78 results about "Quantitative PCR analysis" patented technology

The invention relates to a plasmid control molecule for detection of transgenic soybean and a building method thereof. The plasmid control molecule contains a control gene lectin specific sequence in soybean, a 3' end transformant specific sequence and a 5' end transformant specific sequence of transgenic soybean MON89788. Through designing a specific qualitative PCR primer, the lectin gene specific sequence, the 3' end transformant specific sequence and the 5' end transformant specific sequence of the MON89788 are obtained through amplification. Through molecular cloning techniques such as enzyme digestion, connection, transformation and the like, the three specific sequence segments are built into a plasmid molecule to obtain an artificially recombined plasmid control molecule pMD-LM3M5. According to tests, the plasmid control molecule built in the invention can fully substitute for the positive control of the transgenic soybean MON89788 and is suitable for the transformant specific qualitative and quantitative PCR analysis and detection of the MON89788 soybean.

The invention discloses a standard plasmid molecule to check genetic improved soybean strain GTS40-3-2 and constructing method, which comprises the following steps: incorporating strain special fragment of the genetic improved soybean strain GTS40-3-2 and special fragment of soybean internal standard gene Lectin; analyzing exogenesis inserting carrier by-pass ortho gene sequence of genetic improved soybean strain GTS40-3-2; designing strain special PCR primer; augmenting; getting strain special fragment of genetic improved soybean strain GTS40-3-2 and soybean internal standard gene special sequence; constructing into a plasmid molecule with molecular cloning method; getting artificial retooling plasmid molecule pMD-RRS. This standard plasmid molecule can be fit for strain special quantitative PCR analysis and check of genetic improved soybean strain GTS40-3-2 sample.

The invention discloses a capillary bioanalysis system, and an analytical method and applications thereof, and belongs to the field of medical equipment and biological detection technologies. The capillary bioanalysis system comprises a three-dimensional movement sample injecting platform, a temperature control-optical detection module, a magnetic field control module, a fluid control unit and a capillary array. The three-dimensional movement sample injecting platform and the fluid control unit are arranged on the two sides of the capillary array, and the capillary array is provided with the temperature control-optical detection module and the magnetic field control module. According to the capillary bioanalysis system, the droplet technology and the magnetic bead technology are combined, and bioanalysis processes such as loop-mediated isothermal amplification, fluorescence quantitative PCR analysis and immunochemiluminometry are integrated in capillaries. Advantages of the capillary bioanalysis system are that: volume is small, analysis speed is fast, detecting flux is large, and automation degree of operation is high. The capillary bioanalysis system is flexible in application, is suitable for analysis of single sample, batches of samples and on-site rapid detection, is capable of reducing acquisition cost and operation cost of equipment significantly, and possesses excellent economic benefits.

The invention discloses a method and a device for quantitative PCR multi-wavelength fluorescence detection. N fluorescence wavelength detection passages with the mutual interval as a module standard hole site are arranged, each fluorescence wavelength detection passage is provided with a light source exciting optical fiber path and a fluorescence receiving optical fiber path, the light sources of the n light source exciting optical fiber paths respectively pass through a collimating lens and an exciting light filter so as to obtain exciting light with required wavelength, and then the exciting light is irradiated onto n test tube exciting reagent samples through focusing; the fluorescence of the n test tube exciting reagent samples respectively enters the fluorescence receiving optical fiber paths, the fluorescence with the required wavelength is obtained through the collimating lens and the exciting light filter, is irradiated onto a plane reflecting mirror and is then refracted onto n working surfaces of the same polyhedral reflecting mirror through the plane reflecting mirror, and the fluorescence of the n fluorescence wavelength detection passages is converged onto a photoelectric surface of an optical sensor through the polyhedral reflecting mirror so as to realize the real-time synchronous detection of the multi-wavelength fluorescence. The invention can greatly improve the detection efficiency, no exciting light interference is generated in detection, and quantitative PCR analysis results are exact.

