35 results about "Single copy" patented technology

The invention discloses a system for detecting viral nucleic acid on the basis of a CRISPR (clustered regularly spaced short palindromic repeat) technology and an FET (field effect transistor) chip and application of the system, and relates to a method for detecting the viral nucleic acid on the basis of clustered CRISPR (clustered regularly spaced short palindromic repeat) and related proteins (Cas) and a field effect transistor (FET). The CRISPR-FET (clustered regularly interspaced short palindromic repeats-field effect transistor) detection system comprises a non-amplified CRISPR system, a reporter RNA (ribonucleic acid) and gold nanoparticles (AuNPs) connection system, a separation system and a field effect transistor chip detection system. The CRISPR-FET detection method provided by the invention has high sensitivity and high specificity on virus nucleic acid detection, and the lower limit of detection reaches a single copy (100 copies / [mu] L).

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to sheep fecundity and application of the SNP molecular marker. The SNP molecular marker comprises an SNP1 (Single Nucleotide Polymorphism) and / or an SNP2; the SNP1 is located at a base at the 115747587 site of a chromosome 6 of a sheep reference genome AR-UIRamV2.0 version, and the polymorphism of the SNP1 is T / G; the SNP2 is located at a basic group at the 41419566th site of a chromosome 22 of a sheep reference genome AR-UIRamV2.0 version, and the polymorphism of the SNP2 is A / G. According to the scheme, the SNP molecular marker related to sheep fecundity is provided, sheep with the high fecundity character can be efficiently and accurately identified and screened out, and the marker is high in typing quality, single in copy and high in polymorphism and can be used for sheep fecundity molecular marker assisted breeding.

This invention specifically relates to a de novo 7-DHC-synthesizing *Saccharomyces cerevisiae* genetically engineered strain, its construction method, and its applications, belonging to the field of biotechnology. Using *Saccharomyces cerevisiae* engineered strain SC9 as the starting strain, this invention utilizes multiple copies of tHMG1, DHCR24, and ERG1 via the TY transposon, multiple copies of ERG2, POS5, ERG7, ERG11, IDI1, and ERG3 via the TY transposon, and single copies of POS5, ERG3, and DHCR24 in the genome to increase the intracellular content of reducing coenzyme II and enhance the main pathway of 7-DHC synthesis. Single copies of ACL, ACS1, ERG10, and ERG19 in the genome enhance the utilization of ethanol in *Saccharomyces cerevisiae* and dynamically regulate the synthesis of 7-DHC. The *Saccharomyces cerevisiae* genetically engineered strain constructed by this invention exhibits high product accumulation, laying the foundation for replacing traditional multi-step chemical synthesis routes and realizing industrial production.

The invention discloses a probe, a method for efficiently recognizing licorice chromosomes and application of the probe, and belongs to the field of bioinformatics and molecular cytogenetics. According to the invention, a single copy oligonucleotide sequence of No. 1-8 chromosomes of liquorice is screened by using bioinformatics, and a chromosome specific oligonucleotide probe pool is constructed through an artificially designed universal primer sequence tag splicing strategy. Through multiple rounds of FISH technology, the chromosomes 1-8 of liquorice can be efficiently and accurately identified. The method provided by the invention solves the problem of difficulty in accurate identification caused by tiny morphology and high similarity of the glycyrrhiza chromosome, has the advantages of high resolution, strong specificity, capability of identifying a single chromosome and the like, and provides a powerful tool for follow-up glycyrrhiza chromosome karyotype analysis, evolution research and exploration of a species formation mechanism.

The invention provides a primer combination for identifying a single-copy gene of Australian cyst nematode. The primer combination comprises an upstream outer primer HAUS-F3, a downstream outer primer HAUS-B3, an upstream inner primer HAUS-FIP, a downstream inner primer HAUS-BIP and a downstream loop primer HAUS-LB. The primer combination can specifically distinguish the Australian cyst nematode from other common cyst nematodes such as cereal cyst nematode, Philipse cyst nematode, corn cyst nematode and soybean cyst nematode, the detection limit is low, the detection time is short, and the detection result is identified by means of dye color development or electrophoretogram. The technology is easy to operate, good in sensitivity, high in specificity and high in reaction speed, the result can be directly observed without additional instruments, and the method has important significance in the aspects of rapid detection and early field diagnosis of the Australian cyst nematode.

