58 results about "Recombinase Polymerase Amplification" patented technology

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 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.

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 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 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.

The invention discloses an Alternania alternana RPA (recombinase polymerase amplification) lateral flow test strip detection kit and application thereof in Bo-chrysanthemum disease detection, and belongs to the field of rapid detection of plant pathogenic fungi. The detection kit provided by the invention comprises a reagent for detecting Alt a 1, the reagent comprises a first RPA primer, and the nucleotide sequences of the first RPA primer are respectively SEQ ID NO.1 and SEQ ID NO.2; the reagent for detecting TEF1 alpha comprises an RPA primer II, and the nucleotide sequences of the RPA primer II are respectively SEQ ID NO.4 and SEQ ID NO.5; and an RPA reaction component. Compared with the prior art, the kit disclosed by the invention has the advantages that through mutual verification of double targets, the detection specificity and accuracy are improved, false negative caused by gene variation of a single target is avoided, and the identification capability on a complex sample is improved.

The present disclosure relates to the technical field of molecular diagnosis, and particularly, to a microsphere formulation for nucleic acid amplification, an amplification method, and use in joint detection. The disclosed microsphere formulation can be stored for a long duration at 2-8° C. When the microsphere preparation is used, no solvent is additionally added, and the microsphere formulation is directly mixed with a sample to be detected. On the premise that the concentration of an original system is not changed, the template content in the system can be remarkably improved, so that the detection sensitivity is improved. By using the microsphere formulation in recombinase polymerase amplification (RPA), recombinase-aided amplification (RAA), or a double or multiple detection formed by combining a second reaction on the basis of RPA or RAA, the sensitivity can be remarkably improved, and meanwhile, the amplification specificity is ensured.

The present application relates to the field of molecular diagnosis and nucleic acid amplification, and particularly relates to a recombinase polymerase amplification method based on Twinkle helicase enhancement and application thereof. The present application first introduces Twinkle helicase into the RPA system, utilizes the ATP-dependent strand separation activity and DNA annealing activity of the Twinkle helicase, assists primer binding and template strand separation, and thus significantly improves amplification efficiency and detection sensitivity. The present application is suitable for plasmid DNA, high GC template and clinical nucleic acid sample, and can construct an RPA kit containing Twinkle, and expand the application of the RPA kit in pathogen detection, molecular diagnosis and on-site rapid detection. The present application overcomes the shortcomings of the existing RPA technology in sensitivity and stability, and provides a new solution for developing a new type of nucleic acid self-detection and on-site detection tool.

The invention discloses an immune nucleic acid amplification detection kit and detection method based on MS2 virus-like particles, the kit contains the MS2 virus-like particles, the surface of the MS2 VLP displays an antigen conjugate capable of being specifically bound with an object to be detected, and a nucleic acid template is wrapped in the MS2 VLP; after the MS2 VLP is specifically combined with an object to be detected, detecting nucleic acid fragments wrapped in the combined MS2 VLP by adopting a recombinase polymerase amplification technology or real-time fluorescent quantitative PCR (Polymerase Chain Reaction), so as to improve the detection sensitivity of target protein. By utilizing the method, simple, highly sensitive and specific target molecule detection can be realized, the bottleneck of the traditional immunodetection method in sensitivity is broken through, and the problem of low coupling efficiency of the antibody and nucleic acid is effectively solved; the method is also suitable for the field of food safety (such as cereal toxin detection) and the field of clinical diagnosis (such as detection of low-abundance biomarkers in body fluid), and a new technical solution is provided for molecular detection.

The invention discloses an RPA primer for detecting sclerotium rolfsii, a primer probe combination, a kit and application, and belongs to the technical field of biological detection. The invention provides an RPA (recombinase polymerase amplification) primer for detecting sclerotium rolfsii. The RPA primer comprises primer sequences as shown in SEQ ID NO: 44 and SEQ ID NO: 52. According to the invention, the RPA technology is applied to the molecular detection of the sclerotium rolfsii of the coptis southern blight pathogen for the first time, and the method has the advantages of strong specificity, high sensitivity, simple reaction conditions, short reaction time and the like. According to the method, a PCR instrument, gel electrophoresis and an imaging system are not needed, the detection efficiency is greatly improved, and the problems that an existing conventional detection means needs long time, and the requirements for instrument and equipment conditions and experimenters are high are solved. The invention provides technical support for early detection and on-site detection of sclerotium rolfsii, and provides a basis for preventing and treating coptis chinensis sclerotium rolfsii.

Methods as well as related compositions and kits for recombinase-mediated nucleic acid amplification, such as recombinase-polymerase amplification (RPA), of a nucleic acid template using at least one blocked primer that contains a 5′ domain, at least one nucleotide that is cleavable by an RNase H enzyme, a 3′ domain, wherein the primer is not extendable by a polymerase, and wherein the 3′ domain has a length of 7-100 nucleotides, for example 10-30 nucleotides. These methods and the use of a blocked primer reduce or eliminate non-specific amplification products, such as primer dimers, which are generated in RPA reactions.

