1177 results about "Multiplex pcrs" patented technology

Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.

The present invention provides an efficient way for high throughput haplotype analysis. Several polymorphic nucleic acid markers, such as SNPs, can be simultaneously and reliably determined through multiplex PCR of single nucleic acid molecules in several parallel single molecule dilutions and the consequent statistical analysis of the results from these parallel single molecule multiplex PCR reactions results in reliable determination of haplotypes present in the subject. The nucleic acid markers can be of any distance to each other on the chromosome. In addition, an approach wherein overlapping DNA markers are analyzed can be used to link smaller haplotypes into larger haplotypes. Consequently, the invention provides a powerful new tool for diagnostic haplotyping and identifying novel haplotypes.

The present invention provides a method of amplifying target DNA by PCR or multiplex PCR on a microarray using array-immobilized DNA probes synthesized in a common area on the microarray by a maskless array synthesizer (MAS). The MAS constructed array-immobilized DNA probes include a universal primer linked to a sequence-specific probe, and optionally a calibrated probe for use in quantifying amplified target DNA.

A fast, multiplexed PCR system is described that can rapidly generate amplified nucleic acid products, for example, a full STR profile, from a target nucleic acid. Such systems include, for example, microfluidic biochips and a custom built thermal cycler, which are also described. The resulting STR profiles can satisfy forensic guidelines for signal strength, inter-loci peak height balance, heterozygous peak height ratio, incomplete non-template nucleotide addition, and stutter.

Compositions and methods for nucleic acid amplification are provided that minimize the formation and amplification of spurious products, particularly in multiplex amplification reactions. Linear amplification molecules are provided. A first embodiment comprises a first common primer sequence, a first template homology region, a cleavable site, a second common primer sequence and a second template homology region. Methods employing the linear amplification molecules for nucleic acid amplification are also provided. A first embodiment comprises a template-specific hybridization, a linear amplification, a template-specific intramolecular hybridization, a strand cleavage, a second linear amplification and an exponential amplification using common primers.

The invention is directed to a system for performing multi-color real time PCT, comprising a flexible real time PCT instrument and a specific composition or reaction mixture for performing multiplex PCR: In particular, the present invention is directed to a composition or reaction mixtuer which comprises at least 3, preferably 4-5 and most preferably exactly 4 pairs of FRET hybridization probes. Each pair of said hybridization probes consists of a FRET donor probe carrying a FRET donor moiety and a FRET acceptor probe carrying a FRET acceptor moiety having an emission maximum between 550 and 710 nm.

The invention provides a method for carrying out high-throughput sequencing on a TCR (T cell receptor) or a BCR (B cell receptor). The method is characterized by designing upstream primers according to gene features of a V region of the TCR or the BCR and designing downstream primers according to gene features of a C region or a J region of the TCR or the BCR and obtaining sequences of the of the TCR or the BCR in combination with the multiplex PCR (polymerase chain reaction) technology and high-throughput sequencing, thus analyzing the rearrangement information of the TCR or the BCR. Compared with 25-30 cycles of existing multiplex PCR, two cycles of the multiplex PCR technology provided by the invention can conduce to greatly reducing the sequencing errors caused by primer amplification preference. Besides, the invention also provides a method for correcting multiplex PCR (polymerase chain reaction) primer deviation by utilizing DNA (deoxyribonucleic acid) tag sequences, thus further reducing the sequencing errors caused by primer amplification preference and intrinsic sequencing errors of high-throughput sequencing.

The present disclosure provides methods and kits for performing high multiplex PCR using molecular barcodes. The methods disclosed herein separately extend a set of primers (BC primers) that each comprise a target-specific sequence, a molecular barcode and a universal sequence, and amplify the resulting extension products using another set of primers (LA primers) that each comprise another target-specific sequence and a universal sequence. The methods may further comprise amplification using universal primers (preferably comprising an adapter).

