234 results about "Melting curve analysis" patented technology

This invention concerns a fluorometer preferably combined with a thermal cycler useful in biochemical protocols such as polymerase chain reaction (PCR) and DNA melting curve analysis. The present flourometer features a low heat-generating light source such as a light emitting diode (LED), having a one-to-one correspondence to each of a plurality of sample containers, such as capped PCR tubes in a standard titer tray. The flourometer of the present invention further comprises an optical path between each LED and its correspondingly positioned container, and another optical path between each flourescing sample within the positioned container and an optical signal sensing means. The instrument can be computer controlled.

A reliable and highly sensitive method is provided for detecting methylation status of CpG-containing nucleic acids by nucleic acid amplification and melting curve analysis of amplification products. The methods and compositions employs a novel design of primers. CpG-containing methylation-independent oligonucleotide primers, wherein both unmethylated and methylated alleles of a CpG-containing nucleic acid can be detected by use of only one set of primers after the CpG-containing nucleic acid has been subjected to cytosine to thymine conversion of unmethylated Cytosine. The method is useful for detection of methylation status in for example cancer genes and other disease related genes, wherein methylation influences gene expression.

Disclosed herein are methods for detecting a fungal pathogen in a patient sample, involving isolating the sample, carrying out a PCR reaction on the sample to generate an amplicon that includes a region of the fungal 28S ribosomal RNA gene, and detecting the PCR amplicon. Also disclosed are sequences of primers for specifically detecting a broad range of fungal pathogens in the presence of human ribosomal DNA. In certain embodiments, the amplicon is detected by sequencing or by two-dimensional melt-curve analysis. In yet other embodiments, more than one fungal pathogen is detected in a sample using the methods disclosed herein.

The invention relates to method, wherein the number of repeat sequences which are present in a sample is detemined by means of melting temperature analysis. More precisely, the invention relates to a method for analysis of a target nucleic acid consisting of repetitive and non repetitive sequences comprising (i) hybridization of at least one polynucleotide hybridization probe comprising a first segment which is complementary to a non repetitive region and a second segment which is compementary to an adjacent repetitive region, said second segment consisting of a defined number of repeats and (ii) determination of the melting point temperature of the hybrid which has been formed between the target nucleic acid and the at least one hybridization probe.

Methods for analyzing a target nucleic acid are provided. A fluorescent label attached to a nucleic acid is incorporated into at least one strand of the target nucleic acid and the methods include monitoring change in fluorescence emission resulting from dissociation of the labeled strand of the amplification product from its complementary strand.

A system and methods are provided for melting curve genotyping analysis of nucleic acids. Melting curves are generated by plotting fluorescence of a sample as a function of temperature. In one illustrative example, an exponential algorithm is employed to remove the background from generated melting curves and thereby perform comparative analysis to other melting curves. Additional illustrative examples provide for measuring the differences between two or more melting curves and clustering the genotypes of the provided sample nucleic acids.

The present invention relates to an SNP (single nucleotide polymorphism) combination, detection method and kit for detecting liver damage susceptible genotype of an antitubercular drug and belongs to the technical field of medical molecular biological diagnosis; the SNP combination includes 7 SNP sites, and nucleotide sequences of the 7 SNP sites are shown sequentially as in SEQ ID NO. 1-7; the present invention also relates to an SNP detection method, comprising PCR (polymerase chain reaction) amplification and double-labeled probe melting curve analytical reaction, and primer pairs and double-labeled probe sequences for detection of the 7 SNP sites are shown as in SEQ ID NO. 8-20. The SNP site combination, detection method and kit provided herein enables quick, accurate, simple and high-throughput detection for a patient's genotype and prediction for the liver damage risk due to the patient using the antitubercular drug.

Methods are disclosed for identifying chromosomal aneuploidy using heterozygous SNPs in a melting curve analysis. In one aspect, a panel of SNPs may be used. The heterozygous nature of the SNPs may in some aspects, act as an internal control for the melting curve analysis, and alleviate the need for external controls or competitors. In another aspect, each of the SNP in the panel may have a heterozygocity index of greater than about 30%. While a number of aneuploidies may be identified using the disclosed methods, in one aspect, the chromosomal aneuploidy identified may be trisomy 21.

