170 results about "Recombinase Polymerase Amplification" patented technology

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas-based kit for isothermal rapid visual detection of H1N1 influenza virus and mycoplasma pneumoniae and a detection method thereof. The kit and the detection method provided by the invention can be used for timely, sensitively and specifically detecting a nucleic acid target in a sample. The invention provides a lateral flow analysis (LFA) detection platform of a three-stage amplification system integrating recombinase polymerase amplification (RPA), CRISPR / Cas12a and catalytic hairpin assembly (CHA) circulation, the detection platform is used for sensitively and accurately detecting mycoplasma pneumoniae DNA (MP DNA) and influenza A virus subtype H1N1RNA (H1N1RNA), and compared with other researches based on an LFA-CRISPR / Cas12a system, the detection platform has the advantages that the detection efficiency is greatly improved, and the detection cost is reduced. According to the kit and the detection method provided by the invention, nucleic acids of two pathogens can be visually detected on a single LFA at the same time, and the sensitivity is high.

The invention discloses a primer group, a kit and a method for detecting brucella, and belongs to the technical field of gene detection. The primer group comprises two pairs of specific RPA (recombinase polymerase amplification) primers: a primer pair B1F1 (SEQ ID No.3) and a primer pair B1R1 (SEQ ID No.6) targeting a B1 sequence, and a primer pair B2F1 (SEQ ID No.8) and a primer pair B2R1 (SEQ ID No.11) targeting a B2 sequence. The invention also provides a kit containing the primer group, and a detection method based on the RPA-CRISPR-Cas12a. The invention also provides a kit containing the primer group and a detection method based on the RPA-CRISPR-Cas12a. According to the application, the high efficiency of RPA amplification is combined with the ultrahigh specificity of CRISPR-Cas12a detection, the defects of long time consumption, low sensitivity and easy generation of false positive in the traditional method are overcome, rapid, sensitive and specific detection of Brucella is realized, and the application has important application value in early screening and monitoring of Brucella.

The invention relates to the technical field of rapid detection methods, in particular to a one-step detection method for urinary tract infection escherichia coli with a non-disease diagnosis or non-treatment purpose. According to recombinase polymerase amplification provided by the invention, target DNA is specifically amplified through primers, CRISPR / Cas12a specifically recognizes a target sequence through crRNA, and through double screening of RPA and CRISPR / Cas12a, the target sequence is specifically recognized through crRNA; the rapid, sensitive and specific detection on the Escherichia coli infected with the urinary tract can be realized. Meanwhile, an RPA primer, crRNA and reaction conditions are optimized, so that an RPA reaction system and a CRISPR / Cas12a reaction system can be premixed in a tube, and the reaction can be completed only by a one-step method. The method can specifically detect urinary tract infection escherichia coli and has no cross reaction with common pathogenic bacteria, optimized conditions can avoid the competitive effect of CRISPR / Cas12a and RPA, the reaction rate and sensitivity are guaranteed, and meanwhile, the risk of nucleic acid aerosol pollution is also avoided due to the fact that a cover does not need to be opened.

The invention provides a composition, a kit and a detection method for detecting staphylococcus aureus or a drug-resistant gene thereof based on RPA-CRISPR / Cas12a and application of the composition, the kit and the detection method, and belongs to the technical field of gene detection. A CRISPR / Cas12a system is combined with recombinase polymerase amplification (RPA), an RPA-CRISPR / Cas12a detection method for rapidly detecting the staphylococcus aureus in the wastewater and the drug-resistant gene of the staphylococcus aureus is established, a specific primer and crRNA are designed, the high-specificity nucleic acid cutting capability of a gene editing enzyme CRISPR / CAS system and the isothermal rapid amplification advantage of a nucleic acid isothermal amplification technology are fully fused, and the detection method has the advantages of high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity The fluorescent probe has high-specificity sequence recognition capability, realizes efficient detection of target nucleic acid, is strong in detection specificity, high in sensitivity and simple and convenient to operate, does not need complex equipment and professionals, and is suitable for rapid detection in an on-site environment with limited resources.

