205 results about "Fluorescent reporter" patented technology

The invention discloses a metabolite screening method for promoting expression of porcine intestinal defensin pBD1 and application of the metabolite screening method. The screening method comprises the following steps: 1) constructing a porcine defensin pBD1 gene fluorescence report intestinal epithelial cell line; 2) evaluating the promotion effects of different metabolites based on the pBD1 gene fluorescence report intestinal epithelial cell line; and 3) further evaluating the promotion effects of the metabolites screened in the step 2) based on a porcine intestinal epithelial cell IPEC-J2 model. And 4) further evaluating the promotion effect of the metabolite screened in the step 3) based on the pig intestinal organ. The invention can be used for preparing or screening drugs influencing the expression of the porcine intestinal defensin pBD1. According to the application disclosed by the invention, the effect of the D-malic acid for improving the immune function of the pig intestinal tract by promoting the expression of the defensin pBD1 is found for the first time, verification is carried out on a pig intestinal tract organ-like model, and meanwhile, the optimal treatment concentration of the D-malic acid for promoting the expression of the defensin pBD1 in the pig intestinal tract is optimized.

The invention discloses a kit for detecting salmonella enteritidis and application of the kit, and belongs to the technical field of biomolecular detection. The kit comprises the following components: an allosteric aptamer probe of which the nucleotide sequence is as shown in SEQ ID NO.1, a template chain of which the nucleotide sequence is as shown in SEQ ID NO.3 and a fluorescence report probe of which the nucleotide sequence is as shown in SEQ ID NO.4. According to the kit, salmonella enteritidis can be distinguished from other impurities, and the specificity is high; a visual fluorescence signal can be obtained and is easy to interpret; according to the invention, a template chain can be cyclically utilized in the reaction to generate a Trigger chain to guide the TtAgo protein to efficiently and specifically cut a fluorescent reporter probe, the output signal is increased, the detection time is short, the detection limit is low, the sensitivity is high, the specificity is good, and the method has practical popularization and application prospects.

The invention relates to a construction method of a macrophage specific fluorescence report mouse model, and belongs to the technical field of genetic engineering and genetic modification. According to the invention, a brand-new C1qc-CreChy gene knock-in mouse model is obtained after a C1qc gene locus is edited through a CRISPR / Cas9 genome editing technology for the first time, the expression of Cre and mCherry is directly driven by a C1qc promoter, and flow cytometry verifies that mCherry has very high expression quantity only in microglial cells in the brain of a mouse and macrophages in the spleen of the mouse. And mCherry expression is not detected in other immune cells, so that the two macrophages are traced and used for researching the functions of the macrophages. The invention provides a usable animal model for researching heterogeneity of macrophages, and provides a new animal model for researching complex immune functions in a body.

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 discloses a JEV detection nucleic acid combination based on a CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats-associated 12a) system, a kit and a method. The nucleic acid combination comprises a fluorescent reporter probe, crRNA and a primer group, wherein the nucleotide sequence of the crRNA is shown as any one of SEQ ID NO: 6 and SEQ ID NO: 8. According to the nucleic acid combination, the kit or the method, Cas12a protein specific recognition is guided through crRNA, a target nucleic acid sequence is cut, a fluorescence signal amplification mechanism is combined, rapid, sensitive and specific detection of the JEV is achieved, and a new technical means is provided for rapid diagnosis and monitoring of the arthroviruses.

The invention discloses a D / Yama2019 genetic evolution pedigree influenza delta virus reverse genetic operating system. According to the invention, DNA fragments corresponding to segments of PB2, PB1, P3, HEF, NP, M and NS of a full-length genome of D / JY3002 are amplified by using seven pairs of designed primers, the DNA fragments are seamlessly cloned to a pHW2000 vector and a pCC1-DualPro vector to construct plasmids, the plasmids are co-transfected to a cell line, and the artificially rescued D / Yama2019 genetic evolution pedigree influenza delta virus rD / JY3002 is obtained; the genomic sequence of the rD / JY3002 is completely consistent with the genomic sequence of a naturally separated strain D / JY3002, the infection ability is equivalent, and the rD / JY3002 can be stably subcultured for at least five generations. According to the present invention, the constructed unidirectional expression plasmid pPolI-D / JY3002-PB1-240-GFP-240 is adopted to obtain the artificially rescued recombinant influenza delta virus rD / JY3002-GFP carrying the green fluorescence reporter gene, and after the host cell is infected, the GFP expression quantity is high and stable; the invention establishes an efficient and stable D / Yama2019 genetic evolution pedigree influenza delta virus reverse genetic operating system which can be used for developing an influenza delta virus vector presenting exogenous gene.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas-based kit for isothermal rapid visual detection of tomato mosaic virus and a detection method of the CRISPR / Cas-based kit. The invention belongs to the technical field of plant quarantine, and particularly relates to a CRISPR / Cas-based kit for isothermal rapid visual detection of tomato mosaic virus and a detection method thereof. The composition for identifying the tomato mosaic virus comprises crRNA specific to nucleic acid to be detected, an RT-RPA primer pair, Cas12 protein and a fluorescent reporter probe, the RT-RPA primer pair is composed of a primer ToMV-F and a primer ToMV-R, and the fluorescent reporter probe is a single-chain RNA with one end connected with a fluorophore and the other end connected with a quenching group. According to the present invention, the rapid, accurate and visual detection of the to-be-detected sample can be achieved, the advantages of good specificity, high sensitivity, convenient operation, high detection efficiency and the like are provided, and the basic requirements of field and port detection can be met.