The invention discloses a reagent kit used for detecting CYP2C19 genotypes. Allelic genotypes of CYP2C19*2(681G/A) and CYP2C19*3(636G/A) of a CYP2C19 gene are detected by designing a specific primer pair and a fluorescent probe. Asymmetric PCR and the fluorescent probe melting curve analysis technology are combined, different genotypes are judged through melting peaks of specific temperatures, detection of six genotypes related to CYP2C19*2 and CYP2C19*3 can be achieved in one-tube PCR, subsequent processing is not needed, the possibility of reactant pollution is avoided to the largest extent, operation is easy, consumed time is short, specificity is good, accuracy is high, and results are easy to interpret.

The invention relates to a method for detecting human beta-globin gene mutation, in particular to a method for detecting the beta-globin gene mutation by a modified molecular beacon melting curve. The method comprises the following steps of: designing and preparing a corresponding modified molecular beacon in region of a beta-globin gene needing mutation detection; designing an upstream primer and a downstream primer on the periphery of the designed modified molecule beacon, and performing PCR amplification on fragments containing the region to be detected by using the upstream primer and the downstream primer; and after PCR amplification is finished, analyzing the melting curve, and judging whether a nucleotide sequence to be detected has the gene mutation and possible mutation types according to the change of the melting point of the modified molecular beacon.

This document relates to melting curve analysis of nucleic acids. The method according to first aspect of the invention comprises analyzing a nucleic acid melting curve measured from a sample, the melting curve comprising a sum signal of at least two nucleic acid melt signals and a background signal as a function of temperature. The method further comprises optimizing at least one constant in a temperature-dependent exponential correction function so as to minimize the variation of the nucleic acid melting curve at a temperature region where the target nucleic acids in the sample remain essentially double stranded, and generating a corrected nucleic acid melting curve representative of the nucleic acid melt signal by applying said exponential correction function over the region of the measured melting curve where the strands of the nucleic acids dissociate. According to further aspects, the invention relates to a curve fitting algorithm for precise estimation of melting peak areas and a mathematical transformation for linearization of calibration curve data to enhance the linear measuring range of a competitive PCR assay. The invention provides a powerful tool for analyzing PCR-amplified sample containing two or more different nucleic acids having similar but distinguishable melting temperature.

The invention provides a kit capable of detecting 10 pathogenic vibrios simultaneously based on a fluorescence probe melting curve method and a method capable of detecting the 10 pathogenic vibrios simultaneously. The kit comprises a universal primer, a fluorescence probe and hybridization connecting probes designed according to specific genes of the 10 pathogenic vibrios. The method capable of detecting the 10 pathogenic vibrios simultaneously includes the steps that the DNA of dubious bacteria extracted by a sample to be detected is used as a template, and hybridization connection reacting, fluorescence PCR amplification treatment and melting curve analysis treatment are carried out in sequence through the kit by means of the fluorescence probe melting curve method. According to the kit and the detection method, based on the multiple-connecting probe amplification technology, the 10 pathogenic vibrios can be detected simultaneously through the real-time fluorescence PCR melting curve method, hence, the detection period of the pathogenic vibrios is effectively shortened, and the detection efficiency of the pathogenic vibrios is improved.

The invention discloses a primer and probe composition for detecting polymorphism of human CYP2C9 and VKORC1 genes, a kit and application. The primer and probe composition comprises three pairs of specific primers for amplifying CYP2C9*2, CYP2C9*3 and VKORC1 sites, and three specific fluorescent probes. The primers and the probes, disclosed by the invention, have high sensitivity, strong specificity and strong anti-interference capability; a manner combining an asymmetric PCR (Polymerase Chain Reaction) amplification and fluorescent probe melting curve analysis technologies is used for detecting the gene polymorphism; different gene types can be effectively distinguished according to the quantity and Tm value of a melting peak; a result is convenient, clear and objective to judge and read.Single-tube sampling can be used for simultaneously detecting 6 mutation types of 3 gene sites; the primer and probe composition is simple and convenient to operate and the detection efficiency is improved; a large batch of samples can be detected and clinical operation is facilitated.