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 rapid streptococcus agalactiae detection kit based on RPA (recombinase polymerase amplification) and CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats) and application of the rapid streptococcus agalactiae detection kit. The rapid streptococcus agalactiae detection kit comprises an amplification primer of a recombinase polymerase amplification technology, CRISPR RNA (ribonucleic acid), an ssDNA (single-stranded deoxyribonucleic acid) probe and Cas12a protein Wherein an upstream primer sequence of an amplification primer of the recombinase polymerase amplification technology is as shown in SEQ ID NO: 1; and the sequence of the downstream primer is as shown in SEQ ID NO: 2. According to the invention, rapid, sensitive, accurate and specific detection of streptococcus agalactiae is realized.

The invention discloses a CRISPR / Cas12a-RPA (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 12a-recombinase polymerase amplification)-based sequence combination for rapidly detecting mouse hepatitis virus and a kit thereof, and belongs to the technical field of biological detection The sequence combination comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence; the kit comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence, the kit further comprises a mouse hepatitis virus negative control standard substance, RNase-free water, an RPA reaction buffer solution, an NEB buffer solution, Lba Cas12a nuclease, dry powder RPA reaction microspheres containing recombinase polymerase and magnesium acetate with the concentration of 280 mM. The kit can be used for rapidly detecting the mouse hepatitis virus. The primer sequence combination provided by the invention is good in conservative property and specificity and relatively high in sensitivity, and rapid detection of the mouse hepatitis virus can be realized.

The invention provides an HPV16 and HPV18 nucleic acid detection kit based on a one-step two-channel RPA-CRISPR, and belongs to the technical field of cervical cancer detection. The kit for detecting the HPV16 and the HPV18 by the one-step method, provided by the invention, comprises an RPA (recombinase polymerase amplification) reaction reagent, a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas13a detection reagent and a CRISPR / Cas12a detection reagent. The kit disclosed by the invention can be used for simultaneously detecting the HPV16 nucleic acid and the HPV18 nucleic acid on the basis of a one-step two-channel RPA-CRISPR / Cas12a-Cas13a, so that the multi-step transfer operation and the aerosol pollution risk of a two-step CRISPR reaction are effectively avoided, the HPV16 DNA as low as 2.5 copies / L and the HPV18 DNA as low as 5 copies / L can be detected in 30 minutes, and the specificity is good. The sensitivity of the kit for detecting cervix uteri and vagina swab samples is 94.7%, and the specificity is 100%.

The invention relates to the technical field of biology, and particularly discloses a method for detecting corn leukoderma pathogenic bacteria based on RPA-LFD (Recombinase Polymerase Amplification-Lateral Flow Dipstick), which comprises the following steps: a recombinase polymerase amplification RPA reaction system comprising an upstream primer PA-F2, a biotin-labeled downstream primer PA-R3-biotin and an nfo probe PA3-nfo-2; the lateral flow chromatography analysis LFD test strip is used for detecting an amplification product; a reaction buffer solution and a dilution buffer solution; a rapid and visual corn leaf spot detection method is developed by using a recombinase-mediated isothermal amplification (PRA) technology and combining an nfo probe with a lateral flow chromatography analysis and diagnosis method, and finally, an efficient and rapid corn leaf spot detection, prevention and control system is established by popularizing and applying a novel visual detection method in a field.

The present invention discloses a kit for detecting Fusarium pseudo-verticillium based on RPA-CRISPR / Cas system and its application, wherein the Cas protein is Cas12, the kit includes RPA primers and crRNA, the Fusarium includes Fusarium pseudo-verticillium, and the RPA primers and crRNA of Fusarium pseudo-verticillium are primer combinations FverF1 / FverR1 and Fv-crRNA, respectively. The present invention develops a field visualization rapid detection technology for corn stalk rot pathogens based on recombinase polymerase amplification technology combined with CRISPR / Cas12a for the widely distributed, highly isolated and highly pathogenic corn stalk rot pathogens. The technology can react at a constant temperature of 37-42°C without the need for complex instruments and equipment, and CRISPR / Cas12a can recognize and cut specific nucleic acid sequences, thereby further increasing the specificity and sensitivity of the detection on the basis of RPA.

