978 results about "Nucleic acid detection" patented technology

The invention relates to the technical field of biomedicine, in particular to a primer probe combination for broad-spectrum screening of common respiratory pathogens and application of the primer probe combination. Through screening and optimization, the respiratory pathogen nucleic acid detection primer probe combination for broad-spectrum screening of common acute respiratory infection and community-acquired pneumonia is obtained; the primer probe combination covers 18 respiratory tract pathogens, has the advantages of high accuracy, good specificity, strong anti-interference capability, good inclusiveness, high sensitivity, hyperjoint detection and the like, can realize parallel detection of 20 targets in cooperation with totally-closed single-tube PCR amplification, greatly improves early diagnosis of respiratory tract diseases with complex pathogen structures, and has wide application prospects. The method has very important significance on prevention, control and treatment of infectious diseases.

The invention provides an RPA-CRISPR-Cas12a system-based detection reagent for the drug resistance of a chilo suppressalis organophosphorus insecticide, a detection method and application, and belongs to the technical field of molecular detection. The invention provides a detection reagent for chilo suppressalis organophosphorus insecticide resistance, the reagent comprises a specific RPA2 primer pair designed for chilo suppressalis acetylcholin esterase ace-1 gene A314S resistance mutation site and crRNA, a double screening mechanism of RPA amplification and CRISPR-Cas12a is combined, wild type and mutant type genes can be accurately distinguished, and the detection reagent can be used for detecting the insecticide resistance of the chilo suppressalis organophosphorus insecticide resistance to the chilo suppressalis organophosphorus insecticide resistance to the chilo suppressalis organophosphorus insecticide resistance to the chilo suppressalis organophosphorus insecticide resistance to the chilo suppressalis. The rapid, sensitive and visual nucleic acid detection of the chilo suppressalis ace-1 gene A314S resistance mutation site is realized, and a visual detection means which is high in specificity, simple and convenient to operate, economical and efficient is provided for field on-site determination of chilo suppressalis resistance populations.

The invention discloses application of iron ruthenium nano-enzyme in preparation of nucleic acid detection products, and belongs to the technical field of biological detection and nano-materials. The RIN has stable peroxidase-like (POD) activity, and can catalyze a chromogenic reaction between 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) and hydrogen peroxide (H2O2). The catalytic activity of the nano-enzyme can be selectively inhibited by pyrophosphate (PPi) generated in a nucleic acid amplification reaction, so that visual colorimetric analysis on whether target nucleic acid exists or not is realized. In combination with a polymerase chain reaction (PCR) system, the invention constructs a new nucleic acid detection strategy which does not need fluorescence labeling, is simple and convenient to operate and has high sensitivity. Through verification, the system can be widely applied to rapid on-site screening and visual interpretation of related nucleic acid of drug-resistant pathogens (such as ESKAPE flora) and the like, and has a good application prospect.

The invention relates to the technical field of nucleic acid detection, in particular to a method for regulating and controlling CRISPR-Cas protein activity through temperature and application of the method, a CRISPR reaction system contains Cas protein, guide RNA, a nucleic acid reporter probe and a buffer solution, and then the cis-cleavage activity or trans-cleavage activity of the Cas protein is activated or inactivated by regulating and controlling the temperature of the reaction system; the guide RNA includes a guide RNA targeting a target nucleic acid sequence that does not contain a PAM site. Multiple detection can be realized only by using the same type of Cas protein, the design of PAM-free sgRNA or crRNA facilitates the selection of sgRNA or crRNA, meanwhile, the activity of PAM-free sgRNA or crRNA can be regulated and controlled by utilizing temperature rise, and the Cas protein with lower reaction temperature can be inactivated, so that the Cas protein cannot cause interference to the detection of the second stage.