The invention belongs to the field of plant gene engineering and transgenic breeding, and relates to a method for improving the stress resistance of cut chrysanthemum through trans-CgHSP70 genes. CgHSP70 established plant expression vectors obtained by cloning from Zhongshan purple cinnamon of the chrysanthemum are transferred into the cut chrysanthemum by using an agrobacterium-mediated method for cultivation so as to initially obtain resistant plants, and positively transferred plants are obtained through the screening of hygromycin resistance; PCR (polymerase chain reaction) and fluorescence quantitative PCR analysis is performed on converted plants to prove that endogenous genes are transferred into genome DNA of transgenic plants and transcription occurs; and the resistance analysis on the offsprings of the transgenic plants proves that the resistance to high temperature, drought and high salt is obviously improved. In the invention, the stress resistance of the cut chrysanthemum is improved through the conversion of the endogenous CgHSP70 genes and normal transcription expression as well as the induction of the expression of the genes at an adverse environment; a novel and practical method is provided for selecting and breeding stress resistance varieties of chrysanthemum by using a gene engineering technology; and the breeding progress of the biotechnology of the chrysanthemum can be effectively accelerated.

The invention relates to the technical field of liquor production, particularly to a multi-microbe solid fermentation ethanol and acetic acid production key microbe quantitative analysis method. The method comprises performing quantitative PCR (polymerase chain reaction) analysis on HIS3 genes of saccharomyces cerevisiae, AHD1 genes of schizosaccharomyces pombe and 16S rRNA (ribonucleic acid) fragments of lactobacillus to analyze the change tendency of the saccharomyces cerevisiae, the schizosaccharomyces pombe and the lactobacillus in liquor yeasts and fermented grains and accordingly to predict the yield of ethanol. The multi-microbe solid fermentation ethanol and acetic acid production key microbe quantitative analysis method has the advantages of being high in detecting speed and sensitivity, good in accuracy and specificity and free from cultivation.

A method for revealing and distinguishing paddy field formic acid utilization type methanogenic archaea in situ by adopting DNA-based stable isotope probing technology comprises the followings steps: (1), collecting a paddy soil sample; (2), performing a micro universe cultivation experiment of <13>C-formic acid; (3), performing centrifugation layering on the microbe total DNA of <13>C-formic acid cultivation soil by using an ultracentrifuge; (4), performing real-time quantitative PCR analysis and finger-print analysis on methanogenic archaea genes in layers of multiple buoyant densities after layering to judge whether the paddy soil contains the formic acid utilization type methanogenic archaea with metabolic activity. Therefore, the method can be used for keenly revealing the paddy field formic acid utilization type methanogenic archaea in situ based on the DNA-based stable isotope probing technology, and has great significance on the understanding of the paddy field nutrient cycling process driven by microbes and the cognition of the ecological functions of paddy field methanogenic archaea functional groups.

The invention discloses a plasmid reference molecule which is suitable for the quantitative determination of nucleic acids from a transgenic soybean GTS40-3-2, comprising a structure-specific sequence and a strain-specific sequence of the transgenic soybean GTS40-3-2 and a specific fragment of a soybean endogenous reference gene Lectin. The invention is characterized by designing primers to obtain the structure-specific sequence and the strain-specific sequence of the transgenic soybean GTS40-3-2 and the specific fragment of the soybean endogenous reference gene Lectin by PCR amplification through the analysis of inserting the exogenous source of the transgenic soybean GTS40-3-2 in the gene sequence; constructing by molecular cloning to form a artificial recombinant plasmid molecule pXL02 in a plasmid molecule; detecting the phosphorus content of the plasmid reference molecule by inductively coupled plasma mass spectrometry (ICP-MS) to calculate the concentration of the plasmid reference molecule. According to the invention, the plasmid reference molecule constructed in the invention can completely replace a transgenic soybean GTS40-3-2 positive standard sample, and the plasmid reference molecule is completely suitable for the quantitative PCR analysis and detection of structure specificity and strain specificity of the transgenic soybean GTS40-3-2 sample.