The present disclosure is a cassette for PCR amplification and detection of DNA amplicons, the cassette having a cassette pipette which may be moved vertically and horizontally within the cassette, at least one reagent chamber, and at least one detector, such as a microarray, for the detection of DNA amplified during the PCR amplification steps.

The invention provides a method for typing three sites of key enzyme genes (MTHFR and MTRR) for folic acid metabolism by utilizing a fluorescence labeling probe and combining with a multiple PCR method. An amplimer and a fluorescent probe are designed according to MTHFR and MTRR genes; three sections of DNA sequences are detected in the amplification of a PCR amplifier; fluorescence signals which are released in amplification and dissolution processes in a reaction system are detected; a result is judged in the manner of (1) judging a typing result according to a Tm value shown by a hybrid peak of wild type and mutant type standard plasmids and (2) simultaneously typing according to the magnitude of a fluorescence value of an amplification curve. The primer and the probe adopted by the invention are high in specificity and sensitivity; the detection for three sites is simultaneously performed; the operation is simple and the result is easily judged; the typing result is more accurate and reliable in the manner of twice calibrating typing. The method provided by the invention can be applied to the aspects of guiding the pregnant woman to orally take and supplement folic acid, prompting the high risk in cerebral apoplexy, coronary heart disease and venous thrombus, assessing the metabolic activity of folic acid, and the like.

Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.

The present invention relates to nucleic acid amplification, and is especially multiple PCR process based on selective probe for simultaneous amplification of several nucleic acid segments and its application. The multiple PCR process includes the following steps: 1. constructing selective probe; 2. extension-coupling reaction; 3. PCR amplification; and 4. detecting PCR amplification product. The present invention features the selective probe and the PCR amplification product with difference in length. The process has greatly increased analysis flux, saving in the nucleic acid template consumption for detecting the sample, high compatibility of the detection system and other advantages.

RBC and platelet (Plt) alloimmunization requires antigen-matched blood to avoid adverse transfusion reactions. Some blood collection facilities use unregulated Abs to reduce the cost of mass screening, and later confirm the phenotype with government approved reagents. Alternatively, RBC and Plt antigens can be screened by virtue of their associated single nucleotide polymorphisms (SNPs). We developed a multiplex PCR-oligonucleotide extension assay using the GenomeLab SNPStream platform to genotype blood for a plurality of blood group antigen-associated SNPs, including but not limited to: RhD (2), RhC / c, RhE / e, S / s, K / k, Kpa / b, Fya / b, FYO, Jka / b, Dia / b, and HPA-1a / b.

The invention discloses a fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and a kit. The detection primer set is composed of primer pairs for detecting salmonella, Shigella, Vibrio parahemolyticus, campylobacter jejuni, campylobacter coli, staphylococcus aureus, bacillus cereus, Listeria monocytogenes, yersinia enterocolitica, enterobacter sakazakii, Escherichia coli, vibrio cholerae, Escherichia coli O157, aeromonas hydrophila and internal positive control. A multiplex PCR detection method based on an ordinary PCR platform is built, multiplex PCR reactions are carried out on genomic DNA, extracted from a sample to be tested, of bacteria in the same reaction system through the primer pairs acquired through analysis and design, and whether the food-borne pathogenic bacteria are contained in the sample or not is judged through the electrophoretic analysis of reaction products.

The invention discloses a method and a kit for detecting a non-small cell lung cancer drive gene mutation spectrum, and an application. The method comprises the following steps of: designing 15 pairs of amplification primers for amplifying the exon segments of the seven related genes of non-small cell lung cancer, dividing the amplification primers into 6 groups, and preparing an amplification primer mixed solution, performing multiple PCR (polymerase chain reaction) amplification on the to-be-detected samples by the amplification primer mixed solution respectively, and then performing enzymatic digestion; and designing 39 extension primers used for detecting hotspot mutation sites, dividing the extension primers into 6 groups corresponding to the amplification primer mixed solution, and preparing an extension primer mixed solution, performing extension reaction on the digested PCR product, then performing enzymatic digestion, performing capillary electrophoresis on the obtained product, and making a result judgment via software analysis. The kit provided by the invention comprises the amplification primers for amplifying the exon segments of the seven related genes of non-small cell lung cancer, and the extension primers used for detecting hotspot mutation sites. The method and the kit provided by the invention are simple, high in flux, and short in time consumption.

