57 results about "Single copy" patented technology

The invention belongs to the technical field of living cell imaging, and discloses a living cell nucleic acid imaging system and method based on CRISPR (clustered regularly interspaced short palindromic repeats) and a nano antibody and application, the system comprises: a modified dCas protein expression vector containing n antigen polypeptide tags; the modified sgRNA and / or crRNA expression vector comprises a specific guide sequence; the fluorescent nano-antibody expression vector contains m fluorescent nano-antibody coding sequences which are connected in an operable manner, and fluorescent protein serving as an internal tag is inserted between nano-antibody splitting sites, can be combined with a polypeptide tag carried by dCas protein and can stably emit light; and the fluorescent nano antibody in a free state can be self-degraded, so that the fluorescent nano antibody does not emit fluorescence. According to the invention, the single-copy nucleic acid sequence, especially the imaging marker of the single-copy nucleic acid sequence in a living cell, can be visualized with single-molecule resolution and high signal-to-noise ratio.

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.

The invention relates to the technical field of biology, and particularly discloses a kit for detecting haemophilus influenzae based on PCR (Polymerase Chain Reaction) / RAA-CRISPR-Cas13a. The invention discloses a kit for detecting a haemophilus influenzae gene on the basis of PCR (Polymerase Chain Reaction) / RAA-CRISPR-Cas13a. The kit comprises a primer pair for specifically amplifying haemophilus influenzae genes and crRNA, the sequence of the crRNA is composed of an anchoring sequence used for being combined with Cas13a protein and a guide sequence of a target sequence of the haemophilus influenzae genes, and the guide sequence is shown as the 39-66th site of SEQ ID No.28. The invention constructs a method for detecting haemophilus influenzae genes by combining a PCR or RAA technology with a CRISPR-Cas13a technology, the method is simple, convenient and rapid, has higher sensitivity and specificity, can reach a single copy level in sensitivity, and can be used for clinically and rapidly identifying whether the haemophilus influenzae is infected or not.

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 provides application of an OG7443 gene of trichophyton in identification of trichophyton, a primer pair and an identification method of trichophyton, and relates to the technical field of biology. Based on comparative analysis of genomes of trichophyton and other fungi, the specific marker gene OG7443 which only exists in trichophyton, has single copy and is free of repetitive sequence interference is screened out, and the gene can be used for identification of trichophyton. The primer provided by the invention is designed aiming at the OG7443 gene of trichophyton, has strong specificity and high sensitivity, can accurately distinguish mixed florae possibly existing in a clinical sample, improves the diagnosis accuracy of fungal infection, not only can accelerate the determination of pathogenic bacterium types, but also can improve the diagnosis accuracy of fungal infection. And important technical support can be provided for targeted antifungal treatment and public health prevention and control.

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 application discloses a multiplex method for measuring nucleic acid modification enzyme activity and screening one or more variable elements of enzymatic reaction.The method includes: (i) providing a polynucleotide construct library encoding DNA / RNA target site and variable element to be tested such as nucleic acid modification enzyme variant, (ii) partitioning a single copy of the polynucleotide construct with in vitro transcription and translation (IVVT) reagent, (iii) allowing the IVVT reaction of nucleic acid modification enzyme and DNA / RNA target site to be expressed in each compartment, (iv) depending on the functionality of the nucleic acid modification enzyme encoded, single polynucleotide construct / RNA target is cracked, remains intact or otherwise modified, and (v) by sequencing to cracked, complete or modified polynucleotide construct / RNA target quantification, so as to directly identify the enzymatic activity related to each variable element and to the enzymatic activity quantification.Particularly, nucleic acid modification enzyme is CRISPR associated protein (Cas).

The invention is directed to means and methods for efficient and safe genetic manipulation of a target cell, wherein a knock-out / knock in strategy is performed via HDR in a target gene that is haploinsufficient (and / or in single copy) in the cell.

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 an oligonucleotide-coated fluorescence in-situ hybridization probe for specifically recognizing a tartary buckwheat Chr1-4 chromosome and application, and belongs to the field of cytological markers. The method comprises the following steps: screening a single-copy oligonucleotide sequence of Chr1-4 from a tartary buckwheat whole genome by using Chorus2 software; filtering and removing repetitive sequences to obtain a Chr1-4 chromosome probe sequence library; screening a specific single copy oligonucleotide sequence of the Chr1-4 chromosome; and adding specific primers at the two ends of each single copy oligonucleotide sequence of Chr1-4, and combining the probes into a buckwheat Chr1-4 Oligo-Painting probe mixing pool. By utilizing the constructed Oligo-Painting probe mixing pool and a fluorescence in-situ hybridization technology, accurate identification and karyotype analysis of the buckwheat chromosomes can be realized, the number, morphology, heritable variation and other structural characteristics of the chromosomes can be known, and an important technical support is provided for cytological analysis of buckwheat crossbreeding.