Cas-cleavable reporter systems, CRISPR-Cas systems thereof, and methods of use thereof in CRISPR-Cas based diagnostics. Cas-cleavable reporter systems may generate a luminescent, fluorescent, or other detectable signal upon Cas-collateral cleavage of one or more reporter system components. CRISPR-Cas systems may comprise a Cas protein having collateral cleavage activity, guide molecules, and the Cas-cleavable reporter system. Cas-cleavable reporter systems may be a split luciferase reporter system, at least one element of which is bound to beads. For multiplexing, CRISPR-Cas systems may comprise a Cas-cleavable quenched reporter system, optionally a Cas-cleavable quenched fluorescent reporter system, a Cas protein having collateral cleavage activity, and target-specific guide molecules attached to color-coded beads. CRISPR-Cas systems may further comprise amplification reagents. Methods may apply said CRISPR-Cas systems to flow cell, well-plate, or other devices, and may measure detectable signals by microscopic, plate reader, or other methods.

The invention discloses a jujube genetic transformation method using anther to induce callus as a receptor. The method comprises the following steps: culturing sterile anther to obtain callus; constructing a plant recombinant overexpression vector containing a target gene, and transferring into agrobacterium to obtain agrobacterium dip dyeing liquid; the callus is dip-dyed with an agrobacterium dip-dyeing solution, and then the dip-dyed callus is co-cultured under a dark condition; and then putting the callus on a culture medium containing hygromycin for screening, and screening and culturing for 20-30 days to obtain the homozygous resistant callus. According to the method, the callus induced by the Jing 39 jujube anther is used as a receptor for the first time, the method of the Jing 39 jujube genetic transformation system is established, the transformation frequency is high, meanwhile, screening can be assisted by means of a 35S:: EGFP fluorescent reporter gene marker, and convenience and rapidness are achieved. The method provides convenience for jujube gene function analysis and jujube genetic improvement.

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 provides an EGFR (epidermal growth factor receptor) gene mutation detection system and an EGFR gene mutation detection method based on CRISPR / Cas12a. The detection system comprises an RPA system and a report system; the RPA system comprises restriction endonuclease or a coding gene of the restriction endonuclease aiming at an EGFR mutation site wild type sequence, an RPA primer or a coding gene of the RPA primer aiming at an EGFR mutation site, and an RPA reagent, the report system comprises crRNA (Complementary Ribonucleic Acid) aiming at EGFR (Epidermal Growth Factor Receptor) mutation sites, Cas protein or coding genes thereof and single-chain report DNA ( Wherein the single-chain report DNA is marked with fluorescent report molecules or CRISPR (clustered regularly interspaced short palindromic repeats) lateral chromatography test strip report molecules. The detection method can be used for detecting fresh tissue and paraffin-embedded tissue samples, the whole process is controlled within 2 hours, only a small incubation instrument is needed, and rapid and convenient gene mutation low-detection-limit detection on the samples is achieved.

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 a kit and a method for visually detecting cat parvovirus based on a RAA-CRISPR / Cas12a system. The kit contains an RAA system substance and a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas12a system substance, the RAA system substance comprises an upstream primer and a downstream primer, and the upstream primer and the downstream primer comprise RAA-F1, RAA-R1, RAA-F2 and RAA-R2; the CRISPR / Cas12a system substance comprises Cas12a, a crRNA (Complementary Ribonucleic Acid) sequence and an ssDNA (Deoxyribose Nucleic Acid) fluorescent reporter probe; the crRNA sequence comprises crRNA01 and crRNA02, and the ssDNA fluorescence report probe comprises qPCR-F (quantitative polymerase chain reaction) and qPCR-R (quantitative polymerase chain reaction); the sequences of the upstream primer RAA-F1, the downstream primer RAA-R1, the downstream primer RAA-F2 and the downstream primer RAA-R2 are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 4; the sequence of the crRNA01 is as shown in SEQ ID NO.5, and the sequence of the crRNA02 is as shown in SEQ ID NO.6; the sequence of the qPCR-F is as shown in SEQ ID NO.7, and the sequence of the qPCR-R is as shown in SEQ ID NO.8.

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 present invention relates to the field of biotechnology detection, and in particular to a primer and a probe and a kit for detecting Salmonella pullorum. The present invention provides a primer and a probe for detecting Salmonella pullorum, wherein the primer includes a primer for detecting Salmonella pullorum ( Salmonella Pullorum , SP) citE2 SP-F and SP-R of a gene target sequence, wherein the probe is modified with a fluorescent reporter group and a fluorescent quencher group. citE2 The primer pair SP‑F and SP‑R that specifically bind to the gene target sequence, combined with the probe for atopic recognition, can accurately and sensitively detect Salmonella pullorum.

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.