239 results about "Degenerate primer" patented technology

The invention relates to a respiratory tract common pathogen multiple PT-PCR combined gene chip detection kit. The kit detects one or more of 22 respiratory tract common pathogens by the usage of multiple specific conservative degenerate primer combination and probe combination and provided with endogenous control, positive control and negative control at the same time. According to the detection kit, the coverage rate for a high mutant pathogen subject sequence is increased, the problem of nonspecific cross reaction between the multiple primers and the probe is avoided, one single reaction system can detect more than 20 respiratory tract common pathogens simultaneously, and a detection tool which is simple and sensitive, rapid and large in flux and capable of carrying out multi-index parallel detection is provided for respiratory tract common pathogen detection.

The present invention relates to liquid phase chip method of detecting bird flu virus H5N1 subtype. The liquid phase chip technology includes the homogeny analysis on all the H5 and N1 serum subtype nucleic acid sequence capable of being searched in the nucleic acid sequence bank by means of biological informatic means to find out conservative region, design degenerate primer and specific probe on the H5 and N1 gene fragment and couple the specific probe with fluorescent coding microsphere to make specific detecting microsphere as the liquid phase chip; and recognizing the H5 and N1 gene fragment specifically with the liquid phase chip through two rounds of PCR reaction and reading out the detection result in the chip detection instrument. The method can detect bird flu virus H5N1 subtype quickly for early diagnosis.

The invention relates to a primer, a probe and a method for detecting entomophily or contact transmission pathogens by using a liquid phase chip, which are used for detecting nine clinical common entomophily or contact transmission infectious disease pathogens. The invention can detect the nine clinical common entomophily or contact transmission infectious disease pathogens based on an MASA (multi-analyte suspension array) liquid phase chip technology, homology analysis is performed respectively according to all nucleotide sequences of 9 target viruses which can be retrieved in a gene bank mainly, the degenerate primer and the specific probe are designed, two turns of PCR (polymerase chain reaction) and molecular hybridization are further performed, and a Luminex100 system is further usedfor detection, thereby determining types of the pathogens contained in the sample. The invention is most important for adopting a correct treatment scheme and timely taking measures for preventing disease transmission by detection and early diagnosis of the 9 clinical common entomophily or contact transmission infectious disease pathogens. The invention has the advantages of fast detection speed,simpleness in operation, high sensitivity, good specificity and the like, and is conductive to popularization.

The present invention discloses specific molecular marking process of E chromosome in long spike couch grass with RGA-SCAR primer. The process includes designing degenerate primers in the upstream and downstream of NBS and LRR conservation coding region, PCR amplification to obtain resistant gene analogy segments on different E chromosomes, designing E chromosome RGA-SCAR specifying primers in NBS and LRR conservation region, and determining the different PCR amplification programs and reaction systems for different primers. The present invention makes it possible to identify precisely different chromosomes of diploid long spike couch grass fast, and provides the foundation of auxiliary molecular selection for improving wheat variety with the exogenous gene of long spike couch grass.

The invention discloses a method for amplifying mitochondrial gene total sequences of fishes in Yangtze River based on a degenerate primer combination and application thereof. The primer combination is obtained by designing in a conserved region through comparing the mitochondrial total sequences of fishes of acipenseridae, salangidae, cyprinidae, salmonidae, anguillidae, siluridae, bagridae, cyprinodontidae, hemiramphidae, synbranchidae, serranidae, gobiidae, channidae, bagridae and cobitidae, and the primer combination comprises Mt-1F, Mt-1R; Mt-2F, Mt-2R; Mt-3F, Mt-3R; Mt-4F, Mt-4R; Mt-5F,Mt-5R; Mt-6F, Mt-6R; and Mt-7F, Mt-7R. According to the method, the seven pairs of primers are used for carrying out PCR amplification on corresponding gene fragments, and the mitochondrial gene totalsequences are obtained through connection transformation, sequencing and splicing; the primer combination is less, the amplification effect is good, and the monitored fishes and roe can be effectively classified and identified. According to method provided by the invention, the degenerate primers are designed on the basis of the fish mitochondria conserved region, so that the mitochondrial gene total sequences of various fishes can be obtained; the universality is high, the obtained data can be applied to the research of fish mitochondrial genomes, thus perfecting a mitochondria database.