The invention belongs to the technical field of biological detection, and discloses a nucleic acid detection kit, which comprises the following components: a nucleic acid pre-amplification mixed solution, target capture magnetic beads, a multiple PCR reaction solution, a CRISPR-SERS detection solution and an endogenous control system. The nucleic acid detection kit integrates a nucleic acid pre-amplification mixed solution, target capture magnetic beads, a multiple PCR reaction solution, a CRISPR-SERS detection solution and an endogenous control system, and all the components work cooperatively to achieve whole process optimization. The target capture magnetic beads specifically enrich pathogen nucleic acid through a biotin-streptavidin system, so that host background interference is effectively removed; multiple PCR reaction liquid adopts DPO primer design, non-specific amplification is inhibited through a blocking region, and efficient amplification of a target gene is ensured. Finally, the CRISPR-SERS detection liquid activates trans-cleavage of reporter molecules by using cis-cleavage activity of the Cas12a protein, signal amplification is realized by combining with an SERS technology, so that the detection sensitivity reaches a single copy level, and meanwhile, an endogenous control system can monitor the quality of a sample in real time and avoid a false negative result.

The invention discloses a KASP molecular marker for detecting a rice blast resistance gene Pi65 and application of the KASP molecular marker. The KASP molecular marker comprises SNP1 and / or SNP2; the SNP1 is located at the 28376936bp position of the 11th chromosome of a Nipponbare MSUv7 reference genome, and the polymorphism of the SNP1 is T or C; the SNP2 is located at the 28377512bp position of the 11th chromosome of a Nipponbare MSUv7 reference genome, and the polymorphism of the SNP2 is A or C. The KASP molecular marker provided by the invention has the characteristics of high typing quality, single copy and high polymorphism, can quickly realize genotype identification of the rice blast resistance gene Pi65 at high throughput and low cost, is used for marker-assisted breeding for rice blast resistance breeding improvement, and has wide application universality.

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 provides an expression cassette for reducing lentivirus integration risk and application and a product thereof, and belongs to the technical field of gene engineering. According to the invention, further optimization is carried out based on the prior art, reverse assembly is carried out on an expression cassette, and optimization selection of a polyA sequence finds that the vector disclosed by the invention can improve the gene expression quantity of a single copy and realize the same expression effect with a relatively low lentivirus integration copy number, so that the risk caused by virus integration is further reduced.

The invention provides a sheep-derived component rapid quantitative detection method based on dual real-time fluorescent PCR. According to the method, a single-copy nuclear gene GAPDH is taken as a target spot, a sheep specific primer and a probe are designed in a hypervariable region of the single-copy nuclear gene GAPDH, and an internal reference primer and a probe capable of identifying various common animal-derived components are designed in a conserved region of the single-copy nuclear gene GAPDH. As the target and the internal reference are located in the same gene and coexist in the same cell according to the proportion of 1: 1, quantitative deviation caused by gene copy number difference or expression fluctuation in a traditional two-step method is remarkably reduced. The detection strategy constructed by the invention has double advantages of specific recognition and broad-spectrum reference, is clear in quantitative basis and simple and convenient to operate, is suitable for rapid recognition and quantitative adulteration analysis of sheep-derived components in meat products, and has wide application prospects and supervision values.

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 invention provides a transdermal peptide-epidermal growth factor fusion protein and a preparation method thereof, and belongs to the technical field of biology. According to the present invention, the transdermal peptide TDP1 is fused at the N-terminal of the EGF, such that the EGF transdermal efficiency is significantly improved on the premise of no influence on the EGF biological activity, and the preparation method comprises the following steps: S1, preparing the recombinant plant expression vector plasmid containing the fusion protein gene; s2, preparing a bacterial suspension; S3, carrying out dip dyeing and culturing; s4, obtaining a high-expression homozygous single-copy positive clone strain; and S5, protein extraction. The fusion protein is prepared by utilizing an arabidopsis system, so that the potential sensitization of the EGF is reduced, and the safety of the EGF in use is improved. The problems of low transdermal rate and use safety of exogenous EGF transdermal drug delivery are solved, the pain caused by microneedle transdermal drug delivery of a traditional product is avoided, and the application prospect of EGF in the field of skin care is greatly improved.

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 belongs to the field of biological breeding, and particularly discloses a method for cultivating an insect-resistant and glyphosate herbicide-resistant cotton variety, which comprises the following steps of: hybridizing or backcrossing by taking transgenic cotton GBS8-2 or a derivative line thereof as one of parents and taking a cotton variety with excellent agronomic traits as the other parent for 4-5 generations continuously; a new cotton variety with good insect resistance and high glyphosate herbicide concentration can be bred; wherein the preservation number of the GBS8-2 is CGMCC (China General Microbiological Culture Collection Center) No. 46278. The invention also provides a molecular marker for identifying the GBS8-2 and a filial generation. The insect-resistant gene and the glyphosate-resistant gene in the transgenic cotton GBS8-2 are single insertion sites and single copies, are simple in heredity and are easy to apply in breeding; secondly, the insect-resistant gene provided by the invention is a fusion gene, and compared with a single resistance gene, the insect-resistant gene provided by the invention has the advantage that insects are not easy to generate resistance. In addition, the molecular marker is accurate and reliable and can be used for auxiliary selection in the seedling stage, and the breeding cost is reduced.

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.