59 results about "Molecular beacon" patented technology

The invention discloses a digital single-molecule enzyme multi-activity characteristic cross-scale heterogeneity dynamic evaluation method, which comprises the following steps: diluting a pre-combined enzyme-template-primer ternary complex to a single-molecule level so as to load the enzyme-template-primer ternary complex into a reaction chamber in a micropore array chip for rolling circle amplification, a molecular beacon is combined with an amplification product to release a fluorescence signal; monitoring and acquiring the fluorescence signal in real time to generate a time-resolved fluorescence intensity curve; extracting a slope parameter of the fluorescence intensity curve so as to obtain a catalytic synthesis rate through conversion calculation for evaluating enzyme activity; the distribution of catalytic synthesis rate values is fitted using an n-fold Gaussian mixture model to obtain a standard deviation for assessing population homogeneity and a coefficient of variation for assessing stability. According to the invention, cross-scale analysis of single-molecule resolution, multi-parameter dynamic association and environmental interference processing can be realized at the same time, and resolution limitation of a traditional analysis technology is broken through by integrating single-molecule fluorescence tracking and population statistics.

The invention discloses a method for detecting high sensitivity of monkey pox virus by a one-pot method RPA-CRISPR based on temperature control, and the method comprises the following steps: establishing a temperature-controlled RPA and CRISPR-Cas12b combined detection method, which is called as CRATS for short, by utilizing the reaction temperature difference between RPA amplification and CRISPR-Cas12b cutting; the reaction process in a tube system is regulated and controlled by stages through a temperature switching strategy: target amplification is completed at 37 DEG C firstly, then the temperature is raised to 60 DEG C, Cas12b cutting is started, so that sequential separation of amplification and cutting reactions is realized, and finally detection is realized through a fluorescence signal released by molecular beacon breakage. The problem that Cas enzyme cutting activity interferes with the amplification process in a traditional one-pot method reaction is solved, the detection sensitivity and specificity are improved, and a new technical means is provided for on-site rapid detection of the monkey pox virus.

The invention discloses an engineered artificial vesicle and application of the engineered artificial vesicle in multiple in-situ detection of urine exosome miRNA, and belongs to the field of biosensors. The method comprises the following steps: constructing planar framework nucleic acid simultaneously modified with cholesterol and an aptamer through annealing reaction; a molecular beacon with a fluorophore is designed according to a target gene sequence, and the molecular beacon and a double-strand specific nuclease (DSN) system are jointly encapsulated in an artificial vesicle; the engineered artificial vesicles and a sample to be detected are incubated, the fluorescence modified molecular beacons can specifically recognize target genes, cyclic cutting and signal amplification are achieved under DSN mediation, and the broken molecular beacons release fluorescence signals; all fluorescence signals in the vesicles are collected through a fluorescence imaging technology, and a characteristic fluorescence spectrum of the to-be-detected sample is obtained. According to the present invention, the miRNA heterogeneity analysis at the single vesicle level and the precise multi-target detection of bladder cancer and other diseases can be achieved, and the problems of low throughput, target number limitation and the like of the existing vesicle in-situ detection are effectively overcome.

The invention provides a method and a kit for detecting FecB site polymorphism of a sheep BMPR1B gene based on a molecular beacon-melting curve and application of the method and the kit. And the FecB site polymorphism is generated by A > G mutation at 746nt of a BMPR1B gene coding region. The method comprises the following steps: preparing a fluorescent quantitative PCR reaction system by adopting a specific primer and molecular beacon probe composition, and judging that the genotype is C.746nt AA type when a melting curve peak is 53-56 DEG C; when the peak of the melting curve is 46-49 DEG C, judging that the genotype is C.746nt GG type; and when the two melting curve peaks exist, judging that the genotype is a C.746nt AG type. According to the method disclosed by the invention, whole-course tube closing operation is realized, nucleic acid aerosol pollution caused by uncovering sample application in a gel electrophoresis test is avoided, and the accuracy of a detection result is ensured. The real-time fluorescence PCR instrument is used for monitoring the fluorescence change in the amplification process and the fusion curve process in the whole process, the genotype result judgment of the FecB site can be completed after the test is finished, and the method is simple, convenient and rapid and is suitable for being applied to large-scale detection.