The invention discloses a construction method of a ctDNA ultra-low-frequency mutation detection library, a kit and an analysis method of library detection data. The construction method comprises steps as follows: S1, cfDNA is extracted from whole blood; S2, the terminal of cfDNA is restored and an A basic group is added to the 3'terminal; S3, the terminal of the cfDNA obtained in S2 is connected with connectors containing random label sequences; S4, primers for multiplex PCR (polymerase chain reaction) are designed according to the sequences of the connectors and an object region for object region capturing; S5, a PCR product in S4 is subjected to magnetic bead purification, and small-fragment DNA not subjected to non-specific amplification and primer dimer are removed; S6, a product in S5 is subjected to PCR amplification, an index sequence is introduced, and the ctDNA ultra-low-frequency mutation library is obtained. According to the method, PCR mistakes and sequencing mistakes are eliminated, and the detection specificity is improved.

The invention relates to a multiplex-PCR three-round amplification method, a primer and a kit thereof and application of the method in establishment of next-generation sequencing platform libraries. In the first two rounds of a PCR, a specific primer with the low concentration is consumed as much as possible, so that the differences of different amplicons are reduced and the homogeneity of multiplex-PCR products is improved; when the third round of the PCR is performed, adapter primers carrying different bar code sequences are added to mark different templates, the different adapter primers carry same universal amplification primers, therefore, it is guaranteed that the amplification efficiency of the different templates is consistent, and the differences of different template amplification products are reduced. Accordingly, not only is the problem that the homogeneity of ordinary multiplex-PCR amplification is poor solved, but also establishment of the sequencing libraries can be quickly and conveniently completed.

The invention discloses a method and a device for gene sequencing of a plurality of mixed DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequences, which is mainly characterized by adding random labels at two ends of each DNA or RNA template sequence before establishing a bank. The method comprises the following steps of: mixing a first forward primer containing the random label and a connector, a first reverse primer and the DNA sequences, performing multiple PCR (Polymerase Chain Reaction) amplification on two PCR circulations, and purifying the PCR product to obtain a first DNA product; mixing the first DNA product, a second forward primer containing a sample indexing sequence and the connector, and a second reverse primer, performing PCR reaction to obtain a second PCR product; purifying to obtain a second DNA product; and sequencing the second DNA product to obtain a sequencing result of each DNA sequence. According to the method disclosed by the invention, by introducing the random labels to each DNA molecule, the sequencing precision is improved, the error rate of the sequencing is obviously reduced, and the copy number of each DNA is precisely detected.

The invention discloses a respiratory pathogen multi-detection reagent kit. The respiratory pathogen multi-detection reagent kit has the advantages that the respiratory pathogen multi-detection reagent kit is based on multi-PCR (polymerase chain reaction) technologies, detection results can be determined by the aid of fluorescence resonance energy transfer via the melting temperature ranges, the respiratory pathogen multi-detection reagent kit can be used for qualitatively simultaneously detecting 16 types of respiratory pathogens, the 16 types of respiratory pathogens include 12 types of RNA(ribonucleic acid) viruses (influenza A viruses, influenza B viruses, H1N1 influenza A viruses, type A and type B respiratory syncytial viruses, type -1 / -2 / -3 parainfluenza viruses, type OC43 coronaviruses, type 229E coronaviruses, rhinoviruses and human metapneumovirus), 2 types of DNA (deoxyribonucleic acid) viruses (adenoviruses and bocavirus) and 2 types of bacteria (mycoplasma pneumoniae andbordetella pertussis), the respiratory pathogen multi-detection reagent kit is high in detection sensitivity, and the sensitivity even can reach 1 copy / reaction; the multi-detection reagent kit is good in specificity, and negative results of pathogens which have identical sampling sites and similar pathogenic mechanisms and are not in the detection range of the respiratory pathogen multi-detectionreagent kit can be obtained; the respiratory pathogen multi-detection reagent kit is short in operation time and easy to operate and can be used for quickly detecting the 16 types of respiratory pathogens in a single tube of a reaction system, the results are clear and are easy to interpret, and the like.