The invention relates to the technical field of biology, and particularly discloses a canine single-chain antibody, and a construction method and application thereof. By designing the canine antibody variable region degenerate primers, the canine heavy chain variable region VH and light chain variable region VL genes are successfully amplified. On such basis, a bacteriophage display technique is utilized to successfully construct the canine single-chain antibody scFv library. The scFv single-chain antibody library has diversity and enough storage capacity. A Dot-ELISA method is utilized to screen 3 single chain antibodies capable of being combined with the canine DEA 1.1 blood group antigen from the scFv single-chain antibody library by using the canine DEA 1.1 blood group antigen, wherein Clone 16 has stronger combination characteristic. A pET-32a-Clone16 recombinant protein is constructed according to the Clone 16 gene to perform prokaryotic expression, and the purified antibody has combination activity. The canine single-chain antibody lays solid foundation for preparing canine DEA 1.1 blood grouping reagents.

The invention discloses a degenerate primer for detecting virus belonging to the virus family Arenaviruses and a RT-PCR detection method. The degenerate primer can sensitively detect the virus belonging to the virus family Arenaviridae, and is a universal primer which is specific for the virus belonging to the virus family Arenaviridae, and can be used for detecting lymphocytic choriomeningitis virus which is widely distributed, and also can be used for detecting epidemic strains of other Arenaviruses, thereby preventing foreign epidemic strains from affecting our country, and the primer also can be used for detecting unknown viruses which is homologous to the expansion gene, thereby discovering new virus. Because a codehop primer design method is used, the degeneracy of the primer is reduced and the primer annealing temperature is increased simultaneously, thereby improving the singularity of PCR reaction of the designed degenerate primer.

The invention discloses a method for screening veneridae mitochondria COI (cytochrome c oxidase subunit I) gene amplification primers. The method is characterized by comprising the following steps of: a, logging in National Center for Biotechnology Information, and downloading complete mitochondria sequences of veneridae shellfishes; b, comparing and analyzing the downloaded sequences by using BioEdit, and determining a conserved region of the sequences; c, designing a plurality of pairs of primers by using Primer Premier 5 in the conserved region and synthesizing the primers; d, extracting veneridae shellfish DNA, and performing PCR amplification on the synthesized primers under a corresponding reaction system; and 3, performing electrophoresis detection, and screening a group of cocktail primers with single stripe and high amplification efficiency, wherein the group of cocktail primers consists of a pair of degenerate primers and a pair of common primers. The veneridae mitochondria COI primers can effectively amplify destination fragments of different species of the veneridae, and have significance for COI sequence-based veneridae species classification, phylogenesis, systematicgeography research, veneridae germ plasm resource protection and fishery resource management.

The invention relates to a fluorescent quantitative detecting method for CYP2C19 genotyping fluorescent and a diagnostic kit. At present, CYP2C19 genotyping techniques at home and abroad are mainly RFLP technique and ALA technique which determine genotypes according to the size of a DNA fragment, with the disadvantages of time-consuming operation and low flux. The invention provides a novel point mutating or SNP detecting method which utilizes the ASA combination primer sequence designed for the polymorphic loci of the CYP2C19 gene exons 4 and 5, the referential ASA primer combination or the degenerated primer sequence for quality control, specific TaqMan-MGB probe sequences for amplified products, an ASA amplifying reaction method, the amplifying reaction result of real-time fluorescent quantitative detection, the fast analysis of mutation locus type and genotyping. The quantitative detecting method has the advantages of time saving compared with a conventional ASA method, no need for electrophoresis detection, fastness, accuracy, and the like, and can be used for detecting other drug-metabolizing enzymes, or more extensive genetic variation or mutation.

