149 results about "Fluorescent reporter" patented technology

The invention discloses a rapid detection method for fusarium wilt in corn based on an RPA-CRISPR / Cas12b technology, and belongs to the technical field of nucleic acid detection. The invention provides a specific reagent combination, a kit and a detection method in order to solve the problems of high amplicon pollution risk, complicated operation and the like in the existing technologies such as RPA (recombinase polymerase amplification). The reagent combination comprises an RPA (recombinase polymerase amplification) primer pair with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, and sgRNA (single guide ribonucleic acid) with a sequence as shown in SEQ ID NO.3. The method comprises the following steps: carrying out RPA amplification by taking nucleic acid of a sample to be detected as a template, carrying out mixed incubation on an amplification product, sgRNA, Cas12b protein and fluorescent reporter molecules, and judging a result by detecting a fluorescent signal. The optimized RPA and CRISPR systems are integrated on the centrifugal micro-fluidic chip, so that the full-flow closed tube and automatic detection is realized, the detection can be completed within 30 minutes, the detection sensitivity reaches 1E2 copies / T, the risk of aerosol pollution is greatly reduced, and the centrifugal micro-fluidic chip is suitable for port quarantine and field on-site rapid detection.

The invention discloses a Cb Ago-based TdT detection reagent and a Cb Ago-based TdT detection method. The TdT detection reagent comprises a TdT extension reaction system and a TdT detection system, wherein the TdT extension reaction system comprises a TdT reaction buffer solution, deoxythymidine triphosphate, cobalt ions and an extension reaction detection probe; the Cb Ago system comprises Cb Ago, a Cb Ago buffer solution and a fluorescence report probe. The extension reaction detection probe disclosed by the invention can extend under the action of TdT, and then is in specific complementary pairing with the fluorescent reporter probe to activate Cb Ago to cut the fluorescent reporter probe, so that TdT detection is realized. The high specificity of the TdT extension reaction and the signal amplification capacity of Cb Ago are combined, and the method has the advantages of being easy and convenient to operate, high in sensitivity, low in cost, suitable for on-site detection and the like and is expected to become a powerful tool for detecting TdT.

The invention relates to the technical field of molecular biology and pathogenic microorganism detection, and discloses a Klebsiella pneumoniae and Escherichia coli synchronous detection method based on RPA-CRISPR / Cas12a and application, and the method comprises the following steps: extracting DNA from a sample, carrying out isothermal amplification by adopting an RPA primer designed aiming at a Klebsiella pneumoniae rcsA gene and an Escherichia coli uriA gene, and carrying out amplification by adopting an RPA primer designed aiming at a Klebsiella pneumoniae RcsA gene and an Escherichia coli uriA gene; a detection system containing Cas12a enzyme, crRNA and a fluorescence report substrate is constructed, and a result is interpreted through a fluorescence or lateral flow chromatography test strip. Detection can be completed within 70 minutes at the constant temperature of 37 DEG C, the sensitivity reaches 5 * 10 copies / mu L, the specificity is high, no cross reaction exists, the kit is suitable for clinical early screening, hospital infection monitoring and on-site POCT application, and an efficient and reliable new tool is provided for pathogen diagnosis.

The invention relates to the technical field of biological medicine, in particular to an RNF / CLR / Cas / crRNA compound as well as a preparation method and application thereof. The RNF / CLR / Cas / crRNA compound disclosed by the invention is composed of an RNA nanoflower (RNF), a circular fluorescent reporter molecule (CLR) and a Cas / crRNA compound, and the circular fluorescent reporter molecule and the Cas / crRNA compound are combined on the RNA nanoflower through base complementary pairing; the Cas / crRNA compound is composed of a Cas protein and crRNA, and the crRNA is used for specifically recognizing the crRNA of a nucleic acid molecule. The RNF / CLR / Cas / crRNA compound can be used for nucleic acid molecule specificity detection and nucleic acid molecule base mutation detection, can also be used for fluorescence in situ hybridization tissue imaging, and has a good application prospect.