The invention provides a method for quantitative multiplex PCR of two targets whereby one target is present in at least 100-fold molar excess over that of the other target. The method further comprises the addition of a thermostable DNA polymerase with a final concentration of at least 0.5 units / μl.

The invention belongs to the technical field of food quality and safety detection and particularly relates to a method for rapidly detecting pork, beef, mutton and chicken components in food at the same time through animal genomic DNA (deoxyribonucleic acid) extraction, primer design and UP-M-PCR (universal primers-multiplex-PCR). The four meat components can be distinguished rapidly at the same time according to differential sites of mitochondrial cytochrome b of the animal by using the forward primer sharing and reverse primer specificity strategy and an M-PCR system including five primers. The result shows that the method has the advantages that the detection limit can be up to the pictogram level, the components of the reaction system are free from cross interference and the meat products on market can be detected, verified and distinguished accurately by sampling 80 meat products randomly according to the specificity of the four target meat components. In a word, the invention provides a specific, sensitive and practical multiplex PCR method for rapidly screening four common meat components in food at the same time.

The invention relates to a hereditable deafness susceptibility gene screen test kit. The kit uses mutation from C to T of 1,494 locus of a 12s rRNA gene, mutation from A to G of 1,555 locus of the 12srRNA gene, mutation from A to G of IVS7(-2) locus of an SLC26A4 gene and C(+ / -) of 235 locus of a GJB2 gene as detecting objects, designs and optimizes a set of specific primers respectively againsteach locus to be tested according to the PCR technical principle of a tetra-primer amplification refractory mutation system, amplifies the whole set of the primers in the same reaction tube, and performs primary multiple PCR amplification and primary gel electrophoresis on the four reaction tubes to obtain gene types of four loci simultaneously.

The invention relates to a strain of lactobacillus plantarum and bacteriocins produced by the lactobacillus plantarum and capable of inhibiting Gram negative bacteria. The bacteriocins have the advantages of broad antibacterial spectrum, thermal stability and stable pH, degradability by protease, no residue in a human body and high safety. A lactobacillus plantarum strain is preserved on June 29, 2009 with a preservation number of CGMCC No.3151. A production strain is obtained by separating 'Jiaoke', a conventional dairy product in Inner Mongolia; on a MRS culture medium, colonies are ivory and round with a protruded center and orderly edges; the strain has two blunt round ends and a short and straight stem; and the size of the strain is 0.4 to 0.7 mu m *2 to 3 mu m. The strain is a non-spore Gram positive bacillus, and is identified as the lactobacillus plantarum through an API50CHL sugar alcohol fermentation test, a 16S rRNA sequence homology analysis test and a recA gene multiplex PCR method. The lactobacillus plantarum KLDS1.0391 is used as the production strain and is fermented and purified by using the improved MRS culture medium to obtain the bacteriocins of the lactobacillus plantarum. The bacteriocins are used in food preservatives.

The invention discloses a complex enrichment medium used for salmonella, listeria monocytogenes and staphylococcus aureus and a method for preparing the same. The complex enrichment medium comprises the following components in weight portion: 15 to 19 portions of tryptone, 2 to 4 portions of peptone, 2 to 3 portions of sodium dihydrogen phosphate, 2 to 3 portions of glucose, 0.5 to 1.5 portions of aesculin, 1, 000 portions of distilled water, 1 to 2.5 portions of sodium pyruvate, 3 to 10.0 portions of mannitol, 1.0 to 25.0 portions of sodium chloride, 1 to 2.5 portions of lithium chloride, 0.0001 to 0.0002 portion of potassium tellurite, and 0.005-0.015 portion of nalidixic acid, wherein the pH value is between 7.1 and 7.5. The complex enrichment medium can inhibit the growth of other pathogenic microorganisms while performing enrichment on three target pathogens, thus the complex enrichment medium can be directly applied to the isolation culture and the biological assay test of target bacteria, and can also be directly applied to a detection technology of a plurality of pathogens based on one detection platform for multiple PCR and the like to make a diagnostic report.