The invention relates to a bovine viral diarrhea virus (BVDV) fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit. According to 21 BVDV-I genome complete sequences, 6 BVDV-II genome complete sequences and 34 CSFV (classical swine fever virus) full-length sequences which are published by GeneBank, one pair of degenerate primers specific to BVDV and one probe are designed in a conserved region of BVDV by utilizing software such as DANMAN and Primer Express3.0. By optimizing reverse transcriptase concentration, DNA (deoxyribonucleic acid) polymerase concentration, upstream and downstream primer concentration and probe concentration, the BVDV fluorescent quantitative RT-PCR detection kit is invented. The BVDV fluorescent quantitative RT-PCR detection kit can specifically detect all the strains of BVDV and has no cross reaction with other pathogens; the BVDV fluorescent quantitative RT-PCR detection kit has good sensitivity which is one order of magnitude higher than that of the conventional RT-PCR; tests on repeatability and stability of 20 quality control samples show that the BVDV fluorescent quantitative RT-PCR detection kit has good repeatability and stability; detection results of 94 clinical samples show that coincidence rate of the method and a BVDV RT-PCR detection kit is 97.9%.

The invention discloses a preparation method and an application of a molecular marker of a rape male sterility restoring gene. The preparation method comprises the following steps of A, designing degenerate primers according to PPR gene conservative amino acid sites, B, extracting a lamina DNA of a rape cytoplasm male sterility restoring line through a cetyltrimethyl ammonium bromide (CTAB) method and magnifying the lamina DNA through a touchdown polymerase chain reaction (PCR) process to obtain a specific restoring gene candidate segment, C, carrying out recovery, conversion, sequencing and sequence analysis processes on PCR products, D, carrying out 5 ' RACE and 3 ' RACE processes and splicing a sequencing result to obtain a full-length cDNA sequence, and E, designing specific primers in 5 ' UTR and 3 ' UTR zones of the full-length cDNA sequence according to the full-length cDNA sequence to obtain a molecular marker. The invention also discloses an application of a restoring gene molecular marker in hybrid rape parent breeding. The preparation method has the advantages of improvements of a speed of restorer seeding and an efficiency of verification, simple and easy method, good operability, rapid identification speed of restoring genes, great reduction of necessary workload of test cross and progeny planting observation in a restorative verification process, and improvement of an efficiency of hybrid seeding.

The invention discloses universal primers for identifying bacillus. The universal primers comprise a forward degenerate primer composed of a nucleotide sequence shown as SEQID NO:1 and a reverse degenerate primer composed of a nucleotide sequence shown as SEQID NO:2. The invention also discloses a method for classifying and identifying bacillus, which comprises the following steps: utilizing the universal primers to perform PCR (Polymerase Chain Reaction) amplification; sequencing the PCR amplification product; utilizing sequence comparison software to perform sequence comparison in a Genbank data base; and acquiring the variety of a known strain having the highest sequence similarity, namely the variety of the strain. All varieties of bacillus can be classified by the universal primers, and the classification result is accurate and reliable; and the universal primers can be used for classifying bacillus on a variety level and can be also used for classifying bacillus on a subvariety level. Besides, the classification and identification method is simple and has a high identification speed.

The invention discloses a novel chitinase gene of nematode bio-control fungi lilac paecilomyces with a gene sequence table as seen in SEQ ID NO: 1, and meanwhile discloses a method for cloning the chitinase gene of the lilac paecilomyces. The method comprises the steps of using lilac paecilomyces RNA with highest enzyme activity as a template, designing and expanding a pair of degenerate primers to get the chitinase gene fragments of the lilac paecilomyces, obtaining gene 5' terminal and 3' terminal sequences from a lilac paecilomyces cDNA chain through SMART RACE RT-PCR technology, and connecting the three sequences to obtain a novel cDNA full-length sequence of the chitinase gene (PL Chil). The invention provides important technological base for transforming the chitinase gene of the lilac paecilomyces to other nematode bio-control fungi by using bioengineering technology, realizing the heterologous expression of the gene, and improving the expression quantity of the chitinase gene of the nematode bio-control fungi and the bio-control ability of plant nematodes.

The invention provides a multi-PCR (Polymerase Chain Reaction) primer design method based on Primer 3. The method comprises the following steps: S1, acquiring an original sequence of a target DNA (Deoxyribonucleic Acid) sequence; S2, performing PCR primer design on the target sequence by using Primer 3, and generating candidate primers; S3, evaluating a multi-PCR primer by using a PE (Polyethylene) model or an SE model, and screening a qualified multi-PCR primer; and S4, changing primer screening parameters, and designing and screening target DNA sequences that no primer is designed, thereby finally obtaining multi-PCR primers of all target DNA sequences. By adopting the method, target sequences in a relatively close distance can be recognized, degenerate primers can be also designed, andfurthermore non-specific amplification caused by mutual interference of primers of target sequences in a relatively short distance is reduced; the specificity of the designed primers is systematicallyevaluated, amplification failure caused by factors such as non-specific amplification, primer dimer and hairpin structures can be reduced, and multi-PCR primers with relatively high specificity are designed for multi-PCR experiments.

