131 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 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 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 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 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 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 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.

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 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.

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.

The present invention provides compositions, probes, methods, and point-of-need testing devices for detecting a single nucleotide polymorphism in target DNA sequence. The compositions comprise a probe, a forward primer, a reverse primer, a RNascHII, and a recombinase polymerase amplification reagent mix.

The invention belongs to the technical field of genetic engineering and diagnostic reagents, and particularly relates to a brucella recombinase polymerase amplification (RPA) detection kit and application, and the detection kit comprises a specific primer group and a probe. According to the invention, the RPA amplification product is detected by using the test strip, after amplification is carried out for about 10 minutes, the RPA amplification product is dropwise added into the test strip to read a result, and a PCR (Polymerase Chain Reaction) amplification instrument and a quantitative PCR instrument are not needed. The obtained method has the advantages of strong specificity, good sensitivity, simple operation process, rapid detection and the like, provides a possibility for the detection of brucellosis in the field, and also provides a method for the research of brucellosis which does not aim at the diagnosis and treatment of diseases.

The invention discloses a rapid detection method for three pathogenic bacteria of rice based on an RPA-CRISPR / Cas12b technology, and belongs to the technical field of nucleic acid detection. The method provides a specific RPA (recombinase polymerase amplification) primer pair and an sgRNA (single guide ribonucleic acid) sequence for three quarantine pests, namely BG (rice blight), XOC (rice bacterial leaf streak) and XOO (rice bacterial leaf blight). Recombinase polymerase amplification (RPA) and CRISPR / Cas12b detection technologies are combined and are integrated on a centrifugal micro-fluidic chip, so that high-sensitivity and high-specificity detection on multi-target nucleic acid is realized, integrated detection of'sample input-result output 'can be completed within 30 minutes, uncovering operation is effectively avoided, the risk of aerosol pollution is greatly reduced, and the method is suitable for industrial production. The method is suitable for port quarantine and field on-site rapid detection, and an efficient technical means is provided for early diagnosis and prevention and control of rice diseases.

The present invention discloses a primer-probe combination, a kit and an application for the constant temperature rapid detection of Vibrio coralliiformis. The primers are shown in SEQ ID NO.1 and SEQ ID NO.2, and the fluorescent probe is 5'-GATGCAACGCGAAGAACCTTACC TACTCT(FAM-dT)GAC(THF)(BHQ1-dT)CCTCAGAAGAGACT-3'. The primer-probe combination of the present invention is used to perform real-time fluorescent recombinase polymerase amplification on the sample to be tested, and the test results can be obtained within 20 minutes at 39°C. The detection equipment is small and easy to carry, providing a more convenient detection means for on-site and resource-scarce places. In addition, the detection sensitivity of the kit of the present invention is 10 times that of qPCR, and the reaction speed is faster than qPCR.

The invention provides a primer for detecting bordetella pertussis through RPA (recombinase polymerase amplification)-lateral flow immunochromatography. The primer comprises specific primer pairs NAT-F and NAT-R which are designed aiming at transposase genes of a bordetella pertussis IS1663 family, and specific primer pairs NAT-F and NAT-R which are designed aiming at transposase genes of a bordetella pertussis IS1663 family, the invention also provides corresponding probes, which comprise a gold nanoparticle binding probe, a quality control line capture probe and a bordetella pertussis IS1663 capture probe. Meanwhile, the invention further provides application of the primer and the probe in preparation of a pertussis bordetella detection product, a specific detection kit and a specific detection method. According to the invention, the specific primer and probe are designed aiming at the transposase gene of the pertussis bordetella IS110-like element IS1663 family, and the rapid, high-sensitivity and high-specificity detection of pertussis bordetella is realized by fusing the rapid isothermal amplification technology of RPA and the visual detection advantage of lateral flow immunochromatography, no complex instrument or professional operation is needed, and the kit has the advantages of high sensitivity, high specificity, high sensitivity and high specificity. The method is suitable for multi-scene early convenient diagnosis.