The present application relates to the field of molecular biology, in particular to a primer probe composition, kit and detection method for 23 human papilloma virus genotyping detection, wherein the hydrolysis probe and molecular beacon probe are respectively designed in two different regions of HPV gene sequence E6 and L1, and the fluorescence collection point in the amplification stage is set in the high-temperature denaturation stage, so that the detection in the amplification stage and the melting curve stage is independent and does not interfere with each other, thereby realizing the combined use of real-time fluorescent PCR amplification detection technology and melting curve technology; the technology improves the single-well detection target flux of fluorescent PCR analysis technology, reduces the detection cost, and provides a more optimal detection method for HPV multiplex fluorescent PCR genotyping detection.

The invention relates to the technical field of biology, and discloses a molecular beacon and a high-throughput sequencing library absolute quantification method thereof, and a fluorescent molecular beacon oligonucleotide (Oligo) sequence comprises a nucleotide chain (Loop chain) of a sequencing library recognition region, a first universal sequence region at the 5'upstream of the nucleotide chain (Loop chain) of the sequencing library recognition region, and a second universal sequence region at the 3 'downstream of the nucleotide chain (Loop chain) of the sequencing library recognition region; 5'of the first universal sequence region is modified into a fluorophore, and 3 'of the second universal sequence region is modified into a quenching group; and a nucleotide chain of the sequencing library recognition region is from an Illumina anmena sequencing platform or an MGI Huazai sequencing platform. The invention comprises a fluorescent molecular beacon for high-throughput sequencing library sequencing before-loading quantification, a sequence applied to a high-throughput sequencing platform and a detection method for library absolute quantification based on the molecular beacon, and overcomes the defects of a current gold standard detection method in technology and efficiency. The quantitative precision of the sequencing library is ensured; meanwhile, the economic and time cost of an actual application end is greatly reduced.

The invention provides a molecular beacon probe used in a PCR (Polymerase Chain Reaction) end point detection method as well as a system and a kit thereof, the molecular beacon probe sequentially comprises a 5'end stem sequence, a loop sequence, a flexible Spacer structure and a 3 'end stem sequence, and the whole molecular beacon probe is of a hairpin structure; the 5'end stem sequence and the loop sequence are completely complementary to a to-be-detected target sequence, and the loop sequence and the flexible Spacer structure form a loop structure of the molecular beacon probe; and the flexible Spacer structure is a polyethylene glycol structure. According to the invention, a Spacer structure is introduced into a short-sequence high-specificity probe, so that the problems of background control and structure ring formation of a traditional molecular beacon in complex mutation detection are solved, and the detection accuracy and the application range are remarkably improved. Besides, a fluorescent closed probe is also introduced into the system, and the probe system effectively improves the specific recognition capability of the enzyme digestion dependent molecular beacon probe under a high homologous background, so that accurate detection of a plurality of homologous mutation sites becomes possible.

The invention relates to a kit capable of detecting different types of targets based on DNA multi-pass knots and a detection method. The kit comprises a detection composition and a test strip, the detection composition comprises a molecular beacon and an auxiliary chain, wherein the nucleotide sequence of the molecular beacon (MB) is as shown in SEQ ID No.1; the auxiliary chain is a combination of HBV-alpha1 and HBV-beta1, and / or a combination of miR-21-alpha2 and miR-21-beta2, and / or a combination of ATP-alpha3 and ATP-beta3, and / or a combination of Thrombin-alpha4 and Thrombin-beta4, and / or a combination of TdT-alpha5 and TdT-beta5, and the sequence of the auxiliary chain is as shown in SEQ ID No.2-11. The kit can detect different types of targets, and when a target exists, no matter DNA / RNA, small molecules, proteins or enzymes, alpha and beta auxiliary chains can be connected together and hybridized with MB to form a DNA multi-pass node, so that a test line develops color, the target is conveniently and intuitively determined, and the detection time is shortened. Remodification of nano-particles, re-pretreatment of a pad / film and assembly and cutting of test paper are avoided, and a large amount of manpower, material resources and financial resources are saved.

The invention discloses a rolling circle amplification (RCA) molecular beacon based on DNA synthesized silver nanoclusters (AgNCs / DNA) and application of the rolling circle amplification (RCA) molecular beacon in DNA and RNA virus detection. According to the invention, a padlock template probe PlP with a specific secondary structure is used for carrying out two-enzyme one-step method RCA to generate single-stranded DNA containing a G-rich sequence tandem repeat element. Then H2O2 is added to eliminate the influence of residual dithiothreitol DTT in the RCA reaction, and then AgNCs / DT-GSP-t4 is used as a signal probe to generate a specific fluorescence enhancement signal for a to-be-detected target under the action of a G-rich proximity fluorescence enhancement effect. The RCA reaction does not need primer mediation, so that PlP design and RCA operation steps are simplified; meanwhile, the single-PlP or double-PlP probe is combined with the fluorescent molecular beacon, so that single-base mismatch at a PlP connection point can be recognized, and the probe has the advantage of high specificity.