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 present invention discloses a kit and a detection method for rapidly detecting Fusarium, a pathogen of corn stalk rot, wherein the Cas protein is Cas12, and the kit includes RPA primers and crRNA, wherein the Fusarium includes Fusarium graminearum, and the RPA primers and crRNA of Fusarium graminearum are primer combinations FgraF2 / FgraR2 and Fg-crRNA, respectively. The present invention develops a field visualization rapid detection technology for corn stalk rot pathogens based on recombinase polymerase amplification technology combined with CRISPR / Cas12a for the widely distributed, highly isolated and highly pathogenic corn stalk rot pathogens. The technology can react at a constant temperature of 37-42°C without the need for complex instruments and equipment, and CRISPR / Cas12a can recognize and cut specific nucleic acid sequences, thereby further increasing the specificity and sensitivity of the detection on the basis of RPA.

The invention belongs to the technical field of African swine fever virus detection, and particularly relates to an RPA primer for detecting African swine fever virus, a CRISPR / Cas12 system target, a kit and application. The method comprises the following steps: pre-treating a to-be-detected sample by using a nucleic acid rapid releasing agent; by designing a CRISPR / Cas suboptimal system and screening a target sequence, an RPA (recombinase polymerase amplification) technology is combined with the CRISPR / Cas suboptimal system, and a novel method for rapidly detecting the nucleic acid of the African swine fever virus by a one-tube method is developed. The method does not depend on professional laboratories, instruments and equipment, nucleic acid in a sample is subjected to extraction-free treatment by using a nucleic acid rapid releasing agent, the African swine fever virus nucleic acid is rapidly detected by using a one-tube method, a result is directly observed by naked eyes, and the limitation and the defects of application scenes of traditional ASFV nucleic acid detection are solved.

The invention belongs to the technical field of in-vitro detection, and particularly relates to a bovine parvovirus type I and type II RPA (recombinase polymerase amplification) detection kit and a detection method thereof. The kit provided by the invention comprises a dual RPA reaction system and RPA Basic freeze-dried powder containing recombinase. The dual RPA reaction system comprises a DNA (Deoxyribose Nucleic Acid) template of the virus, an upstream primer pair, a downstream primer pair, a Rehydration Buffer and magnesium acetate tetrahydrate. The kit reacts for 20 minutes at the temperature of 45 DEG C, and an obtained product is subjected to agarose electrophoresis detection. According to the invention, the upstream primer, the downstream primer and the reaction time are screened, so that the bovine parvovirus type I and the bovine parvovirus type II are simultaneously detected, and the problems of long time consumption, high detection limit and dependence on professional equipment and professionals in bovine parvovirus detection are solved. The invention provides an efficient and accurate solution for large-scale bovine parvovirus screening, and has a good application prospect.

The invention provides an aquatic animal source trypanosomes visual detection primer probe set, a kit and a detection method, and belongs to the technical field of parasite detection. The invention provides a detection reagent for aquatic animal source trypanosomes. The detection reagent comprises a recombinase polymerase amplification primer pair and a probe which are designed according to an SSU rDNA gene conserved region of the aquatic animal source trypanosomes. Aiming at SSU rDNA sequence conserved regions of multiple aquatic animal trypanosomes, the invention develops a detection method and a kit based on recombinase polymerase amplification combined with a lateral flow test strip technology, and the detection method and the kit have the advantages of strong specificity, high sensitivity, short time consumption, result visualization and the like, and have low requirements on detection sites, instruments and personnel; the method is suitable for rapid detection of trypanosomes in basic laboratories and farms, and has important significance on prevention and control of trypanosomes of aquatic animals.