The invention provides a kit for realizing multiple amplification of 21 short tandem repeats by adopting degenerate primers, and belongs to a multiple amplification system for simultaneously analyzing a plurality of STR gene loci. 21 gene loci are amplified in a multiple amplification system simultaneously, and are divided into the following four groups: the first group: D19S433, TH01, D21S11, D18S51 and Penta E; the second group: D3S1358, D13S317, D7S820, D5S818, CSF1PO and Penta D; the third group: Amelogenin, vWA, D8S1179, TPOX, D6S1043 and D1S1656; and the fourth group: D16S539, D12S391, D2S1338 and FGA. Specific primers are respectively designed on flanks of a gene loci repetitive sequence, so that the balance of the peaks of the fragments inside the gene loci groups achieves 40% or above, the four gene loci groups are subjected to multiple amplification, and the concentration of the primer of each gene locus is regulated, so that the integral balance of the peaks of the gene loci achieves 30% or above. With the kit, a balanced and stable multiple amplification result is easily obtained, so that the kit belongs to a multiple amplification system with low cost and high efficiency.

The invention discloses an amino acid sequence of beta-mannase gene and coding making method in the biological domain, which is characterized by the following: extracting Armillariella tabescens main RNA of high activity by konjaku flour evoking; using consanguinity sequence of Genebank to design degenerate primer; carrying on PCR expanding to protectorate by primer; designing gene specificity primer to carry on 3'RACE and 5'RACE; cloning total long cDNA of beta-mannase gene; constructing eucaryon expressing carrier with pichia; building-up yeast gene engineering bacterial strain.

The invention discloses a method for detecting lactobacilli in food, which comprises the following steps: (1) extracting the genome DNA (deoxyribonucleic acid) of bacteria in food; (2) performing PCR (polymerase chain reaction) amplification, wherein the used primer pair is a degenerate primer pair; (3) performing electrophoresis detection on the PCR amplification product, wherein if a unique PCR amplification band is detected, the detection result is positive, otherwise the detection result is negative; if the detection result is positive, step (4) does not need to be performed; and if the detection result is negative, the step (4) needs to be performed; and (4) optionally sequencing the PCR amplification product. The invention also provides a primer and the like used in the method. According to the method disclosed by the invention, products of various manufacturers, containing single or multiple lactobacilli, can be directly detected, and the detection result can effectively eliminate false positive, thereby ensuring that the method is convenient to popularize and implement; and sequencing operation can be further performed on the product of which the detection result is negative, thereby ensuring the correctness of the detection result.

The invention relates to a diarrhea-related pathogen detection kit combining multiple RT-PCR with a gene chip. According to the kit, a multiple-specificity conserved degenerate primer composition and a probe composition are adopted to detect one or more of 26 diarrhea-related pathogens, and at the same time, endogenous control, positive control and negative control are arranged. By the adoption of the diarrhea-related pathogen detection kit combining the multiple RT-PCR with the gene chip, the coverage rate of target sequences of high-mutation pathogens is increased, the problem of non-specificity cross reaction between multiple primers and probes is avoided, the purpose can be achieved that a single reaction system synchronously detects more than 20 kinds of diarrhea-related pathogens, and a detection tool which is simple, sensitive, rapid, large in throughput and capable of multi-index parallel detection is provided for diarrhea-related pathogen detection.