The application provides a probe combination, a kit and a detection method for detecting nucleic acid, and belongs to the technical field of nucleic acid detection. The application provides a probe combination for detecting nucleic acid, wherein the probe combination comprises a specific hairpin switch probe HSP and a universal molecular beacon; the HSP is composed of a middle template binding sequence, two end beacon binding sequences and complementary sequences. The universal molecular beacon comprises a short stem structure and a long loop sequence, and the loop sequence is designed to be complementary to the beacon binding sequence region of the HSP. When the temperature is increased or there is a DNA strand complementary to the middle loop sequence, the universal molecular beacon is unfolded from a hairpin structure to a linear structure, the 5' and 3' end groups are far away from each other, and the fluorescence is significantly increased. The HSP and the universal molecular beacon provided by the application are suitable for the detection of qPCR and ddPCR, and can meet the detection of nucleic acid and mutant nucleic acid, and have high sensitivity and strong specificity.

The invention discloses a universal molecular beacon kit for various animal-derived components, and belongs to the technical field of molecular biological detection. Aiming at gene segments with specific structural characteristics in 11 common livestock and poultry animal genomes, the invention designs a composition of a universal primer and a molecular beacon fluorescent probe. The combination can be used for carrying out real-time fluorescent PCR detection and melting curve analysis by using the kit and the method by taking genome DNA of a sample to be detected as a template. By comparing the shape of a melting peak of a detected sample and the characteristic melting temperature (Tm value) of the detected sample, 11 animal-derived components of livestock and poultry can be quickly and accurately identified under the condition of single-tube detection. Technical innovation is achieved in the field of molecular biological detection, an efficient and reliable solution is provided for animal-derived component detection, and the method has important practical value and popularization potential.

The application discloses a functional nucleic acid probe and a preparation method and application thereof. The preparation method comprises the following steps: forming a DNA sheet by self-assembly of a first DNA single strand and a second DNA single strand, and then combining the DNA sheet with a molecular beacon probe to prepare the functional nucleic acid probe; wherein the end of the first DNA single strand and the end of the second DNA single strand are complementary to each other; and the molecular beacon probe comprises an aptamer sequence of foodborne pathogenic bacteria. The fluorescence polarization method based on the functional nucleic acid probe provided by the application has the advantages of high sensitivity, strong specificity, short detection time and the like, and can be used for self-immediate monitoring of the content of foodborne pathogenic bacteria in drinking water or the content of foodborne pathogenic bacteria in environmental water, and has great application value and market value in family health and environmental protection and the like.

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a kit and a method for detecting linear RNA (Ribonucleic Acid). The invention provides a kit for detecting linear RNA (Ribonucleic Acid). The kit comprises a molecular beacon, a hairpin combination for a primer exchange reaction and a reagent for the primer exchange reaction, the hairpin combination comprises an identification hairpin and an extension hairpin. The invention develops a nucleic acid isothermal amplification method for detecting linear RNA (Ribonucleic Acid) based on primer exchange reaction, such as miRNA (Micro Ribonucleic Acid). Compared with the existing technology for detecting miRNA, the kit and the method provided by the invention have the advantages of no need of expensive instruments, simple experimental process, simple design of nucleic acid recognition elements, high sensitivity, extensibility of detection objects and the like, and are high in practical applicability.

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) visual biosensor for diagnosing coagulase negative staphylococcus. The LAMP visual biosensor comprises (1) a coagulase negative staphylococcus aptamer sequence; (2) optimizing reaction conditions of the coagulase negative staphylococcus aptamer molecular beacon fluorescent biosensor; and (3) detecting coagulase negative staphylococcus. And coupling the aptamer and the magnetic bead modified with the complementary chain through complementary base pairing to form a magnetic bead-complementary chain-aptamer compound. When the target exists, the aptamer identifies the target and dissociates from the complementary chain, and signal normalization is completed through magnetic separation. And performing amplification by using the magnetically separated supernatant as an LAMP template to realize signal amplification. In the LAMP amplification process, pyrophosphoric acid generated by decomposition of the dNTP is combined with Mg < 2 + >, and the concentration of Mg < 2 + > is reduced, so that hydroxynaphthol blue is triggered to develop, and visual output of signals is realized. The Ct value is generated based on LAMP amplification, and quantitative detection can be achieved.

