765 results about "Reverse transcription polymerase chain reaction" patented technology

The present inventors have shown that the gene and protein expression profiles of ADAM8, ADAM10 and ADAM12 in different grades and stages of bladder cancer.

ADAM12 gene expression was evaluated in tumors from 96 patients with bladder cancer using a customized Affymetrix GeneChip. Gene expression in bladder cancer was validated using reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR, and in situ hybridization. Protein expression was evaluated by immunohistochemical staining on tissue arrays of bladder cancers.

The presence and relative amount of ADAM12 in the urine of cancer patients were determined by Western blotting and densitometric measurements, respectively.

Particularly ADAM12 mRNA expression was significantly upregulated in bladder cancer, as determined by microarray analysis, and the level of ADAM12 mRNA correlated with disease stage. ADAM12 protein expression correlated with tumor stage and grade. ADAM12 was present in higher levels in the urine from bladder cancer patients than in urine from healthy individuals. Significantly, following removal of tumor by surgery, in most bladder cancer cases examined the level of ADAM12 in the urine decreased and, upon recurrence of tumor, increased.

The present invention provides methods using a gene expression profiling analysis (1) to determine whether a human sample is a tumor using a gene set containing nucleic acid sequences of SEQ ID NOS: 1-7, 8-17 or 1-17; (2) to identify whether a tumor tissue is an adenocarcinoma (using a gene set containing nucleic acid sequences of SEQ ID NOS: 15, and 18-21) or a squamous cell carcinoma (using a gene set containing nucleic acid sequences of SEQ ID NOS: 22-27); and (3) to predict the prognosis of survival and metastasis in humans with tumor (using a gene set containing nucleic acid sequences of SEQ ID NOS:19, and 28-42 or SEQ ID NOS: 19, 29, 31, 40, and 42), particularly for those humans who are at the early stage of lung cancer. The gene expression profiling is preferably performed by cDNA microarray-based techniques and/or Real-Time Reverse Transcription-Polymerase Chain Reaction (Real-Time RT-PCR), and analyzed by statistical means.

The entire process of reverse transcription-polymerase chain reaction (RT-PCR) is simplified by using oligonucleotide-immobilized microplates made of, e.g., polypropylene, to which oligonucleotides are securely immobilized and which can be subjected to thermal cycles of PCR. RT-PCR is preferably conducted in solid-phase. Capturing of mRNA and RT-PCR can be conducted in the same plates. The cDNA synthesized from the mRNA captured on the microplates can be used more than once. Further, in combination with the microplates, a filter plate is used for the preparation of cell lysates wherein target cells are placed on the filter plate, and a lysis buffer is passed through the cell layer on the filter to transfer cell lysate directly to the microplate via well-to-well communication.

The invention discloses root tip detoxification and rapid propagation technology for purple potatoes and belongs to the technical field of plant detoxification and tissue culture rapid propagation. The method comprises the following steps of: (1) preparing a culture medium; (2) performing root tip detoxification and rapid propagation; and (3) performing multi-reverse transcription-polymerase chain reaction (RT-PCR) synchronous and rapid detection on complex virus infection of a purple potato test tube plantlet or a test tube mini-tuber and the like. In the technology, each level of culture medium is adjusted according to the characteristic of the purple potato and conventional stem tip detoxification is changed into root tip detoxification so as to simplify operation process, avoid high pollution rate and improve working efficiency. Moreover, the technology has the advantages of good inoculant differentiation, high test tube plantlet propagation speed, complete detoxification, high mini-tuber growth vigor, monthly mini-tuber reproduction rate of 4 to 6 times and mini-tuber rooting rate and transplanting survival rate of over 98 percent. The technology can be popularized in regions which are suitable for the growth of purple potatoes.

The invention provides a three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as a kit thereof. The method can rapidly and accurately detect the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of enterovirus in a sample. The method comprises the following steps of: (1) acquiring and conveying a sample of an infected patient or a suspected patient; (2) preprocessing the sample and extracting RNA; (3) detecting the sample by adopting a one-step PCR-three-color fluorescent probe in-vitro amplification method; and (4) analyzing the corresponding sample according to the fluorescence intensity of each amplification reaction after the amplification reaction is finished, thereby judging the existence of the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of the enterovirus in the acquired sample and being capable of carrying out accurate quantitation (a figure 3) on the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of the enterovirus. The invention realizes the aim of carrying out rapid and accurate combined detection of the enterovirus 71, the Coxsackie virus A16 and the other subtypes of nucleic acids of the enterovirus.