The invention provides a compound for releasing a urine fluorescent biomarker under induction of mitochondrial DNA mutation as well as a preparation method and application of the compound, and belongs to the technical field of medicines. The compound disclosed by the invention is prepared by the following steps: complementarily pairing a hybrid nucleic acid nano-carrier containing single-stranded DNA (Deoxyribose Nucleic Acid), an annular fluorescent reporter molecule and a Cas12a / crRNA compound through bases of DNA or RNA, and then coating polyethyleneimine and hyaluronic acid. A CRISPR / Cas12a system is selected as a converter of tumor specific mitochondrial DNA mutation information and urine fluorescence signals, a nano delivery system which responds to mtDNA single base mutation and is used for tumor progress monitoring and early metastasis warning is designed and synthesized, and the nano delivery system has a good application prospect in tumor progress monitoring and early metastasis sensitive detection.

The invention discloses application of novel reverse transcriptase and related fusion protein of the novel reverse transcriptase in plant-guided editing. Candidate RT protein is obtained through systematic mining from a public database, novel reverse transcriptase cl1 with activity is screened out in combination with a fluorescence report system, and a guided editing system suitable for plants is constructed. The system tests in wheat protoplast, and verifies the editing activity of cl1 to a plurality of endogenous targets. Further combining with protein structure prediction and rational design point mutation optimization, the cl1 mutant with higher editing efficiency is obtained, and the method can be applied to accurate and effective editing of plant genomes and plant breeding and improvement.

The invention discloses a method for detecting the biological activity of human immunoglobulin ADCP, which is characterized in that CHO-K1 / SPIKESARS2 is taken as a target cell, THP-1 cells (human leukemia monocytes which naturally express CD32a (Fc gamma RIIa) and CD64a (Fc gamma RIIa) and stably transfect NF-kB-Luc luciferase reporter genes) modified by genetic engineering are taken as effector cells, and a fluorescence reporter gene method is adopted for detection. Based on specific target cells and effector cells, the ADCP biological activity of two different human immune globulin preparations, namely intravenous injection human immune globulin and subcutaneous injection human immune globulin, can be accurately and reliably reflected by a drawn dose-effect curve through a detection system matched with specific human immune globulin solution concentration, and the method is simple and easy to implement. Cost is low, and practical application and popularization value is achieved.

This invention discloses a genetic transformation and gene editing method applicable to multiple sesquiterpene species, belonging to the field of plant genetic transformation and gene editing technology. The method uses the cotyledonary nodes of sterile seedlings of the sesquiterpene genus as explants, and introduces an optimized pScEF1α-Cas9 gene editing transformation vector using Agrobacterium infection. Regenerated plants are obtained through recovery culture, selection culture, and rooting culture. The vector contains four gene expression units: pScEF1α:Cas9, pGmU6:sgRNA, pGmUBI:DsRed2, and pCaMV35S:HygR. In vivo screening is further performed using the DsRed2 fluorescent reporter gene, and the gene-edited plants are identified by molecular detection. This method is applicable to common sesquiterpene, stem-nodled sesquiterpene, and spiny sesquiterpene, and has advantages such as strong species versatility, high transformation efficiency, short cycle, visualized screening, and high positive rate, providing efficient technical support for gene function research and genetic improvement of sesquiterpene species.

This invention provides a method for constructing a fluorescently traced mouse model and its application in the sorting of Mcpt4-positive and negative mast cells. First, the Cre gene is knocked into the Mcpt4 gene sequence of C57BL / 6 mice using CRISPR / Cas9 technology. Then, through animal hybridization and Cre-loxP recombination technology, a fluorescently traced mouse model of Mcpt4-positive mast cells is obtained. The fluorescently traced mouse model possesses a dual-fluorescent reporter gene system consisting of the red fluorescent protein tdTomato and the green fluorescent protein ZsGreen. Mcpt4-positive mast cells exhibit red fluorescence, while other Mcpt4-negative cells exhibit green fluorescence. Using this fluorescently traced mouse model, Mcpt4-positive and negative mast cells can be rapidly, accurately, and effectively sorted, providing a reliable technical basis for exploring the roles and mechanisms of Mcpt4-positive and negative mast cells in immune responses.