The invention discloses a selection method of reference genes in quantitative real-time PCR analysis of jerusalem artichoke, and relates to the field of quantitative PCR. The method comprises the following steps: taking different tissues (radicles, immature stems, leaves, stem blocks and petals) of the jerusalem artichoke as materials, and carrying out expression analysis of the reference genes of18S ribosomal RNA gene (18S rRNA), transcription elongation factor gene (Ef-1a), actin gene (Actin and beta-actin), 3-glyceraldehyde phosphate dehydrogenase (GAPDH), 25S ribosomal RNA gene (25S rRNA)and poly-ubiquitin enzyme gene (UBQ)7 by using a q PCR technology; carrying out statistical assessment on the obtained data and analyzing expression change of all housekeeping genes by utilizing GeNorm and NormFinder software, so as to screen out relatively-stable genes as the reference genes of the jerusalem artichoke, which are used for studying the gene dosage changes of the jerusalem artichoke.

The invention belongs to the technical field of biological detection, and in particular relates to a method for evaluating soil health through the quantitative PCR (Polymerase Chain Reaction) analysis of microorganisms. The method of the invention comprises the following steps of: synthesizing a suitable quantitative PCR primer according to microorganism parameters for evaluating the soil health; constructing standard product plasmids of all the parameters and establishing a standard curve; and extracting sample DNA (Deoxyribonucleic Acid) and detecting the copy numbers of all the types of microorganism genes in the samples through quantitative PCR so as to judge the soil health state. The invention integrates various molecular biology methods needed for evaluating the soil health till now into a whole and is rapid, simple, convenient and effective.

The invention provides a method for reprogramming spinal marrow astrocytes into motor neurons through induction. The method comprises steps as follows: (1) primary rat astrocytes are subjected to pure culture; (2) 7 small molecule drugs including SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin and VPA are selected according to substances required in the stages of neuron induction and motor neuron formation and development, and the astrocytes are induced and reprogrammed in vitro through the small molecule drugs; (3) the reprogramming and differentiation conditions of the astrocytes to the motor neurons are analyzed through observation of morphologic change of cells and methods of chemical technology analysis of immunofluorescent cells, real-time fluorescent quantitative PCR analysis and statistical analysis. The rat astrocytes can be successfully induced and reprogrammed into the motor neutrons in vitro through small molecular drug composition, and the transdifferentiation exceeds 75%; in-situ direct reprogramming of the astrocytes to the neurons is a possibly comparatively feasible thought in SCI (spinal cord injury) repairing and functional reconstruction treatment.

The invention discloses a fluorescent PCR (polymerase chain reaction) detection method for identifying the traditional strain and highly pathogenic strain of PRRSV (porcine reproductive and respiratory syndrome virus). In order to accurately and quickly detect the nsp2 gene-deleted PRRSV, an upstream primer and two downstream primers are designed on the nucleotide sequence of the nsp2 gene, wherein one of the downstream primers is designed in a base sequence-deleted area. A PCR method for identifying the traditional strain and the highly pathogenic nsp2 gene-deleted strain is established through condition optimization; and a fluorescent quantitative PCR method for identifying the nsp2-deleted variant strain according to a melting curve is created by applying the optimal condition to the SYBR Green fluorescent quantitative PCR analysis, wherein the sensitivity is enough to detect 3-6 gene copies. Compared with the conventional PCR, the fluorescent quantitative PCR method disclosed by the invention can quickly and accurately detect the traditional strain and the deleted strain, and the sensitivity is 10 times higher than that of the conventional PCR.

The invention discloses a necessary standard plasmid molecular and a construction method of the standard plasmid molecular in the field of transgenic crop detection. The construction method comprises the following steps: modifying an octo-gene plasmid molecular pTLE8 (Patent Application No.201010286884.3), adding a fusion fragment of 3'-transformation event specific sequences of transgenic corn Bt176 strain and Mon810 strain and corn internal reference gene Hmg specific sequence, and modifying into a deca-gene plasmid molecular pTLH10 (Lec1, 35S, NOS, PAT, Sad1, Cry1, Ab/c, Bt176, Mon810 and Hmg). The standard plasmid molecular constructed by the invention can completely replace a positive standard sample, and is suitable for screening transgenic soybean GTS40-3-2 strain, transgenic corn Bt176 and Mon810 strains and transgenic Bt cottons, and qualitative and quantitative PCR analysis and detection of gene specificity and strain specificity.

