238 results about "Recombinase Polymerase Amplification" patented technology

This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of the bacterial RecA and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods has the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods allow amplification of DNA up to hundreds of megabases in length.

This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods may allow amplification of DNA up to hundreds of megabases in length.

The present invention features novel, diverse, hybrid and engineered recombinase enzymes, and the utility of such proteins with associated recombination factors for carrying out DNA amplification assays. The present invention also features different recombinase ‘systems’ having distinct biochemical activities in DNA amplification assays, and differing requirements for loading factors, single-stranded DNA binding proteins (SSBs), and the quantity of crowding agent employed.

This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carry-over contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.

The present invention features novel, diverse, hybrid and engineered recombinase enzymes, and the utility of such proteins with associated recombination factors for carrying out DNA amplification assays. The present invention also features different recombinase ‘systems’ having distinct biochemical activities in DNA amplification assays, and differing requirements for loading factors, single-stranded DNA binding proteins (SSBs), and the quantity of crowding agent employed.

This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carry-over contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.

The invention discloses a quantitative detection method for specific nucleic acid fragments based on the CRISPR technology. The quantitative detection method comprises the following steps: acquiring atreated target nucleic acid sample, and adding the target nucleic acid sample and an aqueous-phase solution into a droplet generation device to generate reaction droplets, wherein the reaction droplets comprise at least one nucleic acid fragment to be tested; amplifying the nucleic acid fragment to be tested in the reaction droplets by using a recombinase polymerase amplification technique, and allowing target gene signals in the nucleic acid fragment to be tested to realize cascade amplification by using a gene editing technique; collecting a positive signal corresponding to a target gene, and converting the positive signal into image data; processing a fluorescence threshold in the image data by using the Poisson distribution principle so as to obtain the test corresponding to the number of the target gene, corresponding to the positive signal, in the nucleic acid fragment to be tested. Thus, rapid efficient quantitative detection of the concentration of the target gene is realizedbased on the CRISPR technology.

The invention discloses a one-step nucleic acid test method based on CRISPR / Cas (clustered regularly interspaced short palindromicrepeats / CRISPR-associated protein) and isothermal amplification and akit. The method is a quick field test method used for testing nucleic acid and displaying visual test results through fluorescent light or lateral side immunochromatographic test paper. The test accuracy is effectively ensured through specificity of crRNA (RISPR-derived ribonucleic acid); at a room temperature, whether a test sample contains test nucleic acid can be determined through the test paper or the fluorescent light within 1-2h; and compared with the traditional test method, the precision, simplicity and convenience in operation of the test method are improved to some extent. The kit with purified freeze-drying LbaCas12a, crRNA and RPA (recombinase polymerase amplification) can be formed; a nucleic acid segment of a specific sequence can be conveniently and quickly tested on site;a complicated temperature control instrument such as a PCR (polymerase chain reaction) instrument and other electrophoresis and centrifugation equipment are not required; and nucleic acid of the specific sequence can be tested quickly and sensitively only by a thermostatic device and fluorescence detection equipment and even visual observation.

A real-time isothermal recombinase-polymerase amplification detection kit for African swine fever viruses is disclosed. A forward primer sequence for detecting the African swine fever viruses through a method provided by the kit is shown as SEQ ID NO:1, a reverse primer sequence is shown as SEQ ID NO:2 and a probe sequence is shown as SEQ ID NO:3. A real-time isothermal recombinase-polymerase amplification method provided by the invention for ASFV detection is simple and convenient in operation, rapid in reaction and low in detection cost, can be used for ASFV detection in a laboratory and on site, particularly ASFV detection in quarantine ports, airports and epidemic disease outbreak sites, and provides a novel and reliable technique support for ASF control in China.

This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of the bacterial RecA and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods has the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods allow amplification of DNA up to hundreds of megabases in length.

The invention relates to a kit for detecting GII type norovirus and applications thereof, concretely a primer pair and a probe for detecting the GII type norovirus, and a kit comprising the primer pair and the probe. The GII type norovirus in a sample cab be successfully detected by a recombinase polymerase amplification reaction, and can be quantified by fluorescence intensity. The kit has advantages of high sensitivity, strong singularity, short detection time, and constant and low reaction temperature, can be used for qualitative screening and quantitative determination for GII type norovirus in the sample, such as a clinic sample, foodstuff, an aquatic product, a water body, etc. accordingly, the kit can be prepared to be a diagnostic kit for GII type norovirus.

The invention discloses a reagent, detection method and application for the detection of African hog cholera virus. The reagent comprises a specific primer pair and a probe, upstream and downstream primers of the primer pair are respectively sequences shown in Seq ID No. 1 and 2, and the probe is a sequence shown in Seq ID No. 3 or its reverse complement sequence; in the probe sequence shown in the SEQ ID No.3, a 28th base modifies fluorescent quenching group-dT, a 31st base is replaced with a base analog, a 33rd base modifies a fluorescent group-dT, and a 3' end modifies C3 Spacer. The reagent is sensitive, specific and efficient for detection of the African hog cholera virus by recombinase polymerase amplification; compared with conventional conventional or real-time fluorescent PCR, thereagent and the detection method have short detection time and simple operation, are especially suitable for on-site testing, and are of great significance for the rapid prevention and control of theAfrican hog cholera virus and guarantee of production safety.