The invention discloses a portable nucleic acid detection micro-fluidic chip, a nucleic acid detection device and a nucleic acid detection method, and belongs to the field of micro-fluidic chips, a detection module comprises a quantitative chamber, a micro-channel, a reaction chamber, a first connecting channel and a color developing module; the reaction chamber comprises an RPA reaction chamber in which an RPA amplification reagent is pre-embedded, a porous filter membrane and a CRISPR reaction chamber in which a CRISPR detection component is pre-embedded, and the RPA reaction chamber, the porous filter membrane and the CRISPR reaction chamber are sequentially positioned at the same position of the three adjacent chip layers; the aperture of the porous filter membrane is larger than the particle size of an RPA amplification product and smaller than the particle size of a core enzyme in a CRISPR detection component; two ends of the micro-channel are respectively communicated with the quantitative chamber and the RPA reaction chamber; two ends of the first connecting channel are respectively communicated with the CRISPR reaction chamber and the color developing module; the flow resistance of the first connecting channel is smaller than that of the micro-channel. According to the invention, the sequential reaction of RPA amplification and CRISPR cleavage can be completed without depending on artificial transfer of amplification products.

The invention relates to the technical field of nucleic acid detection, in particular to a product and a method for detecting bifidobacterium longum subsp. Longum 6-1. Bioinformatics analysis is carried out based on a whole genome sequence of the bifidobacterium longum subspecies longum 6-1, the primer pair and the primer probe combination are obtained through optimization, and the primer pair and the primer probe combination have good specificity and sensitivity in the aspect of detecting the bifidobacterium longum subspecies longum 6-1; the bifidobacterium longum subsp. Longum 6-1 and other strains (including other same subsp. Longum strains, other same strains and other strains) can be quickly and accurately distinguished, and the strain level quantitative detection of the strain in a complex biological sample (such as excrement) can be realized.

The invention relates to the technical field of droplet digital nucleic acid detection and biological materials, in particular to microgel based on acrylic anhydride gelatin and nucleic acid to be detected as well as preparation and application of the microgel. The preparation method comprises the following steps: firstly, dissolving a photoinitiator and acrylic anhydride gelatin, and uniformly mixing to obtain a pre-polymerized solution; uniformly mixing the pre-polymerized solution with a to-be-detected nucleic acid mixed solution to obtain a dispersion phase; oil incompatible with the dispersed phase is used as a continuous phase; building a constant-temperature device, and enabling the dispersed phase to form liquid drops by using a liquid drop microfluidic method; and finally, performing amplification treatment on the liquid drops, and then performing photo-initiation crosslinking to obtain the microgel based on the acrylic anhydride gelatin and the nucleic acid to be detected. The prepared microgel based on the acrylic anhydride gelatin and the nucleic acid to be detected is further applied to detection of target nucleic acid.

The invention relates to a microfluidic nucleic acid detection device, and relates to the field of detection. Comprising a bottom plate, a chip inlet and outlet module erected on one side above the bottom plate, a chip table erected on the chip inlet and outlet module and used for bearing a micro-fluidic chip with a liquid bag, a displacement module erected above the chip table, and a crushing module erected on the displacement module and used for squeezing liquid in the liquid bag into the micro-fluidic chip, the liquid transferring module is erected on one side of the crushing module and is used for transferring liquid in the micro-fluidic chip to flow; the pneumatic module is erected above the front end of the bottom plate and is used for controlling a flow channel of the micro-fluidic chip to be opened and closed; the heating module is erected on the other side above the bottom plate and is used for locally heating the micro-fluidic chip and automatically detecting the temperature; and the fluorescent module is used for acquiring a fluorescent signal of the micro-fluidic chip. The micro-fluidic chip detection device has the advantages that different types of micro-fluidic chips can be rapidly detected, and an accurate result can be obtained.

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 multi-subtype influenza A virus nucleic acid detection kit and a detection method, and relates to the technical field of medical detection.The kit comprises a group of reverse transcription RT-RPA amplification primer pairs used for amplifying influenza A virus M gene target sequences, the reverse transcription RT-RPA amplification primer pairs are designed for specific areas of subtypes H1N1, H2N2, H3N2, H5N1 and H7N9, and the reverse transcription RT-RPA amplification primer pairs are designed for specific areas of subtypes H1N1, H2N2, H3N2, H5N1 and H7N9; the crRNA library comprises a plurality of crRNA molecules which are respectively complementary with different conserved regions in the M gene target sequence and is used for guiding Cas12a protein to carry out specific recognition; a Cas12a protein reagent; the reporter molecule can be subjected to non-specific cleavage by the Cas12a protein; the enzyme preparation, the buffer solution and the nucleotide substrate are required by RT-RPA reaction. The invention aims to realize synchronous, rapid, high-sensitivity and high-specificity detection of various subtype influenza A viruses.