This invention relates to the fields of microfluidic chip technology and molecular diagnostic technology, and discloses a self-driven digital microfluidic chip and its application in digital recombinase polymerase amplification. The chip comprises two layers: an upper PDMS sheet and a lower glass plate. The lower surface of the upper PDMS sheet is etched with a microfluidic channel structure, including a sample loading port, a droplet reactor array, microchannels, and a pump chamber. The upper and lower layers are thermally bonded to form a sealed flow channel. Based on the gas permeability of PDMS material, this invention achieves self-driven sample introduction and digital dispensing by pre-degassing to create negative pressure, eliminating the need for any external equipment. The chip structure of this invention is rationally designed, with a simple preparation process, low cost, and convenient operation. It can automatically complete sample digitization within 5-10 minutes, supports multi-channel parallel detection, is highly compatible with RPA reaction systems, and can achieve absolute quantitative analysis of target nucleic acids. It has advantages such as high detection sensitivity, accurate quantification, and good repeatability, and has broad application prospects in the field of molecular diagnostics.

The invention provides a composition, a kit, an application for detecting human papilloma virus in a sample, a method for detecting the existence or type of the human papilloma virus in the sample, a model construction method for predicting the recombinase polymerase amplification reaction efficiency of a virus template chain, and a prediction method for the recombinase polymerase amplification reaction efficiency. The present invention relates to a composition comprising: a number of different bait oligonucleotides wherein the number of different bait oligonucleotides are configured to hybridize to a number of DNA molecules that are at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of a sequence derived from any one or number of SEQ ID NO: 91-100, and wherein the number of DNA molecules is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of a sequence derived from any one or number of SEQ ID NO: 91-100; the composition can be used for detecting the human papilloma virus with the genotype.

The application discloses a primer, a probe and a kit for detecting Ustilaginoidea virens based on a recombinase polymerase amplification method, the primer comprises a forward primer F-primer shown in SEQ ID NO. 1 and a reverse primer R-primer shown in SEQ ID NO. 2; and the sequence of the probe Probe is shown in SEQ ID NO. 3. Compared with a traditional detection technology for identifying Ustilaginoidea virens based on morphological characteristics and using ordinary PCR, the application has higher accuracy, sensitivity and effectiveness, is convenient to operate, has good practicability, provides a new technical platform for detection of Ustilaginoidea virens, and can be used for high-sensitivity and rapid detection of rice smut caused by Ustilaginoidea virens.

Swift and accurate on-site identification of genomes of pathogens is crucial to curb its spread and contamination. In embodiments disclosed herein, a synthetic biology cascade reaction is presented on a paper substrate using CRISPR-Cas12a and recombinase polymerase amplification (RPA), enabling the programming of a standard toehold RNA switch for a genome of choice through insertion of meticulously designed an oligonucleotide (probe AB).

The invention belongs to the technical field of microbiological detection, and particularly relates to a rapid detection method of pseudomonas aeruginosa RAA-CRISPR / Cas12a and application of the rapid detection method of pseudomonas aeruginosa RAA-CRISPR / Cas12a. According to the invention, based on the combination of recombinase polymerase amplification (RAA) and a CRISPR-Cas12a (clustered regularly interspaced short palindromic repeats-CRISPR-Cas12a) system, the ultrasensitive and rapid detection of the Pseudomonas aeruginosa is realized through constant-temperature nucleic acid amplification and specific gene recognition. The detection limit of the method for detecting the pseudomonas aeruginosa is 5.37 * 10 < 1 > copies / L (genome DNA) or 6.4 * 10 < 1 > CFU / mL (bacterial liquid); the detection time is only 21 minutes, and the method can be applied to rapid detection of various environmental samples.

Systems and methods for detecting the presence of a target nucleic acid in a sample via a recombinase polymerase amplification (RPA) reaction followed by a FEN1 cleavage detection reaction are disclosed. One aspect of the present disclosure relates to systems involving a sample collection device for collecting a sample and performing an RPA reaction on the sample, followed by the detection of the amplified product via a two-step FEN1 cleavage detection reaction which generates a fluorescent signal indicative of the presence of amplified product.

The invention discloses a kit and a method for high-sensitivity detection of nervous necrosis virus of grouper based on RT-ERA-Cas12a. An ERA enzymatic recombinant isothermal amplification technology is combined with CRISPR / cas12a, on the basis of designing and screening appropriate ERA primers, crRNA and ssDNA probes, reverse transcription of RGNNV viruses, recombinase polymerase amplification and a CRISPR / cas12a in-vitro cutting system are integrated in a PCR tube, and the grouper nervous necrosis virus is detected through one-tube type RT-ERA-Cas12a. The method provides a means for detecting the nervous necrosis virus of grouper on site, has the characteristics of rapidness (40-50 minutes), high sensitivity (5 copies / reaction) and no need of expensive instruments at normal temperature (35-40 DEG C), and has practical significance for farmers to timely know the illness state of cultured fishes and take prevention and control measures on site.

The invention relates to the technical field of biological detection, in particular to a primer probe combination, a kit and a method for identifying solenopsis invicta through fluorescent RPA (recombinase polymerase amplification). The primer probe combination for identifying the solenopsis invicta through the fluorescent RPA comprises a primer F1, a primer R1 and a probe P1. Wherein the nucleotide sequence of the probe P1 is SEQ ID NO: 3, and thymine at the position, 29bp away from the 5'end, of the probe P1 is modified by a fluorophore FAM; a G basic group at the position, 30 bp away from the 5'end, of the probe P1 is replaced by dSpacer, thymine at the position, 32 bp away from the 5 'end, of the probe P1 is modified by a quenching group BHQ1, and the 3' end of the probe P1 is connected with ddC. The primer and probe combination is used for fluorescent RPA identification of solenopsis invicta and has the advantages of being good in specificity and high in sensitivity.