The invention provides a dual fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) specific primer for detecting type 1 and type 3 duck hepatitis A viruses. A dual fluorescence quantitative method comprises the following steps: comparing sequences of DHAV-1 and DHAV-3 published on GenBank, and designing a pair of specific detection primers SEQ1 and SEQ2 aiming at DHAV-1 and a Taqman probe (probe1) as well as a pair of specific detection primers SEQ3 and SEQ4 aiming at DHAV-3 and a Taqman probe (probe2) respectively in a region with conservation and relatively large difference between gene sequences of the two viruses; confirming the concentration of the dual fluorescence quantitative RT-PCR specific primer and the Taqman probe aiming at DHAV-1 and DHAV-3. The dual fluorescence quantitative method built by virtue of the group of primers is good in specificity and high in sensitivity, and can be used for quick serum type identification and real-time quantitative analysis of the type 1 and type 3 duck hepatitis A viruses.

The invention relates to the functional genomic and gene expression detection technology and the analysis technology, and discloses a reagent kit for quantitatively assessing long-term recurrence risks of breast cancer. Particularly, a type of genes capable of being used for breast cancer metastasis and prognostic molecular classification is screened within a human functional genome expression profile range, the detection technology is created, and the reagent kit is prepared and applied to breast cancer metastasis and prognostic assessment for a patient. The reagent kit for quantitatively assessing long-term recurrence risks of breast cancer comprises 21 pairs of primers, 21 specific taqman fluorescent probes, 10XRT-PCR (reverse transcription-polymerase chain reaction) buffer solution, 2.5mM of dNTP (diethyl-nitrophenyl thiophosphate) mixed liquor, reverse transcriptase, DNA (deoxyribose nucleic acid) polymerase, 10XPCR buffer solution and RNA (ribonucleic acid) enzyme inhibitor. The reverse transcription PCR technology is combined with the taqman fluorescent quantitative PCR technology, reverse transcription primers, real-time PCR primers and the taqman fluorescent probes are self-designed and optimized, reverse transcription PCR reagent and taqman fluorescent quantitative PCR reagent are integrated to prepare the detection reagent kit, operation is simple and fast, detection results are more stable, and detection cost is lower.

A method is provided for reverse transcription-polymerase chain reaction (RT-PCR) comprising a) amplifying a reverse transcribed cDNA in a mixture comprising Norovirus Genogroup I and Norovirus Genogroup II primers and probes, wherein said Norovirus primers and probes distinguish between Genogroup I and Genogroup II viruses; b) quantifying virus; and c) normalizing data based on a universal internal RNA control. Optionally, the method may also include primers and probes for Enteroviruses. A reaction mixture comprising Norovirus Genogroup I and Norovirus Genogroup II primers and probes, wherein said Norovirus primers and probes distinguish between Genogroup I and Genogroup II viruses and universal internal RNA control primers and probes are also included.

The invention relates to a solidification examination reagent box of one-step real-time fluorescence quantitative reverse transcription polymerase chain reaction for Citrus Tristeza Virus and a method of utilizing the reagent box to detect Citrus Tristeza Virus, which is a fluorescence quantitative detection method and product of especially establishing for Citrus Tristeza Virus. The detection time only need 4 hours and the method has the high specificity, the high sensitivity and the stability and reliability. The method overcomes the shortcomings of the high erroneous recognition and the low sensitivity of serological method for Citrus Tristeza Virus of the conventional diagnosis method according to symptomatology, is suitable for the rapid diagnosis of the viral existence of Citrus seedling and viruliferous insect sector, and can be used in quality inspection of citrus seedling and identification of Citrus Tristeza Virus.