The invention discloses a respiratory infectious disease multi-pathogen nucleic acid detection kit based on a fluorescent PCR detection technology and application of the respiratory infectious disease multi-pathogen nucleic acid detection kit. The invention provides two detection kits aiming at 20 and 6 respiratory tract detection, the two kits can specifically detect respiratory tract pathogens, the detection sensitivity is high, the whole detection system is short in time consumption and simple and convenient in use process, and detection results of 20 pathogenic microorganisms can be reported within 40 min at the shortest; the whole set of detection kit and the detection method are high in sensitivity and good in specificity; the normal-temperature storage and transportation of the reagent can be realized.

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

The invention relates to the technical field of phytopathogen detection, and particularly discloses a primer, a kit and a method for detecting crop root rot fusarium solani and application, the primer comprises a specific RPA primer RPA-F and a specific RPA primer RPA-R; the specific RPA primer RPA-F is as shown in SEQ ID NO. 1, and the specific RPA primer RPA-R is as shown in SEQ ID NO. 2; the kit further comprises a guide RNA primer crRNA, and the guide RNA primer crRNA is as shown in SEQ ID NO. 3. The kit comprises a specific RPA primer RPA-F, a specific RPA primer RPA-R, a Cas12 protein, a T7 transcriptase, a crRNA DNA template, a DNA polymerase and a Cas12 / 13 nucleic acid detection test strip. The kit has the characteristics of good sensitivity, relatively good specificity and enough reliability.

The invention relates to the technical field of biological detection, and discloses a mycoplasma pneumoniae nucleic acid rapid detection method, which comprises the following steps: mixing a sample to be detected with a multifunctional pretreatment buffer solution containing tris (hydroxymethyl) aminomethane, potassium acetate, polyethylene glycol, saponin, spermine tetrahydrochloride and tris (2-carboxyethyl) phosphine hydrochloride, and carrying out cracking treatment; a cracking mixed solution is obtained; redissolving the enzymatic isothermal amplification basic reagent, the specific primer and the probe to obtain a redissolving amplification system; sucking the supernatant of the cracking mixed solution, adding the supernatant into a redissolving amplification system, and adding a magnesium acetate solution to obtain a reaction mixed solution to be detected; and carrying out isothermal amplification and signal acquisition to obtain a detection conclusion. The multifunctional pretreatment buffer solution is used for cracking the to-be-detected sample, sample liquefaction, cracking and nucleic acid release are completed in a single system, the nucleic acid extraction and purification step of centrifugal column adsorption or magnetic bead elution is avoided, and the operation process is simplified.

The invention discloses a preparation method of light-regulated single-stranded DNA and application of the light-regulated single-stranded DNA in CRISPR nucleic acid detection. According to the invention, Lambda excision enzyme and a primer modified with PC-Linker are introduced into a detection system based on RPA-CRISPR / Cas12a, and a one-tube nucleic acid detection system without PAM limitation is constructed. By introducing Lambda excision enzyme, the strict dependence of the traditional CRISPR / Cas12a detection technology on PAM sites is broken through, and the application range of the CRISPR / Cas12a detection technology is remarkably widened. And the system is integrated into one-tube detection, so that the risk of aerosol pollution caused by opening a cover for multiple times and transferring a sample is avoided while the experimental operation process is simplified. According to the research, CRISPR / Cas12a and CRISPR / Cas13a are further integrated into a single-tube reaction system, so that the probability of false negative results is reduced in a double-gene detection manner, and the detection accuracy is improved.

The invention provides a detection method and application of miRNA and analogues thereof, and relates to the field of nucleic acid detection. According to the invention, a probe 1 and a probe 2 are designed for target miRNA and miRNA analogues; the probes are respectively combined with a part of miRNA, the probe 2 can be combined to a chip, a microsphere and other solid phase carriers, and the probe 1 modifies a fluorescent dye or biotin and other signal labeling materials; after the target miRNA or miRNA analogue is mixed with the probe 1 and the probe 2 and incubated, the probe 2 is not hybridized with the miRNA analogue and only hybridized with the miRNA analogue and captures the target miRNA by optimizing the hybridization temperature, so that the detection specificity is high.