The application belongs to the technical field of biomedicine, and relates to a mitochondrion-targeted nano device and a preparation method and application thereof, the nano device comprising a cascade-activated photodynamic molecular beacon and a mitochondrion-targeted delivery carrier, the cascade-activated photodynamic molecular beacon comprising an APE1 cleavage site and a microRNA recognition region. The cascade-activated photodynamic molecular beacon and the mitochondrion-targeted delivery carrier are self-assembled into a complex through electrostatic adsorption, and cascade activation and signal amplification are realized through the dual response of miR-146a / APE1 which is highly expressed in mitochondria of tumor cells. The application triggers precise and controllable cell pyroptosis through tumor-specific signals, reverses the immune-inhibitory microenvironment, converts the 'cold tumor' into a 'hot tumor', significantly improves the effect of cancer immunotherapy, has good biocompatibility and tumor selectivity, and provides a new strategy and tool for precise treatment of cancer.

The application discloses a kind of digital single molecule enzyme multi-activity characteristic cross-scale heterogeneity dynamic evaluation methods, comprising: the enzyme-template-primer ternary complex of pre-combination completion is diluted to single molecule level to be loaded into the reaction chamber in microwell array chip to carry out rolling circle amplification, and utilize molecular beacon and the fluorescence signal released by amplification product combination;Real-time monitoring and obtaining the fluorescence signal to generate time-resolved fluorescence intensity curve;The slope parameter of the fluorescence intensity curve is extracted to obtain the catalytic synthesis rate by conversion calculation for evaluating enzyme activity;The catalytic synthesis rate value distribution is fitted using n times Gaussian mixture model to obtain the standard deviation for evaluating population uniformity and the coefficient of variation for evaluating stability.The application can realize single molecule resolution, multi-parameter dynamic correlation and environmental interference processing cross-scale analysis simultaneously, and break through the resolution limit of traditional analysis technology by integrating single molecule fluorescence tracking and population statistics.

The present invention provides novel primers and sloppy molecular beacons and molecular beacon probes for amplifying segments of different genes in MycobacteriumTuberculosis to identify the presence of M. tuberculosis DNA and / or to identify resistance to anti-tuberculosis drugs.SOLUTION: Oligonucleotide sets are provided for amplifying a portion of an M. tuberculosis region selected from the group consisting of rpoB, gyrA, gyrB, inhA promoter, rrs, eis promoter, embB, katG, dosR, IS6110, and IS1081 genes.SELECTED DRAWING: None

The present application is related to a method for in situ detection of multiple lung cancer-related markers in live circulating malignant cells, and tumor heterogeneity, including accurately detecting at least one lung cancer-related nucleic acid marker in live lung cancer cells by using a lung cancer-targeted nanoprobe loaded with a plurality of molecular beacons to reflect levels of different markers in the lung cancer cells from a single cell level and a heterogeneous state of a tumor of a specific patient. The nanoprobe is a nanoparticle self-assembled from a high polymer material, an electropositive protein, a functional polypeptide and / or a functional aptamer, and a molecular beacon of a lung cancer-related marker. The nanoprobe can be used to target different phenotype circulating malignant cells in whole blood, detect nucleic acid markers in living cells, and reflect levels of different markers in the malignant cells from a single cell level.

Herein, based on Boolean operators, DNA logic gate complexes are constructed to obtain True (fluorescence signal recovery) or False (fluorescence signal quenching) value using a molecular beacon acting as output. A DNA logic gate complex is composed of a DNA board and at least two modular logic units of YES and / or NOT gate. First, the DNA board is designed to accommodate individual YES and / or NOT modular logic units. An OR logic gate is composed of the DNA board and at least two YES modular logic units, and when there are multiple YES modular logic units, they are arranged in a parallel manner. A NAND logic gate is composed of a DNA board and at least two NOT modular logic units, and when there are multiple NOT modular logic units, they are arranged in a parallel manner. An IMPLY logic gate comprises at least one YES modular logic unit and at least one NOT modular logic unit as well as the DNA board. The designed DNA logic gate complexes were tested with biomarker miRNAs of hepatocellular carcinoma and successfully computed True / False fluorescence outcomes, exhibiting great potential for applications in the field of diseases diagnosis.