The invention provides a multiple nucleic acid detection technology based on CRISPR / Gs12 system mediation and application, different Gs12 nuclease are respectively combined with corresponding specific crRNA and single-stranded DNA reporter genes, and then a recombinase polymerase amplification (RPA) method is combined, so that quadruple, triple or double specific accurate detection of target nucleic acid molecules can be realized. Therefore, a nucleic acid detection technology and a kit capable of simultaneously distinguishing the Seneca virus (SVA), the hog cholera virus (CSFV) and the African hog cholera virus (ASFV) are developed. The method provided by the invention can realize rapid multiple detection of target nucleic acid, and has good popularization and application values in the field of nucleic acid detection.

The invention relates to the field of molecular diagnosis and nucleic acid amplification, in particular to a recombinase polymerization amplification method based on Twinkle helicase enhancement and application of the recombinase polymerization amplification method. According to the invention, Twinkle helicase is introduced into an RPA system for the first time, and primer combination and template unlinking are assisted by utilizing ATP-dependent unlinking activity and DNA annealing activity of the Twinkle helicase, so that the amplification efficiency and the detection sensitivity are remarkably improved. The method is suitable for plasmid DNA, a high GC template and a clinical nucleic acid sample, and can be used for constructing an RPA kit containing Twinkle, so that the application of the RPA kit in pathogen detection, molecular diagnosis and on-site rapid detection is expanded. According to the invention, the defects of the existing RPA technology in sensitivity and stability are overcome, and a new solution is provided for developing a novel nucleic acid self-detection and field detection tool.

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 relates to the technical field of biological detection, and discloses a method for double-ring detection of lung cancer L858R mutation based on CRISPR / Cas9 and DNA, and the method comprises the following steps: enriching circulating tumor cells CTC from a blood sample; extracting genomic DNA (deoxyribonucleic acid) of the enriched CTC; carrying out amplification on the DNA fragment containing the L858R mutation by adopting recombinase polymerase amplification RPA (recombinase polymerase amplification); the amplified DNA fragment is hybridized with a recognition ring in a DNA double-ring structure, a DNA double ring is composed of the recognition ring and a report ring, and a part of region of the recognition ring is hybridized with a corresponding fragment of the report ring to form a staggered double-chain region; when L858R mutation exists, the mutated DNA and a recognition ring are hybridized to activate a CRISPR / Cas9 system, and under the guidance of sgRNA, Cas9 cuts hybrid double strands and releases a report ring; carrying out rolling circle amplification RCA under the action of DNA polymerase by taking the released report ring as a template; a fluorescent probe is used for detecting an RCA amplification product, and whether L858R mutation exists or not is judged according to a fluorescence signal. The CRISPR / Cas9 system is combined with a DNA double-ring structure, so that the ultra-sensitive and specific detection on the lung cancer L858R mutation is realized.

This disclosure relates to methods and compositions for detecting a target nucleic acid sequence using a dual-hapten probe. More specifically, the present disclosure relates to using recombinase polymerase amplification (RPA) and a dual-hapten probe to detect a target nucleic acid sequence. In some cases, the detection is on lateral flow strips.

The invention discloses a qualitative detection method and a quantitative detection method of salmonella based on RPA-CRISPR / Cas12a (recombinase polymerase amplification-CRISPR / CRISPR / Cas12a). The qualitative detection method comprises the following steps: reacting a mixed system at least comprising an RPA reagent, a salmonella upstream primer, a salmonella downstream primer, a CRISPR / Cas12a system and a to-be-detected sample to obtain a reaction product of the to-be-detected sample; and detecting the reaction product of the to-be-detected sample by adopting a fluorescence detection method or an electrochemical test paper detection method, and qualitatively detecting the salmonella in the to-be-detected sample according to an absorption peak at 500-520nm of a fluorescence spectrum or a T line and a current value of electrochemical test paper. According to the salmonella detection method provided by the invention, potential pollution risks in a traditional step-by-step reaction process are avoided, and meanwhile, the detection method shows high stability, relatively high specificity, excellent sensitivity and excellent field detection capability.