The invention relates to the technical field of medical diagnosis, and discloses an integrated nucleic acid detection method and device based on a micro-fluidic chip, the integrated nucleic acid detection device comprises a micro-fluidic chip module, a liquid control module, a temperature control module, an optical detection module and a data processing module, and the multiple modules work cooperatively to realize full-process automatic detection of a sample from splitting decomposition to detection; the micro-fluidic chip module comprises a sample treatment area, a nucleic acid extraction and purification area, a nucleic acid amplification area, a nucleic acid detection area and a waste liquid collection area which are connected through a micro-channel, and a valve is arranged at a key node. Through a fluid control mode combining centrifugal force and air pressure driving and valve combination of non-complex micro-pump micro-valves such as the dissolvable membrane valve, the temperature sensitive valve and the capillary force flow limiting valve, full-flow fluid sequential control can be achieved without integrating a plurality of micro-pump micro-valves; the problems of complex structure, high manufacturing difficulty and high cost caused by integration of multiple micro-pumps and micro-valves of the conventional pressure-driven micro-fluidic chip are solved.

The invention relates to the technical field of genetic engineering, and particularly provides a multi-linked quality control product for quality control of a novel coronavirus nucleic acid detection laboratory and a preparation method of the multi-linked quality control product. The multi-linked quality control product is a multi-component composite system and is formed by mixing three substances, namely pseudovirus, target gene RNA (Ribonucleic Acid) molecules and internal standard plasmid DNA (Deoxyribose Nucleic Acid). Wherein the pseudovirus is pseudovirus particles of novel coronavirus ORF1ab gene RNA (Ribonucleic Acid) wrapped by a vesicular stomatitis virus vector and is used for monitoring virus nucleic acid extraction; the target gene RNA molecule is a novel coronavirus N gene RNA molecule and can monitor the reverse transcriptase activity; the internal standard plasmid is plasmid DNA of a human housekeeping gene RPP30, and whether inhibition or failure exists in an amplification link is monitored. According to the method, the whole process of nucleic acid extraction, reverse transcription and amplification is synchronously and hierarchically monitored, the monitoring blind area of a traditional single-component quality control product is solved, and the accuracy and efficiency of laboratory detection quality control are remarkably improved.

The invention belongs to the field of nucleic acid detection and molecular biology, and particularly relates to a method and a kit for eliminating false positive results in nucleic acid amplification reaction. According to the method, after a nucleic acid amplification reaction (taking recombinase polymerase amplification, namely RPA, for example) is completed, a target amplification product is subjected to selective enzyme digestion by utilizing restriction endonuclease, non-target products (such as primer dimers and non-specific amplification products) are not cut, and meanwhile, the non-target products which are not subjected to enzyme digestion are removed by combining solid-phase separation, so that the target amplification product is obtained. Therefore, the false positive result is eliminated. The invention also discloses a kit containing the restriction enzyme. According to the method, the specificity and the signal-to-noise ratio of a nucleic acid amplification reaction, especially RPA, are remarkably improved, the cost is low, operation is easy, the method is compatible with an existing technical platform, the method is suitable for detection scenes such as clinical molecular diagnosis, environmental monitoring and food safety, and the problem of misjudgment caused by false positive signals is effectively avoided.

The invention discloses a nucleic acid detection consumable processing module, a detection system and a use method, and belongs to the field of medical examination and inspection instruments and molecular diagnosis and detection instruments, the technical key points are that the nucleic acid detection consumable processing module comprises a driving module, a temperature control and clamping integrated module, an L-shaped consumable lifting bracket and a flip module; and the driving module is used for driving the temperature control and clamping integrated module to clamp / loosen a detection chip of the consumable, driving the L-shaped consumable lifting bracket to ascend / descend and driving the flip module to rotate at the same time. The invention aims to provide the nucleic acid detection consumable processing module, the detection system and the use method, and the operation of sample processing and detection is simplified.