The invention relates to a plasmid vector capable of easily detecting the copy number of plasmids and a detection kit thereof. According to the plasmid vector, a plasmid replicon ORI gene and a single copy conserved gene of an escherichia coli genome are constructed on the same plasmid. The detection kit contains the plasmid vector or a single-copy quality control product prepared from the plasmid vector. The method has the advantages of high detection result accuracy, high reproducibility and simple detection process, and effectively solves the related problems existing in the traditional escherichia coli plasmid copy number detection method.

The invention relates to the field of genetic engineering and bioengineering, in particular to engineering algae capable of synthesizing alpha-farnesene and a preparation method of the engineering algae. An MEP metabolic network of synechocystis PCC 6803 is modified, through single-copy series recombination and multi-copy combination of dxs, ispA, idi and AFS genes, optimization of a farnesene MEP metabolic pathway is achieved, the carbon flux of oriented synthesis of farnesene is precisely regulated and controlled, under the optimal effect, the yield of alpha-farnesene within 9 days is 83.07 mg / L (25.82 mg / g DCW), and economic application value is achieved.

The invention relates to the technical field of microbiological detection, in particular to a detection method based on specificity of PCR (Polymerase Chain Reaction) and CRISPR-Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Cas12a) for bifidobacterium and application. The method comprises the following steps: amplifying a common single copy core gene of bifidobacterium by adopting a polymerase chain reaction (PCR) technology to obtain an amplification product; and an amplification product is detected by adopting a clustered regular interval short palindromic repeat related protein (CRISPR-Cas12a) system. The detection method is simple and convenient to operate, only comprises two parts of PCR amplification reaction and Crispr reaction, and the evaluation method of the detection result is simple, short in overall time consumption, high in efficiency, capable of detecting various bifidobacteria and definitely free of specificity to eight non-bifidobacteria, and wide in application range.

The invention discloses a rapid detection method for echinococcus granulosus based on RPA-CRISPR / Cas, and belongs to the technical field of molecular diagnosis. The method comprises the following steps: extracting DNA of a sample to be detected; the specific primers are used for RPA isothermal amplification, a reaction system contains a buffer solution, upstream and downstream primers, RPA enzyme, magnesium acetate and template DNA, and reaction is carried out at 39 DEG C for 20 minutes; and mixing the amplification product with LbCas12a protein, sgRNA and a fluorescence report probe, carrying out CRISPR / Cas reaction at 37 DEG C for 30 minutes, and judging the result through a real-time fluorescence signal. Constant-temperature rapid amplification is achieved through RPA, high-sensitivity and high-specificity detection of the echinococcus granulosus can be achieved under the site condition by combining high-specificity recognition and trans-cleavage activity of CRISPR / Cas12a, the sensitivity reaches the single-site copy number, operation is easy and convenient, complex temperature control equipment is not needed, and the method is suitable for primary medical treatment and site quarantine.

The present invention relates to a method for the relative quantification of GM crops using digital PCR without using certified reference materials (CRMs). More specifically, the present invention relates to a primer and probe set composition for digital PCR for the relative quantification of transgenes in GM crops without using CRMs, which comprises a first primer and probe set capable of specifically detecting genes present in a single copy or in 2-3 copies within the genome of GM (genetically modified) crops; and a second primer and probe set capable of specifically detecting transgenes in GM crops, and uses thereof.