In one aspect, a method of treating an NK cell to induce a gene edit. The method may include contacting an NK cell with a transfection composition including poly-L-glutamic acid (y- PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HD AC inhibitor; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I. In another aspect, a method of preparing an engineered NK cell comprising a fluorescent reporter at an endogenous locus. In another aspect, a method of treating an NK cell to introduce an exogenous nucleic acid.

The invention relates to a RAA-CRISPRCas13a method for detecting iridovirus fluorescence of micropterus salmoides, and relates to the technical field of biological detection.The method comprises the following steps that an RAA primer pair and guide crRNA are designed and synthesized based on a virus specific gene sequence; the method comprises the following steps: constructing a single-tube reaction system containing an RAA amplification component, Cas13a protein, crRNA and a fluorescent reporter molecule; after sample nucleic acid is added, constant-temperature incubation is carried out for 15-30 minutes at 37-42 DEG C, an RAA amplification product activates trans-cleavage activity of Cas13a, and a cleavage reporter molecule generates a fluorescence signal; and virus judgment is realized by detecting a fluorescence signal. According to the method, isothermal amplification and CRISPR detection are efficiently integrated through a single-tube reaction system, and the method has the advantages of being easy and convenient to operate, rapid in detection, high in sensitivity and high in specificity and is suitable for on-site high-throughput screening.

The invention discloses a group B streptococcus detection system and method, relates to the technical field of biology, and aims to solve the problem that the existing group B streptococcus detection method cannot give consideration to detection efficiency, sensitivity and specificity at the same time. The detection system comprises a PCR amplification product aiming at a cfb gene specific fragment, crRNA, LbaCas12a and a single-chain DNA fluorescent reporter group, the PCR amplification of the specific fragment of the cfb gene comprises a forward / reverse primer GBS-F / R; wherein the sequence of the crRNA is as shown in one of SEQ ID NO: 1 to SEQ ID NO: 4. When a PCR amplification product of a reaction substrate cfb gene is in a low-concentration condition (10pg), high-sensitivity detection can be realized, and the method has the advantage of low-abundance sample detection.

The application provides a double-stranded walking type DNA nanomachine and a preparation method and application thereof, and relates to the technical field of fluorescent analysis and detection.The double-stranded walking type DNA nanomachine for ctDNA fluorescent detection comprises a gold nanoparticle carrier, an anchor probe, a FAM fluorescent reporter probe, a double-stranded walking arm, a Cas12a protein and a crRNA to form a ribonucleoprotein complex.The walking arm of a conventional CRISPR / Cas12a nanomachine is a DNA single-stranded structure, which is easily cut in trans by activated Cas12a, thereby reducing the stability and sensitivity of the nanomachine.The application introduces a double-stranded DNA structure into the walking arm design to replace the conventional DNA single-stranded walking arm, constructs a double-stranded walking type three-dimensional DNA CRISPR / Cas12a nanomachine, effectively resists the reverse cutting of Cas12a on the walking arm, maintains the structural integrity and functional stability of the nanomachine, and greatly improves the detection sensitivity and specificity of lung cancer ctDNA, thereby having a good clinical application prospect.

This invention discloses a target-gated ratiometric pH sensor based on a dual CRISPR system of tetrahedral framework nucleic acids, belonging to the fields of biomedicine and nucleic acid nanotechnology. The sensor uses tetrahedral framework nucleic acids as a rigid scaffold, precisely assembling Cas12a, Cas13a, and their corresponding fluorescent reporter molecules in a 1:1 stoichiometric ratio. Their orthogonal pH responses form the basis of the ratiometric sensing. Target recognition-pH sensing logic gating is achieved through target-specific crRNA design. Cholesterol modification allows for stable anchoring on the lipid membrane surface. This invention also discloses the sensor's fabrication, membrane anchoring, pH monitoring, and target-gated function implementation methods. This sensor possesses advantages such as high-precision ratio measurement, accurate spatial arrangement, high specificity, stable membrane anchoring, and a wide pH response range, and can be used for precise monitoring of pH dynamics at biomembrane interfaces and during extracellular vesicle fusion.