The invention discloses an enrichment culture method of whole-process nitrifying bacteria. The enrichment culture method comprises the following steps of S1, filtering a water body sample by adoptinga sterile filter membrane; S2, culturing the sterile filter membrane in an enrichment culture medium added with a substrate to obtain a filter membrane subjected to enrichment culture, wherein the substrate comprises ammonia or urea; S3, adopting a new sterile filter membrane as a transfer filter membrane, culturing the transfer filter membrane and the filter membrane subjected to enrichment culture in a new enrichment culture medium added with the substrate, and making the transfer filter membrane contact with the filter membrane subjected to enrichment culture, so that the whole-process nitrifying bacteria are transferred to the transfer filter membrane from the filter membrane subjected to enrichment culture; S4, taking out the transfer filter membrane, and culturing the transfer filtermembrane in a new enrichment culture medium added with the substrate; and S5, continuously transferring the whole-process nitrifying bacteria on the transfer filter membrane for multiple times by adopting the filter membrane, so as to achieve the purpose of enrichment culture of the whole-process nitrifying bacteria. In the culture process, part of the filter membrane is taken out regularly to extract DNA, and quantitative PCR analysis is carried out to confirm the enrichment condition of the whole-process nitrifying bacteria.

The invention provides a method for analyzing hypoxia stress on shrimps by detecting HURP1 gene expression. The method comprises the steps as follows: step one, acquiring the sequence of an open reading frame of an HURP1 gene of litopenaeus vannamei, and designing and synthesizing a sequence-specific fluorescent quantitative PCR (polymerase chain reaction) primer; step two, acquiring to-be-detected blood lymphocytes of litopenaeus vannamei, extracting the total RNA (ribose nucleic acid) of the blood lymphocytes, reversely transcribing the total RNA into cDNA, and preparing a fluorescent quantitative PCR template; step three, performing fluorescent quantitative PCR analysis to analyze expression of HURP1 in the blood lymphocytes of litopenaeus vannamei. With the adoption of the method, whether litopenaeus vannamei is in a low-oxygen stress state is judged more conveniently, more quickly and more accurately by detecting expression of the HURP1 gene.

The invention discloses application of brain cell-derived exosome circular RNA in serum as an Alzheimer's disease diagnostic marker. The invention relates to application of a reagent for detecting any one or more of hsa_circ_0029426 and hsa_circ_0044837 in preparing an Alzheimer's disease auxiliary diagnostic reagent. High-throughput sequencing and quantitative PCR analysis in nearly 100 patients finds that two kinds of circular RNA in exosome in serum can effectively distinguish Alzheimer's disease patients from normal people and non-Alzheimer's disease patients with clinical symptoms similar to Alzheimer's disease, and the area under an ROC curve is greater than 0.90. Therefore, the two kinds of circular RNA or a combination thereof can be used as detection markers for Alzheimer's disease diagnosis.

The invention discloses an HRM primer, a kit and a method for rapidly identifying bactrocera cucurbitae and South Asia fruit flies. The method comprises the steps of firstly, designing and synthesizing a HRM specific primer for distinguishing two kinds of fruit flies based on mitochondrial genomes of bactrocera cucurbitae and South Asia fruit flies; respectively extracting genome DNAs of bactrocera cucurbitae and South Asia fruit flies; carrying out high-resolution real-time fluorescent quantitative PCR analysis by taking the extracted DNAs as templates and EvGreen as a dye; and distinguishingtwo types of bactrocera cucurbitae according to peak difference of melting temperature curves. According to the invention, the problem of rapid identification of two kinds of bactrocera cucurbitae and South Asia fruit flies which are co-hosted and have similar larvae is solved; the HRM method for identifying the two kinds of bactrocera cucurbitae based on mitochondrial genes is established; bactrocera cucurbitae and South Asia fruit flies which are mixed on melon vegetables can be quickly, efficiently and simply distinguished; and the HRM primer, the kit and the method have the advantages ofhigh accuracy, high flux, good repeatability and low cost.