The invention relates to the technical field of bioengineering, in particular to a high-yield Ishwarane saccharomyces cerevisiae engineering strain construction method based on non-targeted transcriptomic analysis, saccharomyces cerevisiae is used as a host strain, and non-targeted transcriptomic sequencing is performed on the saccharomyces cerevisiae growing in a basic culture medium Ori and an optimized culture medium ZR1 respectively to obtain a high-yield Ishwarane saccharomyces cerevisiae engineering strain. The differential genes are analyzed and screened, the genes with the transcriptional level higher than that of the basic culture medium Ori in the optimized culture medium ZR1 are concluded as functional genes, and the genes with the transcriptional level lower than that of the basic culture medium Ori in the optimized culture medium ZR1 are concluded as competitive genes; carrying out single-copy integration and overexpression on the functional gene; carrying out accurate knockout on the competitive gene; the saccharomyces cerevisiae engineering strain with high yield of Ishwarane is constructed by the steps; and the continuous, efficient and safe production of the Ishwarane is realized.

The invention belongs to the technical field of environmental biological safety monitoring, and particularly relates to an RPA-CRISPR method for rapidly detecting pathogens in air. According to the invention, a two-step detection strategy integrating RPA isothermal amplification and CRISPR detection is established; the method specifically comprises the following steps: firstly, acquiring genome DNA of an air sample, and carrying out specific amplification on a trace target by utilizing an RPA technology; then, a product is introduced into a CRISPR-Cas12a system, and the trans-shear activity is recognized and activated by utilizing the specificity of crRNA, so that signal cascade amplification is realized. Pseudomonas aeruginosa is used as a model pathogen, and a primer and probe combination is designed aiming at a specific gene of the pseudomonas aeruginosa. Results show that the method can complete detection within 10-15 minutes at the soonest, the lowest detection limit can reach a single copy level (100 copies / L), and the method has the advantages of high environmental interference resistance, high sensitivity and no need of large instruments, and can meet the on-site rapid screening requirements of air pathogen aerosol in public places, medical environments and other scenes.

The invention discloses a mustard SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof. The mustard SNP molecular marker combination is composed of 1694 SNP molecular markers, and the physical positions of the 1694 SNP molecular markers are determined based on sequence alignment of a mustard reference genome Bju.gene. According to the scheme, the mustard SNP molecular marker combination marker is a co-dominant marker, and has the characteristics of high quality, single copy, high polymorphism, high specificity, high sensitivity and high resolution; the marking is not influenced by environmental conditions, seeds or any type of plant tissues can be used, the detection result is accurate, and the repeatability and the stability are good; different detection laboratories and different data results can be compared and verified, and the data has universal comparability, can be accurately used for background detection of mustard breeding materials, and has wide application universality.

The invention discloses a HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus) and TP (total protein) hypersensitive detection kit and detection method based on a digital PCR (polymerase chain reaction) platform. The kit comprises a primer and a probe, the design of the primer and the probe is optimized, and the molecular detection system has higher sensitivity by utilizing high affinity and strong mismatch recognition capability of the LNA probe, so that a single copy can be detected. The method possibly has huge potential in the aspects of large-scale screening and clinical diagnosis of blood donors and epidemiological research of pathogens.

The application discloses a crRNA target point and a CRISPR-Cas13a system for detecting pigeon paramyxovirus. The CRISPR-Cas13a system comprises a Cas13a protein and a crRNA, or a complex formed by the two; the crRNA comprises an anchor sequence for being combined with the Cas13a protein and a guide sequence for targeting a pigeon paramyxovirus target point sequence; and the pigeon paramyxovirus target point sequence is sequence 1. Experiments prove that the crRNA can realize high-sensitivity and high-specificity detection on the nucleic acid of the pigeon paramyxovirus by activating the Cas13a, and the sensitivity reaches a single copy (1 copy / µL).

The invention provides a gene chip for identifying bolete species and application of the gene chip, and belongs to the technical field of bolete identification. According to species with published reference genomes, Chinese saprophytic bolete genomes are combined as test objects, and on the basis of selecting single-copy orthologous genes, the gene chip for identifying bolete species is obtained. Further selecting a conserved segment in the gene sequence, and detecting the SNP difference between the bolete species; according to the present invention, the gene chip for identifying the species of the bolete family is designed, the gene chip comprises 11075 SNP sites, and the gene chip can efficiently and accurately identify the species of the bolete family; the topological structure of the evolutionary tree constructed based on the SNP sites of the gene chip is consistent with that of the evolutionary tree constructed based on single-copy orthologous genes, and the branch support rate is reliable. The universal gene chip for the bolete family developed by the invention has important significance on classification and identification of species of the bolete family.