Presented herein is a sensor platform which can be used for the detection of arbitrary molecules of interest or for clinical diagnosis using test solutions. The sensor may include an array of environmentally sensitive fluorescent reporter probes. Each probe is placed on a separate pixel or several pixels of the sensor array and has a different coating, such that it responds slightly differently to environmental stimuli than every other pixel upon addition of the test solution.

The application discloses a method for synthesizing reverse transcriptase: obtaining a target reverse transcriptase expression sequence based on database primary screening; obtaining a candidate reverse transcriptase expression sequence by using a fluorescent reporter system to rescreen the target reverse transcriptase expression sequence; and determining a synthetic reverse transcriptase expression sequence by using the fluorescent reporter system after optimizing the candidate reverse transcriptase expression sequence. The application further screens a plurality of natural or engineered small reverse transcriptases in view of the bottleneck of the prior art, and verifies the applicability of the reverse transcriptases in prime editing. The technology is expected to break through the application limitation of the existing prime editing system, and provide more efficient and safer precise editing tools for the fields of genetic disease treatment, agricultural breeding and synthetic biology.

A vibrationally enhanced fluorescence microscopy system includes a narrowband pulsed mid-infrared laser configured to excite selected chemical bond vibrations in a sample. A continuous-wave visible laser is coaligned with the mid-infrared laser and configured to induce fluorescence from at least one fluorescent reporter bound to the sample. An external pulse generator temporally synchronizes emission of the mid-infrared pulses with emission of the visible laser. A reflective objective is arranged to deliver the mid-infrared pulses to a focal plane within the sample. A water-immersion objective is coaxially aligned with the reflective objective and arranged to collect the fluorescence from the focal plane. A photon-counting detector is coupled to receive the collected fluorescence. Control electronics are configured to output a vibrational-contrast signal derived from counts produced by the photon-counting detector.

The invention discloses an influenza reporter virus carrying a fluorescent RNA aptamer and a construction method and application thereof, and the influenza reporter virus carrying the fluorescent RNA aptamer is obtained by introducing a termination codon TAA into the 328th nucleotide of an ORF region of an NS1 gene on the basis of a genome of an influenza A virus and terminating in advance, then introducing a terminating codon TAA into the 328th nucleotide of the ORF region of the NS1 gene of the influenza A virus; original nucleotides at the 328-480 sites are replaced by forward or reverse RNA aptamers, and the RNA aptamer is obtained. The influenza reporter virus carrying the fluorescent RNA aptamer constructed by the invention also has the characteristics of good virus replication ability, high RNA imaging sensitivity and good specificity on the basis of stable inheritance, can be used for real-time visual observation of virus RNA, and is used for research on virus replication cycle and molecular mechanism related to the virus RNA. Besides, the construction method of the influenza reporter virus carrying the fluorescent RNA aptamer can also be applied to construction of RNA fluorescent reporter viruses of other subtype influenza viruses such as H3N2 and H5N1, and an important tool is provided for real-time tracing of corresponding virus RNA.

This invention relates to the field of biomedical detection technology, and discloses a bitetrahedral framework nucleic acid TDN cascade amplification system and its application, including TDN-1 and TDN-2. TDN-1 has a hairpin H1 containing a fluorescent reporter group attached to its surface, and TDN-2 has a hairpin H2 attached to its surface. The sequences of H1 and H2 are designed to undergo an alternating hybridization chain reaction triggered by target mRNA. This scheme relies on the synergistic effect of TDN structural anchoring and precise hairpin probe sequence design to overcome the technical defects of traditional free HCR hairpins in living cells, such as poor stability, low reaction efficiency, and non-specific triggering, achieving more efficient and specific intracellular signal amplification. Furthermore, it enables highly specific and sensitive in-situ imaging and dynamic tracking of low-abundance mRNAs such as c-Myc in single living cells, effectively solving the problems of insufficient detection sensitivity and false positives caused by the inability of traditional detection methods to effectively enrich targets and the low efficiency and high background of traditional HCR and other signal amplification technologies in intracellular applications.

