention is completely matched with that of a sequencing method, and the liquid phase chip has good signal-noise ratio.

The invention discloses a kit and method for staphylococcus aureus CRISPR locus detection. The kit comprises three pairs of specific primers designed according to three CRISPR locus gene sequences of staphylococcus aureus. The invention further discloses the method for staphylococcus aureus CRISPR locus detection. The three pairs of specific primers are utilized to amplify corresponding CRISPR fragments from sample genome DNAs. The kit and the detection method are simple to operate, high in sensitivity and specificity and low in detection cost and have good popularization and application values.