The invention discloses a herbicide-resistant alfalfa transformation event TWMS2-17 and a detection method thereof, an exogenous DNA fragment is inserted into an alfalfa genome, and herbicide-resistant plants TWMS2-17 are obtained through herbicide spraying treatment and single-copy plant screening. Through analysis and verification of a flanking sequence of a T-DNA insertion site, the position of an exogenous gene inserted into a genome is a No.3 chromosome, a 270bp genome sequence is replaced, and after asexual cuttage planting, excellent glyphosate and glufosinate resistance is shown, and normal growth is realized. The DNA molecule of the transformation event and the detection method have remarkable application value on breeding of herbicide-resistant alfalfa.

The application discloses a primer pair and a probe for detecting duck liomia morbid bacteria, the nucleotide sequence of the primer pair is shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3. The application finds that the conserved site of duck liomia morbid bacteria is located on the ggt gene by analyzing the whole genome sequence of 399 duck liomia morbid bacteria, and the primer and probe sequence designed on the ggt gene are detected in specificity and sensitivity, which shows that the primer and probe sequence can achieve the detection sensitivity of single copy, and the specificity is strong, and the primer and probe sequence is the optimal primer and probe design. The primer pair and the probe can be used to quantitatively detect duck liomia morbid bacteria more quickly and more specifically.

The application belongs to the technical field of live cell imaging, and discloses a live cell nucleic acid imaging system, method and use based on CRISPR and nanobody, which comprises: a modified dCas9 protein expression vector containing n ALFA polypeptide tags; a modified sgRNA expression vector comprising a specific guide sequence; and a fluorescent nanobody expression vector containing a bivalent or trivalent fluorescent nanobody coding sequence, which are operatively connected, wherein a fluorescent protein is inserted between nanobody split sites as an internal tag, can be combined with the ALFA polypeptide tag carried by the dCas9 protein and stably emit light, and the free state fluorescent nanobody is self-degraded and does not emit light. The application can visualize single copy nucleic acid sequences with single molecule resolution and high signal-to-noise ratio, especially the imaging marking of single copy nucleic acid sequences in live cells.

The invention belongs to the technical field of molecular biology and medical detection, and discloses a universal trypanosomes detection primer pair and a digital PCR detection method. According to the invention, two pairs of high-specificity universal primers are designed based on highly conserved 18S rRNA sequences among trypanosomes, and the sequences are shown as SEQ ID NO: 1-2 or SEQ ID NO: 12-13. Meanwhile, the invention provides an optimized digital PCR detection method, and the detection sensitivity and specificity are remarkably improved by accurately optimizing key parameters such as annealing temperature and primer concentration. Experiments prove that the system can stably detect a whole blood simulation sample with the trypanosomes content as low as 5 / ml or a trypanosomes sample with the DNA content as low as 0.01 pg (single reaction system), and the detection limit can reach a single copy level. The invention provides a powerful technical tool for early diagnosis, precise medication guidance and epidemiological monitoring of trypanosomiasis.

The invention discloses a double-gating detection method for nucleic acid sequence analysis and a detection kit thereof, and belongs to the technical field of molecular diagnosis. The invention provides a double-gating detection method integrating nano-enzyme visual preliminary screening and double-strand specific endonuclease auxiliary verification. A preliminary screening module can detect nucleic acid as low as a single copy level; the verification module utilizes the characteristic that DSN only cuts completely complementary double chains, can distinguish single base mismatch, and ensures extremely high specificity of detection. According to the double-gating detection method, rapid screening and category identification detection of nucleic acid sequences can be realized, synchronous differential diagnosis of multiple pathogens in a single tube can be carried out, and a technical solution with great prospects is provided for on-site diagnosis and treatment and rapid and accurate diagnosis of the pathogens in an environment with limited resources.