The invention discloses a primer group, a kit and a method for rapidly and visually detecting silver salmon components, belongs to the technical field of nucleic acid detection of marine fish products, and particularly relates to a method for rapidly and visually detecting the silver salmon components by using an LAMP (loop-mediated isothermal amplification) technology. The invention provides a primer combination suitable for rapid nucleic acid detection and a corresponding kit, the primer combination comprises an outer primer pair and an inner primer pair, and the kit integrates the primer combination and an optimized LAMP reaction mixed solution. The detection means has a plurality of prominent characteristics that during detection, only DNA of a sample to be detected needs to be added into an LAMP reaction mixed solution prepared in advance, and the detection work can be carried out by means of common constant-temperature equipment. After the whole detection process is about 60 minutes, the result interpretation can be completed by observing the color development condition with naked eyes, and when the reaction mixed solution is yellow, the reaction mixed solution is judged to be positive; and if the color is red, determining that the color is negative. Due to the design of closed tube detection, the hidden danger of aerosol pollution caused by uncovering operation is effectively avoided. The method is excellent in specificity and relatively high in detection sensitivity, and can detect 10 <-1 > ng of genome DNA or a sample with the mass fraction of 1% at least. The method has the advantages of good result reproducibility, simplicity and convenience in operation and low cost. The method is particularly suitable for being applied to scenes with relatively limited resources such as basic units and field detection, the cost investment in the aspect of supervision can be remarkably reduced, and the efficiency of detection work is improved.

The invention discloses a multiple nucleic acid detection device and a use method, the multiple nucleic acid detection device comprises a card box and multiple chips which are communicated with each other, multiple detection of the whole process is realized in a form of integrating the extraction card box and the multiple detection chips, the sample transfer step is reduced, the pollution risk is reduced, the detection sensitivity is improved, and the detectable target range is widened. A plurality of independent functional chambers are arranged in the card box, under the cooperation of a near-end flow channel opening and a far-end flow channel opening, a reaction chamber is communicated with other functional chambers and sample injection holes required by nucleic acid reaction, and tedious magnetic bead method nucleic acid extraction, nucleic acid release and removal of impurities in a sample are realized in the card box to obtain pure nucleic acid. Meanwhile, the nucleic acid concentration is improved, the subsequent detection sensitivity can be improved, the internal part of the extraction card box, the internal part of the detection chip and the connection between the card box and the chip are fully sealed, and cross contamination among multiple reaction chambers and aerosol pollution to the environment during amplification are prevented.

Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.

The invention discloses an amplification chip and an amplification analysis device, and belongs to the field of nucleic acid detection, the amplification chip comprises a chip main body provided with a flow channel, the amplification chip also comprises a temperature control assembly, the temperature control assembly comprises at least one heating member, the at least one heating member heats a part of the flow channel, so that the flow channel forms at least two areas with different temperatures, and the heating member is used for heating the flow channel. The reagent reciprocates in two regions with different temperatures, so that the reaction time of the reagent is shortened, and the detection sensitivity of the amplification chip is improved.

The invention belongs to the field of nucleic acid detection, and discloses a method for detecting mycobacterium paratuberculosis based on a CRISPR-Cas12a technology. Aiming at the problems that an existing MAP detection method is time-consuming, high in cost and low in sensitivity and specificity, and an existing CRISPR technology is poor in adaptability, insufficient in detection of low bacterium loading amount and the like, the method is combined with recombinase polymerase amplification (RPA) and nested polymerase chain reaction (PCR), takes an MAP high-conservative genome sequence as a target, and fuses high sensitivity of nucleic acid amplification and high specificity and signal amplification capability of CRISPR-Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-CRISPR-CRISPR-CRISPR-CRISPR-CRISPR-Cas12a). The lowest detection limit of the kit reaches 1020M, which is far better than the single use effect of the traditional PCR or CRISPR, and the kit realizes ultra-sensitive, high-specificity, rapid and low-cost detection, and is suitable for paratuberculosis early infection or low-bacterial-loading sample detection.

The invention provides an RPA-PAND detection kit and detection method for canine parvovirus and canine adenovirus, and belongs to the technical field of canine virus detection.The kit comprises PfAgo protein and a gDNA and MB combination used for detecting canine parvovirus MVC, canine parvovirus CPV and canine adenovirus CAV. The RPA-PAND rapid nucleic acid detection method for detecting canine parvoviruses MVC and CPV and canine adenovirus CAV is successfully established on the basis of PfAgo protein mediated target detection and in combination with the RPA technology, and the method has the advantages of high sensitivity, high specificity, low cost, relatively low position targeting limitation, simple and convenient reaction system, no aerosol pollution, capability of performing multi-channel nucleic acid detection and the like; and a new reagent and a new way are provided for clinical diagnosis and epidemiological monitoring of viruses MVC, CPV and CAV.