The invention discloses a kit for detecting the reproductive capacity of sheep and a using method thereof. The kit is internally provided with primer nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 in a sequence table, probe nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6 as well as respectively placed FTA (Fault Tree Analysis) card and relevant reagents. The using method of the kit provided by the invention comprises the following steps: adding the primer nucleotide sequences, probe nucleotide sequences and relevant reagents in the kit into the DNA (Deoxyribose Nucleic Acid) of the sheep to be detected; then carrying out PCR (Polymerase Chain Reaction) amplification; carrying out fusion curve analysis on an amplification product in a high-resolution fusion curve analyzer; and forecasting the lambing situation of the sheep to be detected according to G, A, T and C peaks in the fusion curve.

The present invention relates to a method and a kit for detecting nucleic acid sequence variation using melting curve analysis, especially relates to a method and a kit for detecting nucleic acid sequence variation by melting curve analysis using self-quenched probe. Said method provides the characteristics of the self-quenched probe employed, as well as the corresponding nucleic acid amplification conditions, so that the probe can bind to the amplified target sequence, and variations of the target sequence can be detected by melting curve analysis. The present invention also encompasses a kit assembled according to the method described.

The invention provides a method for screening low enrichment cadmium peanut varieties by a fluorescent quantitative PCR technique, which relates to a method for screening the low enrichment cadmium peanut varieties. The method comprises the following steps of: 1, culturing peanut seedlings by using nutrient solution; 2, designing specific different cadmium treatment levels; 3, performing a method for detecting and analyzing expression differences of AhMT2a genes of peanuts; and 4, screening cadmium sensitive varieties by using the method for detecting the expression differences of the genes by using the fluorescent quantitative PCR technique according to the screening bases of culturing the peanut seedlings by using the nutrient solution, and using sequences of the AhMT2a genes of leaf blades of the seedlings. The method has the advantages of simple detection method, no need of probe design, low cost and good universality, and can perform melting curve analysis to detect the specificity of amplified reactions.

The invention relates to low-abundance gene mutation detection. Particularly, the invention provides a method for detecting whether mutation exists in the nucleic acid molecules of a nucleic acid sample to be detected through self-quenching probe melting curve analysis. Besides, the invention also provides a kit comprising a self-quenching probe and primer pairs, and the kit can be applied to implementing the method.

The invention discloses a two-color fluorescence PCR primer, probe and method for quickly distinguishing canine parvovirus vaccine strain from wild strain. The real-time fluorescence PCR technique and the melting curve analysis technique are combined, and canine parvovirus vaccine strain and wild strain are identified according to the peak pattern of a melting curve and Tm value difference; operation is easy, and identification and detection of three kinds of wild strain with different genotypes and two kinds of vaccine strain can be achieved through one reaction; detection speed is high, and flux is high; the whole operation process can be finished within 3 h, cell cultivation of viruses is not needed, and time for identification and detection of three kinds of wild strain with different genotypes and two kinds of vaccine strain is shortened greatly; accuracy, specificity and repeatability are high, accurate, quick and high-flux analysis can be achieved, and application and popularization in clinical practice can be achieved easily.

The invention discloses a multiplex PCR (Polymerase Chain Reaction) assay method for simultaneously assaying multiple respiratory viruses, which includes the following steps: design of probes, design of upstream and downstream primer sequences, multiplex PCR reaction and dissociation curve analysis. By designing Taqman probes for the conserved group sequences of the respiratory viruses and adopting different fluorophores when Tm phase difference is less than or equal to 6 DEG C, the method can assay a variety of respiratory viruses with high throughput and high accuracy. The invention further provides a multiplex PCR assay probe set and a corresponding kit for simultaneously assaying multiple respiratory viruses, which are convenient to apply and have a great market and application prospect.