The invention belongs to the technical field of agricultural biology, and particularly relates to a cotton multi-character synchronous improved molecular pyramiding breeding method which comprises the following steps: constructing an artificial haplotype construct containing minimum functional units of an insect-resistant gene Bt and a fiber quality gene GhPEL, and introducing an FRT sequence at a preset safe port site of a chromosome 5 by utilizing CRISPR / Cas9, so as to obtain a molecular pyramiding breeding sequence; and then the screening marker is cut off through agrobacterium transformation and an FLP / FRT system, so that double-gene orientation, no linkage burdens and single-copy integration are realized. According to the method, a homozygous strain with stable inheritance can be obtained without backcross, and multi-character efficient superposition and modular breeding are supported.

The present invention provides a novel method that is specifically designed to statistically start from a single copy of a nucleic acid molecule and requires only a single encapsulation step to provide a clonal microcompartment library that represents a high concentration of encoded polypeptides. The method of the present invention makes it possible to perform in vitro uHTS by one-step encapsulation of linear DNA constructs containing candidate sequences to be tested in a molecular mixture, allowing for simultaneous specific gene amplification and protein expression.

The invention provides a qualitative and quantitative detection method for deer-derived components and a corresponding kit. According to the qualitative detection method and the kit, universal primers and probes of sika deer and red deer are designed by taking a mitochondrial gene as a target aiming at the sika deer and red deer with the most medicinal value, and the probes are high in specificity and have no cross reaction with other common meat. The method for identifying the deer-derived components with high sensitivity is established, and true and false identification of the deer-derived components in the functional food is realized. The detection method is accurate and efficient, the total detection time can be controlled within 2 hours, and the sensitivity reaches the pictogram level. According to the quantitative detection method and the kit, quadruple digital PCR primers and probes are designed by taking a single copy gene as a target aiming at four components of sika deer, red deer, reindeer and muntiacus criniacus, and the four sets of primers and probes are good in specificity and free of cross reaction. According to the method, four sibling species of cervidae can be identified simultaneously, the content of target deer species in a mixed sample can be measured and calculated, deer-derived component adulteration discrimination is realized, and the limit of quantitation can reach 0.1%.

The invention belongs to the technical field of gene engineering, and relates to a method for efficiently integrating a target gene into a pichia pastoris genome in a multi-copy manner, which comprises the following steps: S1, constructing a pPIC9K recombinant plasmid containing a single-copy target gene or a pPICZ alpha Amut + recombinant plasmid containing a multi-copy target gene; s2, constructing and integrating a pichia pastoris strain containing the multi-copy target gene by utilizing the pPIC9K recombinant plasmid containing the single-copy target gene or the pPICZ alpha Amut + recombinant plasmid containing the multi-copy target gene; and S3, electrically transforming the recombinant plasmid constructed in the S1 into the pichia pastoris strain integrated with the multi-copy target gene constructed in the S2, and carrying out resistance screening. According to the invention, the contradiction between copy number and screening pressure in the traditional gene integration technology is broken through, and an engineering bacterium construction scheme with higher economical efficiency and stability is provided for industrial production of recombinant protein with high expression quantity.

The application discloses a SNP molecular marker for identifying the gender of sheep and application thereof. The SNP molecular marker comprises one of SNP1, SNP2, SNP3, SNP4 and SNP5. The application provides a SNP molecular marker for identifying the gender of sheep, and the SNP molecular marker has high typing quality, single copy and high sample detection rate, and can be used for molecular marker assisted breeding of the gender of sheep. The SNP molecular marker can realize early gender typing identification of sheep embryos in a quick, high-throughput and low-cost manner, and the gender identification can be completed before embryo transplantation, so that pregnancy, delivery and feeding consumption of non-target embryos can be effectively avoided, and the breeding cycle can be directly shortened, and the selection efficiency is improved.