The invention provides a genetic engineering bacterium capable of promoting the synthesis of antibiotic medermycin and a method for producing the medermycin by using the same. The method comprises the steps that by taking deoxyribonucleic acid (DNA) of a genome of Streptomyces nashvillensis AM-7161 as a formwork and utilizing degenerate primers to conduct polymerase chain reaction (PCR) amplification, a new complete sequence of genes of ribosome recycling factors (RRFs) of the Streptomyces is obtained; the genes are downstream placed on an efficient promoter PtipA of the Streptomyces, so that a plasmid pHSL56.2 capable of efficently expressed in the Streptomyces is constructed; the pHSL56.2 is led into the host cell Streptomyces AM71-61, and then an engineering bacterium AM71-61/pHSL56.2 (with a preservation number of CCTCCNo:M2012093) is obtained; and when the engineering bacterium AM71-61/pHSL56.2 is used to conduct solid fermentation and liquid fermentation, the accumulation of the antibiotic medermycin can be effectively promoted. The efficient expression plasmid pHSL56.2 of the genetic engineering bacterium can also be directly led into a Streptomyces strain cell for producing other antibiotics of a benzoisochromanequinones family or other aromatic polyketide antibiotics, so as to obtain corresponding antibiotic high-yield engineering bacteria. By utilizing the method, the antibiotic high-yield bacteria can be genetically bred, so that the synthetic capability of the antibiotics is enhanced.

The invention discloses a degenerate primer which consists of a forward primer and a negative primer. The nucleotide sequence of the forward primer is 5'-GTITGYGTKGAYGAYTTYAAYAA-3', and that of the negative primer is 5'-GTRTGBCKYTCIGTRTYYTC-3', wherein Y=C/T, K=G/T, R=A/G, B=G/T/C, and I=hypoxanthine. The invention further discloses a method of detecting Y potyvirus virus of potato by using the degenerate primer. The method comprises the following steps: by taking total RNA (Ribonucleic Acid) of a sample as a template, carrying out Touch-down RT-PCR reaction by means of the primer; after reaction, detecting an amplifying product by gel electrophoresis; judging that the sample contains Y potyvirus virus of potato when an electrophoretic band exists in a position about 1700bp. The primer disclosed by the invention is good in universality and quick and accurate in detection method.

The invention relates to an internal reference gene capable of stable expression in different tissues of Sogatella furcifera, and a screening method and application thereof, belonging to the technical field of biology. The nucleotide sequence of the internal reference gene RPL9 is disclosed as SEQ ID NO:7. The method comprises the following steps: designing degenerate primers according to other insect related gene sequences disclosed in NCBI, carrying out PCR (polymerase chain reaction) to obtain 6 common internal reference gene partial nucleotide sequences of Sogatella furcifera, and carrying out cloning, sequencing and NCBI database Blast comparison to determine the partial sequences of the Sogatella furcifera internal reference gene; and designing specific quantitative primers on the basis of the partial sequences, establishing an RT-qPCR (reverse transcription-quantitative polymerase chain reaction) process based on an SYBR Green I dye technique, and screening out the internal reference gene RPL9 capable of stable expression in different tissue parts of Sogatella furcifera by using a fluorescent quantitative PCR technique. The internal reference gene lays solid foundation for researching gene expression level of different tissue parts and gene functions of Sogatella furcifera in future.

The invention provides a method for the cloning of a pleurotus djamor HP1 laccase gene and the dye decoloration of recombinase. According to the invention, a pair of degenerate primers is designed according to a conserved region of several white-rot fungi laccase gene copper ion binding site amino acids, a laccase cDNA gene core segment is amplified through RT-PCR, and finally a laccase cDNA full-length sequence is amplified by a 3'/5' RACE technology. The pleurotus djamor cDNA is connected with an expression vector pPICZB to construct a recombinant plasmid pPICZB/lac, and the pichiapastoris SMD1168H is converted by an electric conversion method to obtain an engineering strain. The purified laccase is obtained by DEAE-sepharose CL-6B ion exchange column chromatography, polyethylene glycol concentration and SephadexG-75 gel filtration chromatography. The unique degradation ability of the purified laccase against alizarin red can be applied to the wastewater treatment after dye pollution.

The invention relates to an RT-LAMP detection kit and a detection method of swine influenza virus (SIV). According to different subtypes of SIV gene sequences published by a GenBank, 6 SIV RT-LAMP degenerate primers are designed aiming at a relative conserved domain of a PA sequence, and an RT-LAMP method for detecting all the subtype SIV is established by taking H1, H3, H5 and H9 subtype strains of the swine influenza as templates. An LA-320 LAMP Tubidimeter is employed to analyze the reaction process and judge the results; the reaction at a temperature of 63 DEG C for 45min is displayed in the LA-320 LAMP Tubidimeter; RNAs of four subtypes of SIV samples used in all the tests are all subjected to high efficient specific amplification. Sensitivity tests prove that the method can still effectively amplify the RNAs of SIV samples after carrying out 10<-4> dilution on the RNAs, thus showing high sensitivity of the method; through specific tests and RT-LAMP detection of SIV pathogeny of 19 clinical samples, SIV nucleic acid in 5 samples is detected to be positive, which is identical with the results of RT-PCR and isolation of chicken eggs; typing and identification of gene chips verify that the accordance rate of the comprehensive result is 100%. The method is specific, sensitive and quick and is suitable for SIV detection under various test conditions.