To provide RPA compositions and methods for detecting the presence or absence of a target nucleic acid using a dual-hapten probe.SOLUTION: This disclosure relates to methods and compositions for detecting a target nucleic acid sequence using a dual-hapten probe. More specifically, the present disclosure relates to detection of a target nucleic acid sequence using recombinase polymerase amplification (RPA) and a dual-hapten probe. In some cases, the detection is on lateral flow strips.SELECTED DRAWING: Figure 4

The invention discloses a pathogenic microorganism detection method based on RPA-THz (recombinase polymerase amplification-THz). The method comprises the following steps: constructing a rapid and efficient RPA reaction system and an enrichment method of main related pathogenic microorganism DNA (deoxyribonucleic acid); establishing a terahertz detection method for the RPA product of the main related pathogenic microorganism DNA; and a terahertz high-specificity, high-sensitivity and rapid detection normal form process of main related pathogenic microorganisms is established. The method can be used for rapidly, sensitively and specifically detecting pathogenic microorganisms threatening human safety. On the basis of the terahertz wave technology, RPA amplification and probe capture are combined, so that pathogenic microorganisms are recognized and detected through spectral information, possibility is provided for intelligent recognition of the pathogenic microorganisms, an important basis is provided for diagnosis and subsequent treatment of infectious diseases, and the method has important significance in treating patients, controlling epidemic situation development, reducing casualties and the like.

The invention discloses a detection kit based on orthogonal dual-channel label-free CRISPR-Cas and application of the detection kit, relates to the technical field of nucleic acid detection, and constructs an orthogonal dual-channel response system by utilizing the differentiated cleavage activity of Cas12a targeted DNA and Cas13 targeted RNA to a substrate. A Cas12a pathway is reported by utilizing protoporphyrin IX to be compounded with G-quadruplex G4DNA, and a Cas13a pathway is reported by utilizing DFHBI to be compounded with a Broccoli RNA structure, so that label-free signal transduction is realized. Through accurate screening of fluorophores, channel specificity is ensured, and optical signal crosstalk is eliminated. Mycobacterium tuberculosis (MTB) and respiratory syncytial virus (RSV) are used as model pathogens for verification, and a multiple recombinase polymerase amplification (RPA) technology is combined, so that the detection sensitivity on a synthetic target reaches a single molecule level. The system has high specificity, and even if the concentration of a non-target pathogen is increased by 100 times, no cross reaction exists.

The invention belongs to the technical field of microbiological detection, and particularly relates to a rapid detection method for listeria monocytogenes RAA-CRISPR / Cas12a and application of the rapid detection method for the listeria monocytogenes RAA-CRISPR / Cas12a. According to the present invention, based on the combination of the recombinase polymerase amplification (RAA) and the CRISPR-Cas12a system, the ultra-sensitive and rapid detection of the Listeria monocytogenes is achieved through the constant temperature nucleic acid amplification and the specific gene recognition; the sensitivity reaches 3.6 CFU / mL (bacterial liquid) and 3.78 copies / L (genome DNA); the detection time is shortened to 26 minutes, and the method can be applied to rapid detection of various environmental samples.

The invention discloses a quick freezing probe fixed CRISPR (clustered regularly interspaced short palindromic repeats) cyclic cleavage mediated signal enhanced vibrio parahaemolyticus biosensor and application thereof. The vibrio parahaemolyticus biosensor comprises an RPA (recombinase polymerase amplification) isothermal amplification primer pair, a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a system and an electrochemical three-electrode system, the RPA isothermal amplification primer pair comprises a tdh primer pair and a trh primer pair, the tdh primer pair comprises a tdh upstream primer and a tdh downstream primer, and the trh primer pair comprises a trh upstream primer and a trh downstream primer; the CRISPR / Cas12a system comprises tdh-crRNA (Ribonucleic Acid) aiming at a tdh gene, trh-crRNA aiming at a trh gene, and Cas12a protein. The vibrio parahaemolyticus detection method provided by the invention can be completed within 30 minutes, has the characteristics of simplicity, rapidness, strong anti-interference capability and the like, is suitable for the field of rapid detection of aquatic products, and opens up a new way in the aspect of promoting safety supervision of the aquatic products.