The invention provides a detection reagent and a kit for detecting type 1 bovine viral diarrhea virus and type A bovine rotavirus based on a dual RT-ddPCR technology and application, and belongs to the field of molecular biology, the detection reagent comprises: (a) a first primer pair and a first probe for specific amplification of a type 1 bovine viral diarrhea virus target gene; (b) a second primer pair and a second probe for specifically amplifying the A-type bovine rotavirus target gene; (c) reverse transcriptase, DNA (deoxyribonucleic acid) polymerase and ddPCR reaction premix liquid for the dual RT-ddPCR amplification; according to the invention, the absolute quantitative detection of BVDV-1 and BRV-A in a sample can be realized through dual RT-ddPCR in combination with dual-channel fluorescence detection and Poisson distribution analysis; the sensitivity of the method can reach a single copy and is improved by more than 10 times compared with the traditional qPCR, and the method has no non-specific reaction to other common pathogens.

The invention discloses a rapid detection method for echinococcus multilocularis based on RPA-CRISPR / Cas, and belongs to the technical field of molecular diagnosis. The method comprises the following steps: extracting DNA of a sample to be detected; the specific primers are used for RPA isothermal amplification, a reaction system comprises a buffer solution, upstream and downstream primers, RPA enzyme, magnesium acetate and template DNA, and reaction is carried out at 39 DEG C for 20 minutes; and mixing the amplification product with Cas12a protein, sgRNA and a fluorescence report probe, carrying out CRISPR / Cas reaction at 37 DEG C for 30 minutes, and judging the result through a real-time fluorescence signal. Constant-temperature rapid amplification is achieved through RPA, high-sensitivity and high-specificity detection of echinococcus multilocularis can be achieved under the site condition by combining high-specificity recognition and cleavage activity of CRISPR / Cas12a, the sensitivity reaches the single-site copy number, operation is easy and convenient, complex instruments are not needed, and the method is suitable for primary medical treatment and site quarantine.

The application provides a CYP21A2 gene mutation detection method, system and storage medium, comprising the following steps: identifying difference sites and homologous intervals by reference genome self alignment, and constructing a paralog characteristic site fingerprint library; constructing a Masker reference genome according to the homologous intervals on the reference genome, and obtaining a candidate variation list; using the physical link information of a double-end sequencing read pair and the characteristic site fingerprint library to determine the true or false gene source of each candidate variation; quantitatively calculating the true gene copy number based on re-alignment sequencing depth, background reference library and characteristic site allele frequency; and determining whether a recombination event occurs between the true gene and the false gene based on the physical link information of the double-end sequencing read pair across multiple characteristic sites. The application effectively distinguishes different copy states such as gene deletion, single copy, normal double copy and duplication, and assists in clinically determining gene deletion or duplication events.

The invention discloses a one-tube method mycoplasma rapid detection primer combination based on RPA-CRISPR / Cas12a and application of the one-tube method mycoplasma rapid detection primer combination, and relates to the technical field of mycoplasma detection. The invention provides a product for rapidly detecting mycoplasma by a one-tube method. The product comprises an RPA (recombinase polymerase amplification) reaction system and a CRISPR (clustered regularly interspaced short palindromic repeats) gel system which are positioned in the same reaction tube and are layered up and down, the RPA reaction system comprises a forward primer Myco-RPA-F and a reverse primer Myco-RPA-R, and the reverse primer Myco-RPA-F is a positive primer; the CRISPR gel system comprises a Cas12a enzyme, gRNA (guide ribonucleic acid) and a fluorescence labeling probe. According to the product provided by the invention, the whole-course single-tube closed reaction is realized, aerosol pollution can be avoided, and the detection reliability is improved; the total duration from sample processing to result output is less than or equal to 1h, no thermal cycle equipment is needed, and the detection efficiency is higher; the lower limit of detection reaches single copy nucleic acid, and a mycoplasma conserved sequence is targeted through gRNA, so that no cross reaction with bacteria and fungi exists. In practical application, a user only needs to add a sample and incubate at constant temperature to directly detect samples such as a cell suspension, a supernatant and the like, and a conventional nucleic acid extraction step is not needed.