The invention discloses a primer, a probe and a method for detecting a respiratory infectious disease pathogen by using a liquid chip, which are used for detecting nine clinically common respiratory infectious disease pathogens. In the invention, the nine clinically common respiratory infectious disease pathogens are detected by a multi-analyte suspension array (MASA) liquid chip technology and are subjected to homology analysis mainly according to all nucleotide sequences of nine targeted viruses which can be searched in a gen bank; a degenerate primer and a specific probe are designed; and a polymerase chain reaction (PRC) and molecular hybridization are performed for two times, and then a Luminex 100 system is used for detection, so that the types of the pathogens in a sample are determined. The detection and early diagnosis of the nine clinically common respiratory infectious disease pathogens provided by the invention are extremely significant in the aspect of preventing diseasesfrom propagating by correct treatment schemes and timely responding measures. The primer, the probe and the method have the advantages of high detection speed, high sensitivity, high specificity and the like, are easy to operate and are suitable for large-scale popularization and application.

The invention discloses primers for screening and parting enterovirus viruses and a detection method. The sequences of the primers are the CODEHOP outer primer sequence, the CODEHOP inner primer sequence and the primer sequences of 20 viruses. The detection method includes the steps of extraction of RNA; one-step RT-PCR; nest type PCR; 1.5% agarose gel electrophoresis to judge the positivity and parting result of enteroviruses. A pair of enterovirus consistent-degenerate primers is designed, a pair of short-fragment consistent-degenerate primers and parting primers of 20 different enteroviruses are designed inside amplified fragments of the primers, and in combination with the nest type RT-PCR technology, a degenerate primer nest type RT-PCR method capable of being used for screening and parting different genotype viruses of the enteroviruses is established. The advantages of being high in sensitivity and specificity and suitable for monitoring and variety identifying of enteroviruseses of low-virus-content samples are achieved.

The invention relates to the fields of agricultural biotechnical engineering and wheat disease resistance genetic breeding, and in particular relates to a molecular marker interlocking with a wheat stripe rust resistant gene, an acquisition method and application thereof. The marker interlocking with the wheat stripe rust resistant gene is obtained through amplification by degenerate primers: an upstream primer 5-'AAGTGGAACAAGGTTACG-3', and a downstream primer 5'-GGIGGIGTIGGIAAIACIAC-3'. The marker can be used to assist selective breeding of wheat stripe rust resistance, and overcome the shortcomings in wheat stripe rust resistance identification in conventional breeding, such as vulnerability to environment impact, poor stability and repeatability, heavy workload, time and labor consumption and long breeding cycle, so as to speed up the breeding process of a wheat stripe rust resistance variety.

The invention relates to clone and recombination of a cotton mitogen activated protein kinase gene as well as salt resistance function analysis and application thereof, belonging to the fields of molecular biology and biotechnology. In the invention, total RNA is extracted from primary leaves of the cotton, and reverse transcription is carried out to obtain obtains cDNA. A degenerate primer is designed according to a conservative amino acid sequence of the mitogen activated protein kinase gene in the other plants to carry out a conventional polymerase chain reaction, a PCR product is connected with a pMD18-T carrier to transform a colibacillus DH5 alpha competent cell, a recombinant is selected to carry out sequence analysis, and the full-length cDNA is obtained through a rapid amplification technology of 3'end and 5' end. A plant positive expression vector is further constructed to transform the tobacco cultivated variety NC89, the transgenic tobacco has the obvious salt resistance capacity so as to transform the gene into crops such as cottons, wheat, corns, and the like, thereby improving the salt resistance capacity of the crops and increasing the yield and the quality of the crops, and having important economy value and social value.