The invention provides a human brain organoid drug screening platform related to Huntington's disease, and the platform comprises: (1) a first brain organoid, which is formed by differentiation culture of a visual cell line containing a plurality of CAG repetitive sequences of HTT, and (2) a second brain organoid, which is formed by differentiation culture of the visual cell line containing a plurality of CAG repetitive sequences of HTT; and (2) a second brain organoid which is formed by carrying out differentiation culture on a quantifiable cell line, wherein the quantifiable cell line contains a plurality of CAG repetitive sequences of HTT. The human brain organoid drug screening platform related to the Huntington's disease is an organoid model with a fluorescence reporter gene d2GFP and luciferase, and an enhanced synaptic active response element (E-SARE) promoter is used for marking neuron activity; in addition, secretory luciferase can detect subtle changes of neuron activity.

The invention discloses a rapid detection method for Xanthomonas oryzae pv. Oryzae on the basis of RPA-CRISPR / Cas12b. The reagent combination comprises a specific RPA primer pair and sgRNA, and the sequences of the specific RPA primer pair and the sgRNA are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. The detection method comprises the following steps: taking nucleic acid of a sample to be detected as a template, and performing RPA isothermal amplification by utilizing the primer pair; the amplification product is mixed with sgRNA, Cas12b protein and fluorescent reporter molecules for incubation, and the trans-cleavage activity of Cas12b is activated; a result is judged by detecting a fluorescence signal. According to the invention, the optimized RPA and CRISPR systems are integrated on the centrifugal micro-fluidic chip, so that the full-flow closed tube and automatic detection is realized, the detection can be completed within 30 minutes, the sensitivity reaches 0.156 pg / reaction, no non-specific reaction is caused to five related bacteria such as Xa and Xap, the sensitivity to XOO3 subtype is high, but the kit is not suitable for XOO2 subtype, and the kit has the advantages of rapidness, sensitivity, specificity and low equipment dependence degree, and can be used for detecting the related bacteria of XOO3 subtype and XOO2 subtype. The method is suitable for non-diagnostic detection of field environments, port environments and the like.

The invention discloses a dermatophyte nucleic acid rapid detection kit, which comprises: a CRISPR / Cas12a reaction system, which comprises a Cas12a protein and a corresponding crRNA; the fluorescent reporter probe is provided with a 5 '-end FAM label and a 3'-end biotin label; an anti-FAM antibody-fluorescent quantum dot conjugate; the quantum dot immunochromatography test strip comprises a sample pad, a nitrocellulose membrane and an absorption pad, and the nitrocellulose membrane is provided with a test line and a control line; the control line is coated with streptavidin, and the test line is coated with goat anti-mouse IgG. The invention further discloses a corresponding detection method. Detection can be completed within 1 h, operation is easy and convenient, detection sensitivity and specificity are high, and the method is suitable for rapid detection and field application of dermatophyte.

The invention provides a CRISPR-Cas12a-based target pathogen rapid detection method, which comprises: S1, determining an LAMP primer group, and carrying out isothermal amplification on a target sequence of a target pathogen in a to-be-detected sample through the LAMP primer group; s2, determining crRNA, a fluorescent reporter probe and Cas12a protein matched with the target sequence, and detecting the target sequence through the crRNA, the fluorescent reporter probe and the Cas12a protein to judge whether the target sequence of the target pathogen exists in the target sample or not; the LAMP primer group comprises sequences as shown in SEQ ID NO.1-SEQ ID NO.12, and the crRNA comprises sequences as shown in SEQ ID NO.13-SEQ ID NO.15. According to the invention, high-sensitivity and high-specificity detection of target nucleic acid is realized, the detection accuracy is high, the operation is simple and rapid, and the requirements on instruments and equipment are low.

The present invention relates to a fluorescent reporter plasmid system for making Bacillus strains fluoresce, and to a method for visualizing the state of Bacillus strains.