Neisseria gonorrhoeae is one of the most common bacterial sexually transmitted infections (STI). Diagnosis depends on standard nucleic acid amplification testing (NAAT), which is impracticable in most low-resource settings, where the prevalence of this STI is highest. Consequently, such areas utilize syndromic management, which misses a high proportion of cases and leads to antibiotic overuse, contributing to the troubling rise of resistance to the commonly prescribed ciprofloxacin, cefixime and ceftriaxone antibiotics for the treatment of N. gonorrhoeae infections. A specific, highly sensitive and cost-effective lateral flow assay is disclosed that utilizes CRISPR-Cas orthologs, multiplex SHERLOCK technology and isothermal amplification via recombinase polymerase amplification (RPA) for the rapid detection of antibiotic resistance to ciprofloxacin, cefixime and / or ceftriaxone at the point of care. This approach has the potential to increase treatment efficacy in the field and mitigate the spread of antibiotic resistance.

The application discloses a TdT signal amplification PAM-independent Cas12a lateral chromatography nucleic acid detection method and application. The method comprises the following steps: performing a first reaction on a first reaction system comprising a to-be-detected nucleic acid, a recombinase polymerase amplification reaction component, a recombinase polymerase amplification primer pair and an exonuclease, so as to obtain a single-stranded target nucleic acid; performing a second reaction on a second reaction system comprising the single-stranded target nucleic acid, a CRISPR / Cas12a reaction component, a reporter probe, a terminal deoxynucleotidyl transferase and a deoxynucleotide triphosphate substrate, so as to obtain an extension product; and contacting and hybridizing the extension product with a gold nanoparticle-labeled detection probe in a lateral chromatography process, and when a visual detection signal is obtained, it is judged that the to-be-detected nucleic acid is a target nucleic acid. The method of the application can significantly improve the detection sensitivity and signal contrast while getting rid of the PAM restriction, the detection process does not need complex instruments, the result can be judged by naked eyes, and the method is suitable for on-site rapid visual detection of pathogenic microorganism nucleic acid.

The invention discloses a kit and a method for rapidly and visually detecting grapefruit moth based on RPA-CRISPR / Cas12a. The kit comprises a recombinase polymerase amplification primer and a targeting crRNA sequence, and the amplification primer can specifically amplify a target nucleotide sequence derived from a grapefruit botryosphaeria maculata mitochondrial genome; the primer comprises an upstream primer and a downstream primer, the sequence of the upstream primer is as shown in SEQ ID NO: 1, and the sequence of the downstream primer is as shown in SEQ ID NO: 2; the target crRNA sequence can be specifically combined with a target nucleotide sequence generated by amplification of the RPA primer and activate the bypass cleavage activity of Cas12a protein, and the sequence of the target crRNA is as shown in SEQ ID NO: 3. The detection kit disclosed by the invention has the characteristics of strong specificity, high speed and high sensitivity, and can be used for detecting grape fruits at the initial stage of being infected by the grapefruit botryosphaeria hemsleyana.

This invention discloses a reaction tube for recombinase polymerase amplification (RPA), belonging to the field of nucleic acid detection. The reaction tube of this invention includes a tube body and a cap separate from the tube body. The tube body contains lyophilized bulbs or lyophilized powder containing premixed nucleic acid detection reagents, and the cap contains lyophilized powder containing an activator. The tube body and cap are sealed with a thin film. The lyophilized bulbs or lyophilized powder containing the premixed nucleic acid detection reagents include: recombinase, single-stranded DNA-binding protein, strand displacement DNA polymerase, buffer components, ATP, ATP regeneration components, and dNTPs. The lyophilized powder containing the activator includes: potassium ions, magnesium ions, and polyethylene glycol. This invention enables magnesium acetate to stably adhere to the cap, solving the problem of easy spillage of the lyophilized activator in the cap. In use, only the nucleic acid of the sample to be tested needs to be added; the operation is simple, easy to promote and apply, and suitable for batch detection.