The invention relates to the technical field of white spirit brewing, in particular to a quantitative analysis method of important saccharomycetes Zygosaccharomyces bailii in a white spirit solid-state fermentation process. According to the invention, a specific primer is adopted to carry out quantitative PCR analysis on 26S rRNA of Zygosaccharomyces bailii, so as to analyze the variation trend ofZygosaccharomyces bailii in Daqu and fermented grains and predict the alcohol yield. The method disclosed by the invention has the advantages of high detection speed, high sensitivity, good accuracy,good specificity and no culture.

The invention discloses an essential standard plasmid molecule used for detecting genetically modified crops and a building method thereof. The standard plasmid molecule contains specific fragments of a soybean endogenous gene Lectin and specific fragments of an exogenous gene EPSPS in the genetically modified soybean strain GTS40-3-2. The standard plasmid molecule and the building method have the following beneficial effects: the two target gene fragments are obtained by analyzing sequences, designing primers and amplifying the genes; the standard artificial recombinant plasmid molecule pTLE2 is obtained through such gene cloning technologies as fusion PCR (polymerase chain reaction), link, transform and the like; the standard plasmid molecule built in the invention can absolutely replace the positive controls and is suitable for qualitative and quantitative PCR analysis and detection of the specificity of the structural genes in the genetically modified soybean strain GTS40-3-2; and new exogenous gene fragments can be added on the basis of the original standard molecule if new genetically modified materials occur, thus meeting the increasing detection requirements of the genetically modified crop materials.

The invention belongs to the technical field of gene expression analysis, and concretely relates to application of CL5547.Contig2 gene to pumpkin gene expression real-time fluorogenic quantitative PCR analysis as a reference gene. The gene expression level and stability are compared at whole genome level by utilizing pumpkin transcriptome data, 10 genes with stable expression are selected, also 2 commonly-used conventional reference genes in pumpkin are selected as contrast, and the expression stability of the 12 genes in different tissue organs of two pumpkin varieties under conditions of abiotic stress, biotic stress and plant-growth regulator processing (GA3, ABA, NAA and ETH), and CAT1 gene and CAT2 gene are used to perform verification, so that the CL5547.Contig2 gene is confirmed to have the highest expression stability, and is an optimum reference gene for pumpkin gene expression real-time fluorogenic quantitative PCR analysis.

The invention discloses application of GAPDH, UBQ, Actin7, PP2A and Actin1 genes as reference genes and special primers thereof in real-time fluorescent quantitative PCR (Polymerase Chain Reaction) analysis of plum blossom. The invention also discloses a screening method of the reference gene in real-time fluorescent quantitative PCR analysis of plum blossom, which can provide theoretical basis and technical guarantee for molecular level research of plum blossom.

The invention provides a method for screening a reference gene in the real-time quantitative PCR analysis of a heterodera glycines infected wild soybean root tissue, and relates to the fields of biology and genetics. A high-resistance high-sensitive wild soybean germplasm is taken as a test material, the condition of gene expression of 24 candidate housekeeping genes in a wild soybean root system in different heterodera glycines infection periods is detected, the expression abundance and the expression stability are evaluated so as to screen the reference gene suitable for studying the expression of the gene in the heterodera glycines infected wild soybean root system, and the method is used for exploring the resistance mechanism of the wild soybean to the heterodera.