The application discloses a method and application of a Cas12a sensing system based on an sgRNA blocking strategy for single nucleotide polymorphism typing. The method comprises the following steps: pre-annealing sgRNA and blocking chains to form a complex; obtaining a single-stranded DNA target by using an asymmetric recombinase polymerase amplification; constructing a detection system comprising a Cas12a protein, the complex, a target and a fluorescent substrate; activating the trans-cleavage activity of Cas12a through a strand displacement reaction to generate a fluorescent signal, so as to realize SNP typing. The method combines the sgRNA blocking strategy with the Cas12a system for the first time, does not need to depend on a PAM sequence, has the advantages of high universality, high specificity, simple operation, rapidness, low cost and the like, and is suitable for ApoE gene typing and other SNP related detection.

The invention discloses a primer and probe composition and a kit for detecting a chrysanthemum B virus, and a detection method and application of the primer and probe composition, the kit, the detection method and the application of the primer and probe composition, the rapid detection of the chrysanthemum B virus is completed within 5 minutes through the combination of recombinase polymerase amplification (RPA) and lateral flow test strip (LFD) technologies and the combination of isothermal amplification and visual detection advantages, and the sensitivity is 1000 times of that of a conventional RT-PCR method. And the method does not depend on complex instruments and equipment and is suitable for field and basic laboratory operation. The primer and the probe are designed for a conserved domain of a chrysanthemum B virus genome, do not have cross reaction with common chrysanthemum viruses such as chrysanthemum dwarf viroid CSVd, chrysanthemum chlorotic mottle viroid CChMVd, tobacco mosaic virus TMV and the like and host plants, and are high in specificity. A detection result is directly interpreted through color development of the test strip, and the stability is high. The method is highly effective in chrysanthemum seedling detoxification identification, early warning of diseases and large-scale field screening, and provides technical support for accurate prevention and control of chrysanthemum virus diseases and healthy seed industry development.

The application discloses a RPA-CRISPR / Cas12a-based rapid visual detection method for manning's paragonimus. The method takes the mitochondrial cox1 gene as a target, screens a conservative region through sequence alignment, designs specific crRNA and RPA primers, optimizes Cas12a, crRNA, a fluorescent reporter molecule and a buffer system, and constructs a recombinase polymerase amplification combined CRISPR / Cas12a trans-cleavage detection system. Two visual detection strategies, visual fluorescence and lateral flow chromatography, are adopted, so that instant detection can be realized without large instruments and professional personnel, and the method has the advantages of high sensitivity and good specificity, can accurately detect manning's paragonimus DNA, cat and dog feces and blood samples, and brain schistosomiasis cerebrospinal fluid samples, and is suitable for on-site rapid screening.

The invention relates to the technical field of biology, in particular to a reagent and a detection method for real-time nucleic acid detection by a semi-quantitative one-pot method. Aiming at a one-pot method recombinase polymerase amplification (RPA) and CRISPR detection (CRISPR-Dx) reaction, a series of one-pot method RPA-CRISPR-Dx systems with different detection limits are constructed by redesigning an RPA primer, screening a CRISPR target PAM and combining the RPA primer and the CRISPR target PAM. A detection array constructed on the basis of the detection systems can output semi-quantitative results which do not need to be calculated and processed and can be read directly, and a powerful tool is supplemented for improving the real-time nucleic acid detection quantitative capacity.

The invention provides an RPA (recombinase polymerase amplification) primer group, a kit and a method for detecting the alternaria brassicae of broccoli, which are used for detecting the alternaria brassicae of broccoli. The primer group comprises specific primers F1 / R1, F1 / R1 and F2 / R1, and the sizes of theoretical amplification products of the primer group are 204bp, 187bp and 189bp. Specific target genes are screened out through comparative genomics, RPA primers are designed, and a constant-temperature (39 DEG C) and rapid (20 minutes) detection method is established. The primer provided by the invention has strong specificity, is only effective to the brassica oleracea var. Brassicae, and has no cross reaction with sibling species; the sensitivity is high, the detection lower limit is 0.08 ng / mu L, and the sensitivity is 100 times higher than that of conventional PCR. The actual sample verifies that the detection accuracy reaches 100%. The method is easy and convenient to operate, rapid in detection and suitable for field early diagnosis, plant quarantine and disease monitoring of the pathogenic bacteria.