This invention relates to a reagent container for detection and/or quantitation of at least one biological or chemical analyte from a sample, said reagent container comprising an inner surface enclosing a volume, wherein volume analytical reactions of at least one analysis for detection and/or quantitation of at least one analyte take place, and at least two reagents of an analysis of an analyte have been dried onto said inner surface and at least a first said reagent has been dried onto a first area of the inner surface distinctly separate, i.e. without any overlap, from a second area of the inner surface onto which at least a second said reagent has been dried. Characteristic for the reagent container is that the first reagent and the second reagent form a pair wherein the first reagent is an enzyme and the second reagent is a substrate of said enzyme, and said pair consists of a nucleic acid polymerase and substrate thereof. This invention also relates to a method for stabilising onto an inner surface of a reagent container dried assay reagents for the detection and/or quantitation of a biological or chemical analyte from a sample, said method comprising the steps of dispensing at least two reagents, a first reagent and a second reagent, needed in the detection of the analyte onto the inner surface of the reagent container; and removing excess water from the reagents, wherein first reagent is dispensed onto a first area of the inner surface distinctly separate, i.e. without any overlap, from a second area of the inner surface onto which the second reagent is dispensed. Characteristic for the method is that the first reagent and the second reagent form a pair wherein the first reagent is an enzyme and the second reagent is a substrate of said enzyme, and said pair consist of a nucleic acid polymerase and substrate thereof. This invention further relates to the use of the reagent container to perform an assay selected from the group consisting of a polymerase chain reaction (PCR) assay, an assay that utilizes a reverse transcriptase, a reverse transriptase polymerase chain reaction, an immuno-PCR assay, a nucleic acid sequence based assay (NASBA), a proximity ligation assay, a ligase chain reaction (LCR) assay, a rolling circle amplification (RCA) assay, and a strand displacement amplification (SDA) assay.

The invention relates to a high-precision nucleic acid quantitative detection kit for hepatitis C virus (HCV) applied to the field of biomedical clinical diagnosis. The kit comprises a nucleic acid extraction kit based on a paramagnetic particle method and an HCV nucleic acid amplification box, wherein the nucleic acid extraction kit based on the paramagnetic particle method comprises a splitting combining liquid, a rinsing liquid A, a rinsing liquid B, a rinsing liquid C, an eluting liquid and a magnetic bead liquid. The HCV nucleic acid amplification box comprises an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) reaction liquid, enzyme mixed liquor, an HCV-interior label, an HCV quantitative reference 1-4, a negative quality control product, a critical positive quality control product and a positive quality control product. The splitting combining liquid in the nucleic acid extraction agent based on the paramagnetic particle method comprises 0.5-2.5% of lauryl sodium sulfate, 0.5-2.0ml/100ml of TritonX-100, 2-6mol/L of guanidinium isothiocyanate and 1-10mM of EDTA (Ethylene Diamine Tetraacetic Acid) (PH 7.5). The kit provided by the invention is simple and fast to operate, low in cost, high in coverage of primer and probe genotype, high in conservative property, strong in specificity, high in detection sensitivity and good in repeatability, and can better simulate the extraction state of real viruses.

The invention discloses a method for obtaining sogatella furcifera carrying SRBSDV (southern rice black-streaked dwarf virus) and an application sogatella furcifera. The method for obtaining the sogatella furcifera carrying the SRBSDV, comprises the following steps of: feeding the sogatella furcifera by utilizing rice infecting the SRBSDV or cryopreserved rice diseased plant so that the sogatella furcifera obtains the SRBSDV, transferring the infected sogatella furcifera to a healthy rice to be fed till passing through a circulation period of virus, and detecting the sogatella furcifera by utilizing an RT-PCR (reverse transcription-polymerase chain reaction) technology and a serology method to obtain the sogatella furcifera carrying the SRBSDV, wherein the carrier rate of the sogatella furcifera can reach more than 50% through a manual feeding manner based on the experiment. Through utilizing the method for obtaining the sogatella furcifera carrying the SRBSDV, the obtained sogatella furcifera carrying the SRBSDV can be applied to screening of SRBSDV-resisting monoclonal antibody to carry out a rice inoculation experiment to identify the resistance of the rice, screen disease-resistant variety and provide a service of the breeding for disease resistance. The invention can be further applied to researching mutual relation between the SRBSDV and a vector insect sogatella furcifera and provides powerful theoretical evidence for prevention and treatment on the viruses.

The invention relates to a method for detecting miRNA (micro Ribose Nucleic Acid) by an improved stem-lop primer qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction). The method comprises the steps: (1), designing a miRNA detection primer, wherein a stem-loop primer comprises two parts of a general stem loop sequence and a specific primer sequence which is in complementary pairing with a 3' end of a target miRNA, a base number which is in complementary pairing with the target miRNA is 11bp, a downstream primer needle of qPCR is specific to a general stem loop sequence, an upstream primer needle is specific to a 5' end of the target miRNA, a GC tail is added in the 5' end of the upstream primer so that the annealing temperatures of the upstream primer and the downstream primer are close; (2), performing inverse transcription on the target miRNA into cDNA (complementary Desoxvribose Nucleic Acid); and (3), performing a quantitative PCR amplification and detecting by adopting an SYBR Green qPCR detection method. The method disclosed by the invention is good in specificity, and high in sensitivity; and one downstream primer is only synthesized, and an SYBR Green I fluorochrome method is adopted, and thus the cost is greatly saved.