The invention discloses a method for promoting brown adipose tissue differentiation through SFRP4 and an application. The method comprises treating precursor adipose cells for differentiation by usingSFRP4 active proteins (with different concentrations of 0, 1, 10, 100ng/mL), when the cells grow to 80%, culturing with a cell differentiation induction culture medium I until the cells are fused, adding an induction culture medium II into the cells, inducing for 2-8 days, and replacing the culture medium in the culture bottle with an induction culture medium III, inducing for 2-8 days, observingunder a microscope, after dyeing with oil red O, observing under the microscope again, collecting cells with different differentiation days, extracting total RNA of the cells, detecting expression levels of marker genes UCP-1 and SFRP4 of brown adipose cells by using a real-time quantitative PCR analysis technology after the purity and integrity of the RNA are detected to be qualified, and finally, carrying out statistical analysis. The experimental process is uniform in sampling, and the result reliability is high.

The invention discloses a Wnt family gene which is amplified for the first time from Schistosoma japonicum 19-day schistosomulum by utilization of RACE technology, wherein, sequence analysis indicates that: a complete coding frame of the gene comprises 1311bp, 436 amino acids coded; and theoretical molecular weight of 49.6kD. Homology analysis results indicate that: amino acid sequences of the gene have typical Wnt family protein characteristics; similarity of the amino acid sequences with amino acid sequences of Dugesia japonica and human Wnt4 is relatively 43 percent and 37 percent; the gene is presumed to be a Wnt4 gene of schistosome and named as Sjwnt4 (GenBanK Log-On No.: DQ643829). Real-time quantitative PCR analysis indicates that: the gene is expressed all in 14-day schistosomulum, 19-day schistosomulum, 31-day imago, 44-day female worms and 44-day male worms. The invention constructs a pronucleus expression vector pGEX-4T-2-Sjwnt4 of the gene and applies an Escherichia coli system for expression; expression proteins exist in the form of inclusion bodies; Western blottings indicate that expression products can be identified by crude antigen immune serums of Schistosoma japonicum imago.

The invention discloses a method for rapidly judging brassica plants with low cadmium accumulation by detecting the up-regulated expression of cadmium inhibition genes. Mustard type rape with low cadmium accumulation is taken as the material, the transcriptomes of roots and leaves of the cadmium processed material and a control material are subjected to sequencing analysis, the mRNA sequences, whose expression amount in leaves and roots is increased by 2.00 times or more, are selected, the functions of the genes corresponding to the mRNA sequences are analyzed, and the genes that can inhibit the absorption and transportation of heavy metals such as cadmium are screened out, namely cadmium inhibition genes. Quantitative PCR is used to analyze the relationship between the gene expression change and the cadmium processing concentrations, 5 cadmium inhibition genes are determined, and the expression amounts of the five genes in the leaves and roots of all processed groups are all increased by 2.00 times or more. The primer of the five genes is utilized to detect the change of gene expression of a brassica plant in a MS culture medium under the induction of cadmium with a concentration of 5-25 mg/kg. If the expression amounts of the five cadmium inhibition genes in roots and leaves are all increased by 2.00 times or more compared with the control group, the brassica plant is a plant with low cadmium accumulation.

The invention belongs to the technical field of biological detection, and particularly relates to a standard plasmid molecule for genetically modified maize strain Mon810 detection and a construction method thereof. The standard plasmid molecule comprises a strain specific sequence, a construction specific sequence and a maize internal standard gene zSSIIb specific segment of genetically modified maize strain Mon810; by analyzing an exogenous insertion vector border sequence of the genetically modified maize strain Mon810 and a sequence of a joining region of two elements Hsp70 and cryIA (b), strain specific and construction specific PCR (Polymerase Chain Reaction) primers are designed, amplification is performed to obtain the strain specific segment and the construction specific segment of the genetically modified maize strain Mon810 and the maize internal standard gene specific sequence; and an artificial recombinant plasmid molecule pMHZ is obtained by constructing the sequences and the segment into the plasmid molecule through a molecular cloning method. A positive standard sample of the genetically modified maize strain Mon810 can be fully substituted by the constructed standard plasmid molecule; and the plasmid molecule is used for quantitative PCR analysis and detection of the genetically modified maize strain Mon810 sample.