The invention belongs to the technical field of nucleic acid molecule detection, and particularly relates to an enzyme-free isothermal strand displacement cascade amplification reagent for acute respiratory pathogen detection and application of the enzyme-free isothermal strand displacement cascade amplification reagent. The detection method based on the amplification reagent is based on a nucleic acid reaction system composed of an initiation double strand and two molecular beacons, cyclic amplification is performed through a toehold-mediated strand displacement reaction trigger signal under a constant temperature condition, and end point interpretation is performed through a fluorescence signal. The method does not need enzyme and thermal cycle, is short in detection time, adapts to high-throughput operation of a porous plate, and realizes specific recognition and signal cascade amplification of target nucleic acid under a constant temperature condition, so that rapid, accurate and high-throughput detection of acute respiratory pathogens is realized on the premise of not depending on enzymatic reaction and thermal cycle equipment. In addition, the kit has the advantages of low cost, good stability and wide application range, and is suitable for rapid detection of various acute respiratory pathogens.

The invention discloses an aptamer-mediated EXPARs-molecular beacon fluorescent biosensor as well as a preparation method and application of the aptamer-mediated EXPARs-molecular beacon fluorescent biosensor. The construction and detection process of the sensor comprises three key steps: protein binding aptamer, exponential isothermal amplification (EXPAR) and molecular beacon fluorescence resonance energy transfer (FRET). Through the ingenious design of the EXPAR system, the system realizes triple signal amplification, the detection sensitivity is remarkably improved, and Abeta42 and Abeta40 amyloid proteins as low as 100fM level can be detected. Besides, based on the sequence specificity of the aptamer and the highly specific design of each nucleic acid sequence involved in the detection system, the method can be used for simultaneously detecting the Abeta42 amyloid protein and the Abeta40 amyloid protein or other substances in the same system (such as a plasma sample), so that the detection efficiency is improved.

This invention discloses a method and kit for quantitative detection of circularized rolling circle amplified miRNAs. The method utilizes T4 RNA ligase to ligate the 3'-OH and 5'-phosphate ends of the target miRNA to form a circular miRNA. Using the circularized miRNA as a template, isothermal rolling circle amplification is performed using phi29 DNA polymerase to generate a large number of single-stranded DNA sequences with repetitive sequences. A universal molecular beacon is used to monitor the fluorescence signal of the amplification products in real time, achieving highly sensitive quantitative detection of miRNAs. This method has advantages such as high sensitivity, high specificity, and a wide linear detection range. It eliminates the need to design specific reverse transcription primers for each miRNA, can be extended to multiplex detection, and is suitable for clinical applications such as early tumor diagnosis, drug efficacy evaluation, and single-cell miRNA expression profiling.

The invention discloses an addressable nanoliter reactor chip and application of a nucleic acid signal amplification integrated system in single cell level detection markers. The nucleic acid signal amplification integrated system comprises aptamer DNA molecules and probe DNA molecules of a hairpin structure. According to the invention, the programmable characteristic of nucleic acid molecules is utilized to construct an aptamer-probe DNA synergistic signal amplification system; through combination of molecular beacon design and endonuclease reaction, in-situ signal multiplication of EpCAM protein is realized on a single cell level. After the aptamer recognizes the target epitope, the cascade hybridization reaction of the probe DNA is triggered, so that a single antigen binding event is converted into hundreds of fluorescent signal molecules to be released, and the signal gain is remarkably improved compared with that of a traditional immunofluorescence method.

The invention discloses a multi-modal nucleic acid detection method (multi-modal LAMP, M-LAMP) of an LAMP (loop-mediated isothermal amplification) auxiliary molecular beacon system based on connection start. In M-LAMP, aiming at different targets to be detected, only target binding sites can be changed, and primer binding sites and signal probe binding sites are fixed, so that single, double and triple detection can be simultaneously realized by using one set of LAMP primer and signal probe, and the tedious and time-consuming design and optimization process of the primer and signal probe is avoided. The two major problems, namely the primer design and optimization complexity and the false positive risk, existing in the current carbapenemase detection based on LAMP are effectively solved, and the reliability and timeliness of a detection result are remarkably improved.

The invention relates to the technical field of biosensors, in particular to a transgenic corn CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor. According to the invention, a systematic aptamer rational cutting and conformation adaptation strategy is constructed, the strategy obtains an initial aptamer through magnetic bead-SELEX screening, and the CP4-EPSPS protein aptamer is precisely cut and optimized by adopting the steps of loop region active core positioning, stem region structure optimization and base binding domain identification, so that the binding performance and the structural stability are remarkably improved. Subsequently, on the basis of stable conformational characteristics, a complementary chain competition sensing mechanism is designed, and a transgenic corn CP4-EPSPS protein aptamer molecular beacon fluorescent biosensor is successfully constructed, so that amplification-free high-sensitivity rapid detection of CP4-EPSPS protein is realized, and a solution is provided for supervision of transgenic crops.