The invention discloses a triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for African swine fever viruses, swine fever viruses and respiratory syndrome viruses and a preparation method and application thereof. Three sets of specific primers and Taqman probes as well as positive controls specific to African swine fever virus CP530R genes, swine fever virus 5 minute-UTR genes and swine respiratory syndrome virus NSP2 genes respectively are designed and synthesized, and a rapid, easy and convenient triple fluorescent RT-PCR detection system with high specificity and high sensitivity is established by using the three sets of primers and probes, so that nucleic acids of the African swine fever viruses, the swine fever viruses and the respiratory syndrome viruses can be detected synchronously from a detected sample within 3-4 hours in a rapid, accurate, specific, safe, easy and convenient way. The detection reagent can be applied to synchronous detection of the nucleic acids of trace African swine fever viruses, swine fever viruses and respiratory syndrome viruses in hogs and relevant samples thereof.

The invention discloses a citrus fruit fly odorant binding protein-based attractant screening method belonging to the technical field of bioengineering. The citrus fruit fly odorant binding protein-based attractant screening method comprises the following steps of: collecting the total RNA (Ribonucleic Acid) of antennae of a citrus fruit fly; obtaining the overall length of a citrus fruit fly odorant binding protein through RT-PCR (Reverse Transcription-Polymerase Chain Reaction); constructing a prokaryotic expression vector of the citrus fruit fly odorant binding protein; inducing the expression of a citrus fruit fly recombinant odorant binding protein through IPTG (isopropyl-beta-d-thiogalactoside), and purifying the citrus fruit fly recombinant odorant binding protein through a nickel sepharose gel affinity column; obtaining a conjugation reaction spectrum of the citrus fruit fly recombinant odorant binding protein and a host fruit odor volatile matter through a competitive fluorescence combing method, wherein a dissociation constant (KD) is lower than below 10 mu mol/L host fruit smell; and the IC 50 value of fluorescence competition is less than 30 mu mol/L, determining a host fruit smell attractant suitable for the citrus fruit fly. The invention provides a new strategy for screening and designing a citrus fruit fly odorant host fruit smell odor information attractant formula.

The invention discloses a method for detecting an FMDV (Foot and Mouth Disease Virus) group and O type and AsiaI type thereof through real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction). By carrying out specificity and sensitivity verification on the method established by the invention, the result shows that the FMDV can be well set apart from other Artiodactyla infectious viruses as well as O type and AsiaI type, and the sensitivity can be up to 10 copies of a template. The method established by the invention is characterized in that the whole reaction process is a closed pipe operation, thereby maximally reducing the pollution to the system. The whole process sequentially including the acquisition of a sample to be detected, the extraction of nucleic acid, the preparation of a fluorescent quantitative RT-PCR system and the representation of a reaction result can be completed within 2.5 hours, and the time required for detection is greatly shortened on the premise of ensuring the detection result.

The invention discloses an efficient virus-induced phytoene desaturase gene silence system for Chinese pink. A specific primer is designed according to a coding gene sequence of PDS (phytoene desaturase), and about 500bp cDNA (complementary deoxyribonucleic acid) fragments are amplified in the Chinese pink through RT-PCR (reverse transcription-polymerase chain reaction). Obtained PDS cDNA fragments are connected to TRV2, and acetosyringone concentration, seedling age and preculture temperature after infection as well as light conditions for plants after preculture are integrated in an assorted manner by optimizing an agrobacterium-mediated virus-induced gene silence system according to photobleaching frequency, bleached leaf area and bleached conditions of the plants, so that the rapid efficient virus-induced silence system is established, virus-induced gene silence character can be obtained, and further, the problem that Chinese pink gene functions are verified effectively is solved.