There is provided a method for designing primers for multiplex PCR, in which, as a result of designing primers in candidate amplification regions for which priorities are set, if the number of candidate amplification regions in which primers are successfully designed does not reach a desired value, primers can be redesigned while a broad feature of previously set priorities is maintained.

The invention discloses a Multiplex PCR detection kit for nucleic acids of twelve respiratory viruses. The kit includes primers for amplifying the specific gene loci of the following twelve respiratory viruses: influenza A virus (Inf A), influenza A H1N1 (2009), influenza A H3N2, human parainfluenza virus (HPIV1), human parainfluenza virus (HPIV2), human parainfluenza virus (HPIV3), human parainfluenza virus (HPIV4), human metapneumovirus (hMPV), respiratory adenovirus (AdV), respiratory syncytial virus (RSV), bocavirus (BoV) and coronavirus (CoV). The kit allows multiplex detection, has high sensitivity and is convenient and quick to use. Specific primer sequences ensure reliability of detection results. A detection method is simple in operation, save time and labor intensity, has high detection throughput, is low in cost of reagent and consumable, and can directly detect the nucleic acids extracted from a respiratory pathogen sample, is low in requirement on detection platform and operation technology, and can be widely promoted in common detection.

The invention belongs to the technical field of marine organism pathogen detection, and in particular relates to a multiple-PCR detection method and a kit for simultaneously detecting WSSV (white spot syndrome virus), AHPND (acute hepatopancreatic necrosis disease), EHP (enterocytozoon hepatopenaei) and IHHNV (infectious hypodermal and hepatopancreatic necrosis virus) in prawns. With the application of the multiple-PCR detection method, amplification of four pathogen DNAs can be simultaneously conducted in one time by virtue of special detection primers, namely WSSV F and WSSV R, AHPND F and AHPND R, EHP F and EHP R as well as IHHNV F and IHHNV R of the WSSV, the AHPND, the EHP and the IHHNV. The multiple-PCR detection method provided by the invention is convenient and rapid, low in detection limit, strong in specificity, high in sensitivity and high in accuracy rate and is capable of simultaneously detecting the white spot syndrome virus, the acute hepatopancreatic necrosis disease, the enterocytozoon hepatopenaei and the infectious hypodermal and hepatopancreatic necrosis virus in the prawns.

The invention discloses a primer system for PCR (polymerase chain reaction) identification for a deer, a pig, a cow, a sheep, a horse, a donkey, a rabbit and a chicken, and can detect eight animal species in one time so as to achieve a purpose that common animal species can be almost covered. The primer of the primer system only carries out specific amplification on a respective species target segment without reacting with other species, and therefore the primer can be suitable for multiple PCR reaction characterized in that at least two primer pairs are induced in one-time PCR reaction so as to effectively shorten experiment time. In addition, when the PCR is carried out, an optimized specific PCR reaction system and reaction program can be used, a detection flow is simplified due to the adoption of a uniform PCR detection method, a quick and efficient identification mode with the high specificity is established so as to effectively identify whether the deer blood product is true and false and identify the adulteration mode, and a modern molecular biology detection means is provided for deer blood quality control. In addition, the primer system can be cooperated with relevant reagent to be prepared into a kit, thereby being convenient to use. Meanwhile, possibility is provided for the industrial production and application, and the primer system has an excellent application prospect.

Subject of the present invention is to provide an apparatus, an instrument, and a method particularly useful in multiplex PCR applications permitting short sample measuring times of many samples combined with high sensitivity.