The invention discloses a miRNA (micro Ribonucleic Acid) detection system and a detection method based on CRISPR-Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats) and split DNAzyme coupling, and belongs to the technical field of biological detection. The composition provided by the invention comprises a magnetic bead modified with a recognition unit, crRNA, Cas12a nuclease and a fluorescence reporter, the recognition unit is obtained by hybridizing and compounding DNAzyme 1, DNAzyme 2 and a substrate chain; the sequence of the DNAzyme 1 is as shown in SEQ ID NO. 1; the sequence of the DNAzyme 2 is as shown in SEQ ID NO. 2; the sequence of the substrate chain is as shown in SEQ ID NO. 3; and the sequence of the crRNA is as shown in SEQ ID NO. 4. According to the present invention, the split DNAzyme is combined with the CRISPR, such that the high-sensitivity, pre-amplification-free and high-specificity miRNA detection is achieved, and the conditions are mild (37 DEG C).

The invention discloses a green fluorescence report system for screening a Listeria monocytogenes quorum sensing inhibitor as well as a construction method and application of the green fluorescence report system. The report system is constructed through the following steps: synthesizing a DNA fragment according to a listeria monocytogenes AGR system pII promoter sequence, connecting the DNA fragment with a green fluorescent protein pEGFP plasmid, amplifying an EGFP-pII target fragment through PCR, and connecting the target fragment to a pKSV7 shuttle vector plasmid through homologous recombination to obtain a pKSV7-EGFP-pII plasmid, and then transferring the gene into a competent cell of listeria monocytogenes ATCC BAA-679, and screening to obtain a report system. The report system disclosed by the invention can be used for intuitively evaluating the expression condition of the quorum sensing system of the listeria monocytogenes and screening out the quorum sensing inhibitor.

The application relates to a detection method based on a nano-addressable DNA origami platform combined with a CRISPR technology, in particular, a crRNA chain and a DNA fluorescent reporter molecule chain are hybridized at a preset spatial site through an address sequence, Cas12a is combined with the crRNA at a specific region, the activated Cas12a cuts a substrate, the fluorescent signal on the DNA origami changes, and thus the target nucleic acid is detected. The method of the application is suitable for various targets, and realizes simultaneous detection of multiple targets in a one-pot method.

The invention discloses a method for visually detecting mandarin fish iridovirus and infectious spleen and kidney necrosis virus based on RPA-CRISPR Cas12a / Cas13a, and belongs to the technical field of biological diagnosis. According to the method, a CRISPR / Cas system is combined with RPA amplification, a fluorescent reporter and a test strip are used for naked eye observation, and the method combines the advantages of different isothermal amplification technologies, has the advantages of short detection time, high specificity, no cross reaction, high sensitivity and the like, realizes rapid and accurate diagnosis of the two pathogens, and has a good application prospect. Important technical support is provided for prevention and control of mandarin fish diseases.

The application discloses a thermus butyricum transcriptional regulatory protein TrpR mutant screening and application, and belongs to the technical field of genetic engineering. Through screening of the TrpR mutant, favorable modification of the thermus butyricum transcriptional regulatory protein TrpR is realized, so that the specificity of the response of the thermus butyricum transcriptional regulatory protein TrpR to 5,6-dihydroxyindole is enhanced, a special biosensor is constructed by combining with a fluorescent reporter gene, effective detection of the 5,6-dihydroxyindole content is realized, and the efficiency and purity of extraction of a target component are ensured, thereby laying a solid foundation for subsequent application and research.

The invention belongs to the field of synthetic biology and metabolic engineering, and discloses a screening method and application of a microbial strain with high yield of ergothioneine, a promoter (such as PECL1, PBSC5, PEDC2 and the like) responding to ergothioneine is screened through transcriptomics analysis based on saccharomyces cerevisiae, and the promoter can present high sensitivity and linear response under 0-1000 mg / L of ergothioneine. The strain is used for driving fluorescent protein gene expression, a fluorescence report strain is constructed, the strain can rapidly and sensitively detect the concentration of ergothioneine in fermentation liquor by virtue of fluorescence signal change, and high-yield strains are efficiently screened. The method overcomes the problems of low flux, long period, low sensitivity and the like of traditional screening, and the fluorescence signal intensity is in significant linear correlation with the ergothioneine concentration. And the perforated plate and the microplate reader are used for automatic high-throughput screening, so that the efficiency is improved, the resource consumption is reduced, effective support is provided for production optimization of ergothioneine, and the application prospect is wide.