The invention discloses recombinant oral protein TAT-GH of tilapia, a preparation method for the recombinant oral protein TAT-GH and application of the recombinant oral protein TAT-GH. The method comprises the following steps of: extracting total messenger ribonucleic acid (mRNA) from a pituitary gland of male tilapia, performing reverse transcription-polymerase chain reaction (RT-PCR) to obtain complementary deoxyribonucleic acid (cDNA), performing PCR amplification to obtain a growth hormone (GH) gene of the tilapia, and amplifying a TAT-GH gene by a PCR overlap extension method; constructing a recombinant plasmid pET-32a(+)-TAT-GH; and transforming Escherichia coli BL21 (DE3) by using the recombinant plasmid pET-32a(+)-TAT-GH, efficiently expressing fusion protein TAT-GH in the form of an inclusion body under the induction of isopropyl thiogalactoside (IPTG), and purifying and renaturing to obtain active protein. The protein can effectively promote the growth and development of fries of the tilapia after being fed to the fries of the tilapia.

The invention discloses quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and a method capable of simultaneously detecting multiple chili viruses. The method adopts four pairs of primers disclosed as SEQ ID NO:1-SEQ ID NO:8; and one RT-PCR process can simultaneously detect four chili viruses: Broad bean wilt virus 2 (BBWV 2), Pepper vein yellows virus (PeVYV), Chilli veinal mottle virus (ChiVMV) and Pepper mildmottle virus (PMMoV). Compared with the existing single RT-PCR and duplex RT-PCR detection methods, the method disclosed by the invention can simultaneously detect the four chili viruses by one RT-PCR process, and is capable of detecting more virus varieties, greatly enhancing the detection efficiency and obviously lowering the detection cost.

The invention relates to a primer and a method for detecting GI type and GII type norovirus by utilizing the primer, belonging to the technical field of biological detection. The sequence of the primer is as follows: Nov-F: 5-GTGAATGAWGATGGCGTCKA-3, Nov-R: 5-GTHAGWATCCAGGGGTCAAT-3. The invention focuses on the design of the sequence of the primer, the composition of an RT-PCR (reverse transcription-polymerase chain reaction) system, the selection of reaction conditions and the judgment of reaction results can be performed according to the conventional methods in the field. By adopting the primer and the method provided by the invention, a pair of primers can simultaneously detect and differentiate the GI type and the GII type norovirus in a same reaction tube, thereby not only simplifying the operation steps, but also shortening the detection time.

The invention provides a method for detecting swine epidemic diarrhea by reverse transcription-loop-mediated isothermal amplification. According to the conserved domain gene of PEDV (Porcine Epidemic Diarrhea Virus) N protein, three pairs of primers aiming at six areas on a target gene are designed, two RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primers are combined, and PED (Porcine Epidemic Diarrhea) viruses are identified by the RT-LAMP (Reverse Transcription-Loop-Mediated Isothermal Amplification) technology; RT-LAMP reacts; reverse transcription is performed on obtained RNA(Ribose Nucleic Acid); and the obtained cDNA is used for PCR reaction. The method provided by the invention ensures amplification specificity, has high amplification speed and can obtain a result within 30-60 minutes, and people can observe amplification effect with eyes without electrophoresis. Reverse transcription and nucleic acid amplification can be realized in 1 hour at the constant temperature of 65 DEG C by using enzyme mixture; RT-LAMP detection is carried out on the basis of LAMD amplification DNA; a reverse transcription enzyme is added to realize amplified detection of RNA; and reverse transcription and amplification are finished in one step so as to omit the reverse transcription step which is firstly carried out by the traditional RT-PCR.

The invention provides a pseudovirion containing hepatitis c virus (HCV) RNA (Ribonucleic Acid) fragment and preparation method thereof which are applied in the field of biomedical clinical vitro diagnostic. The pseudovirion is a RNA-protein complexes that MS2 bacteriophage capsid protein wraps the hepatitis c virus, and the complexes is icosahedral; the method comprises the following steps of designing and synthesizing primer to obtain target gene MS2 by overlapping and splicing PCR (Polymerase Chain Reaction) method; connecting the target gene MS2 to plasmid pET-32a (+) to obtain recombinant plasmid pET-MS2; connecting the recombinant plasmid pET-MS2 and the HCV fragment to obtain pET-MS2-HCV recombinant plasmid; the obtained pET-MS2-HCV recombinant plasmid is introduced into escherichia coli for prokaryotic expression; releasing virus-like particles by adopting ultrasonication and the obtained virus-like particles are the pseudovirion containing the hepatitis c virus RNA fragment. The pseudovirion preparation method provided by the invention has the advantages that the preparation method is simple, the operation is easy, the obtained pseudovirus is high in purity, and the pseudovirion can be used as standard and quality control material of detection of RT-PCR (Reverse Transcription-Polymerase Chain Reaction), is good in stability, has no infectivity, has RNase-resistant, and the like.