The invention discloses a technology for detecting target nucleic acid and gene mutation by using a Taq DNA polymerase large fragment (Taq KF enzyme) without 5'exonuclease activity, heat-resistant ribonuclease HII (RNase HII), and a Taqman probe or a molecular beacon with an RNA single basic group in the middle. Comprising the following steps: step 1, preparing a Taqman probe or a molecular beacon with an RNA basic group in the middle; 2, establishing a PCR amplification reaction system; 3, carrying out a PCR conventional amplification reaction and a fluorescence signal amplification reaction of target nucleic acid; 4, quantifying the concentration of the target nucleic acid in real time; and 5, determining a gene mutation type. The method has the beneficial effects that compared with the existing nucleic acid detection and gene mutation typing technology based on the conventional Taqman probe or molecular beacon, the method has obvious progress in the aspects of accuracy, sensitivity and the like.

The present application relates to a kind of DNA adenine methyltransferase activity detection method, according to substrate hairpin in the double enzyme coupling of DNA adenine methyltransferase and restriction endonuclease obtains a truncated hairpin structure, and design contains RNA cleavage type DNA mimic enzyme complementary sequence's rolling circle probe, with truncated hairpin structure as primer, with rolling circle probe as template to carry out rolling circle amplification, the RNA cleavage type DNA mimic enzyme cleavage molecular beacon obtained, so that the fluorescent group and quencher group modified in the two ends of molecular beacon are far away, release fluorescence signal, realize the detection of DNA adenine methyltransferase activity, detection limit is as low as 3.09x10 ‑4 U / mL, provide a specific, high sensitivity method for DNA adenine methyltransferase activity analysis.

This invention belongs to the medical field and provides a calcium carbonate manganese nanoprobe for in situ imaging detection of vascular epidermal growth factor, its preparation method, and its application. In this strategy, a DNA strand (P1 and P2) containing a protein recognition antibody, a protein recognition sequence, and a deoxyribonuclease, along with a molecular beacon DNA strand (H1), is stirred in a DMEM cell culture medium containing calcium chloride and manganese chloride to prepare the calcium carbonate manganese nanoprobe. In acidic tumor cells, this nanoprobe releases P1, P2, H1, and manganese ions. P1 and P2 can simultaneously recognize proteins, forming a complete deoxyribonuclease center, which catalyzes the cleavage of H1 under the action of the cofactor manganese ions, generating a fluorescent signal. This strategy achieves specific in situ dynamic imaging of low-abundance proteins in vivo and has great potential in biomedical research.

The invention provides a digital PCR (Polymerase Chain Reaction) detection kit for detecting multiple mutation sites of an ESR1 (Enhanced Session Receptor 1) gene. According to the kit, the mutation sites of L536Q, L536H, L536P, L536R, Y537N, Y537S, Y537C, Y537D, D538G and E380Q of the ESR1 gene are detected through a single-tube digital PCR reaction tube; wherein each site is detected by using a respective spacer molecular beacon detection probe. Aiming at the problems of specific interference and fluorescence background accumulation confronted by simultaneous detection of a plurality of mutation hot spots in the same reaction system, a molecular beacon probe based on a Spacer structure is adopted, and the Spacer structure enables the probe to maintain more consistent structural stability and background inhibition capability; therefore, high-throughput and high-sensitivity mutation site distinguishing detection is realized.

The invention discloses a bioluminescence kit for detecting myeloperoxidase in whole blood as well as a use method and application of the bioluminescence kit. The bioluminescence kit comprises a nucleic acid hairpin probe, H2O2, NaCl, a PBS (Phosphate Buffer Solution), a Cas12a / crRNA binary complex, a bioluminescence type molecular beacon, an NEBuffer 2.1 and a luminescent substrate Furimazine. The 5'end and the 3 'end of the bioluminescence type molecular beacon are respectively marked with Cy3 and cpHNLuc, and a bioluminescence resonance energy transfer phenomenon (BRET) can occur. A BRET signal does not need an external excitation light source, interference of autofluorescence of a blood sample and environmental factors can be effectively avoided, meanwhile, the excellent signal amplification capacity of a CRISPR / Cas12a system is combined, rapid and ultra-sensitive detection of MPO in a whole blood sample can be achieved, and therefore the method has a great application prospect in the fields of biosensing and medical examination.