The utility model relates to the technical field of biological detection, in particular to a fluorescent PCR (Polymerase Chain Reaction) nucleic acid detection reagent strip for detecting Listeria monocytogenes, which is characterized in that after a sample to be detected is added into the reagent strip through a sample adding hole, the sample flows into a nucleic acid extraction area for nucleic acid extraction, and the extracted nucleic acid enters an amplification reaction area for amplification reaction; when a target nucleic acid sequence is amplified and then enters fluorescence detection to generate a fluorescence signal, a sensor in a fluorescence detection area captures the fluorescence signal in real time and transmits the signal to a control chip, the control chip processes and analyzes data, and a result is transmitted to external equipment (such as a computer, a mobile phone and the like) through a data transmission module. The rapid detection on the listeria monocytogenes is realized. The reagent strip disclosed by the utility model integrates the functions of nucleic acid extraction, amplification reaction and detection, is simple and convenient to operate, does not need complex sample pretreatment and professional experimental equipment, and is suitable for on-site rapid detection and basic application.

The invention discloses a primer group, a kit and a method for rapidly identifying components of salmon red, belongs to the technical field of nucleic acid detection of marine fish meat products, and particularly relates to a method for rapidly identifying the components of salmon red by using an LAMP (loop-mediated isothermal amplification) technology. The invention provides a primer group for rapid nucleic acid detection and a matched kit, the primer group is composed of an outer primer pair, an inner primer pair and a single loop primer, and the kit comprises the primer group and an optimized LAMP reaction mixed solution. The detection method has the following remarkable characteristics: the DNA of a sample to be detected only needs to be added into the pre-prepared LAMP reaction mixed solution, the detection can be completed by using common constant-temperature equipment, the whole process is about 60 minutes, the color development result can be observed by naked eyes to realize interpretation, if the reaction mixed solution is yellow, the reaction mixed solution is positive, and if the reaction mixed solution is red, the reaction mixed solution is negative. According to the closed tube detection design, the aerosol pollution risk caused by uncovering operation is avoided. The method is excellent in specificity, detection sensitivity (the lowest 10 <-1 > ng genome DNA can be detected or the mass fraction is 1%) and result reproducibility, has the characteristics of simplicity and convenience in operation and low cost, is particularly suitable for resource limited scenes such as grassroots units and field detection, and can remarkably reduce the supervision cost and improve the detection efficiency.

This invention provides a CRISPR / Cas12a fluorescence detection method for FocTR4 based on magnetic bead-ssDNA-HRP probes, belonging to the field of nucleic acid detection technology. This invention involves isothermal amplification of the FocTR4 target sequence using RPA primers; then, a magnetic bead-ssDNA-HRP probe is prepared; and the Cas12a / crRNA complex, RPA amplification product, and probe are co-incubated. The activated Cas12a cleaves the probe, releasing HRP. After magnetic separation, a colorimetric solution containing OPD is added, and the presence and concentration of FocTR4 are determined by detecting the fluorescence signal. This invention combines the high efficiency of CRISPR / Cas12a cleavage with the high sensitivity and strong specificity of RPA technology, achieving highly sensitive and specific detection of FocTR4, with a linear detection range of 10. 2 ~10 7 With a detection limit as low as 70 CFU / mL, it can quickly and accurately detect Foc TR4 in soil samples, enabling rapid on-site detection and providing reliable technical support for the early diagnosis and control of banana wilt disease.

The invention relates to a normal temperature amplification-CRISPR Cas12a one-pot method detection system and a nucleic acid detection method based on the detection system. The detection system consists of a normal-temperature nucleic acid amplification system (unit I) which focuses on target signal amplification and a CRISPR Cas12a system (unit II) which focuses on target recognition and signal output. By means of a self-driven and deoxyribozyme-mediated reaction equilibrium transfer strategy, the equilibrium in the initial stage of the reaction tends to the unit I, and the equilibrium in the middle and later stages of the reaction tends to the unit II, so that the compatibility of the two units is remarkably improved. By means of the system, high-sensitivity and high-specificity portable one-pot detection of nucleic acid targets can be achieved.