The invention discloses an RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting a high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof. The kit comprises a pair of primers and a probe, the sequences of the primers are shown as SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the probe is shown as SEQ ID NO.3. It is proved through experiments that the kit can detect adverse effects of the high-pathogenicity porcine reproductive and respiratory syndrome virus (HP-PRRSV), a hog cholera virus, a C-type porcine reproductive and respiratory syndrome virus, a porcine circovirus type II, a porcine pseudorabies virus and a foot and mouth disease virus in a specificity mode. It is proved through experiments that the kit can detect out templates of at least 70 copies at the temperature of 40 DEG C on the condition of 20 min amplification, and the conformity between the kit and RT-qPCR is high. This shows that the kit can detect HP-PRRSV fast, efficiently and sensitively and provides an effective technological means for differential diagnosis of HP-PRRSV.

The invention belongs to the technical field of nucleic acid detection, and relates to a mycobacterium tuberculosis complex detection kit and method based on a CRISPR-Cas12a system. According to the kit, the detection sensitivity is improved by adopting a recombinase polymerase amplification technology, CRISPR-Cas12a is used for specifically targeting a mycobacterium tuberculosis complex target sequence and then activating the bypass cutting activity of Cas12a, and a mycobacterium tuberculosis complex can be sensitively and specifically detected from sputum. The kit and method have the advantages of being non-invasive, capable of detecting the mycobacterium tuberculosis complex frequently and repeatedly, high in detection speed and the like. Compared with an existing PCR detection method,the method based on the CRISPR-Cas12a system does not need a thermal cycler with heating and cooling functions, can detect the mycobacterium tuberculosis complex in sputum through fluorescence reading, has the advantages of being low in cost, easy and convenient to operate, high in sensitivity, good in specificity and the like and is suitable for clinical large-scale application.

The invention discloses an RPA (recombinase polymerase amplification) primer and a detection kit for rapidly detecting African swine fever viruses. Target genes can be effectively amplified, specificity is 100%, detection sensitivity is 102 copy / reaction, and sensitivity is equivalent to that of fluorescent quantitative PCR (polymerase chain reaction). Cross reaction between the RPA amplificationprimer and one of classical swine fever viruses, vesicular exanthema swine viruses I, porcine reproductive and respiratory syndrome viruses, porcine circoviruses and the like is omitted. A RPA isothermal amplification system is rapidness in reaction and wide in temperature range, effective amplification of the target genes can be achieved at the temperature of 38-46 DEG C, and the detection kit can rapidly, efficiently and sensitively detect the African swine fever viruses and has the advantages that the kit is simple to operate, high in specificity, safe and free from pollution, reaction results are easily observed and the like. Effective technical means are provided for on-site rapidness detecting and screening of infection nucleic acid of the African swine fever viruses, and the RPA amplification primer has great significance for control of infection spreading of the African swine fever viruses in China and inspection and quarantine in infected areas and entry and exit ports.

The invention provides a method for detecting Vibrio parahaemolyticus. The sequence of the forward primer is SEQ ID NO:1, the sequence of the reverse primer is SEQ ID NO:2, and the sequence of the probe is SEQ ID NO:3. A real-time RPA (recombinase polymerase amplification) process is utilized to detect the food-borne pathogen Vibrio parahaemolyticus, thereby implementing safe, specific, quick, sensitive and simple field quick detection on the Vibrio parahaemolyticus, and further overcoming the defects in the existing traditional detection technique. The method is suitable for field detection, can effectively inhibit the epidemic situation caused by Vibrio parahaemolyticus food poisoning in time, and perfects the food safety control system.

The invention belongs to the field of molecular biological detection, and relates to an RPA (recombinase polymerase amplification) primer and an RPA probe for detecting influenza B viruses, a kit andapplication of the RPA primer. The sequences of upstream and downstream primers are B-F and B-R, and the sequence of the probe is represented by SEQ ID. NO. 2. The RPA primer and the RPA probe for detecting the influenza B viruses can be used for rapidly detecting the influenza B viruses or simultaneously detecting influenza A and B viruses; the RPA downstream primer for the influenza B viruses can serve as a PCR amplification primer for the influenza B viruses. When the RPA primer and the RPA probe are used for detecting the influenza B viruses, the detection time is short, sensitivity and specificity are high, and the influenza A and B viruses can be simultaneously detected rapidly, accurately and specifically.

The invention discloses a universal primer and probe combination for detecting foot and mouth disease viruses through RPA (recombinase polymerase amplification)-lateral flow assay technology. The forward primer sequence is shown as SEQ ID No.1, the reverse primer sequence is shown as SEQ ID No.2, and the probe sequence is shown as SEQ ID No.3. The invention also discloses a kit for detecting foot and mouth disease viruses. Detection with the primers and probes involved in the invention only needs virus RNA extraction on clinical samples and isothermal amplification after one-step process reverse transcription into cDNA, thermal cycling reaction is not needed, amplification in a PCR instrument is unnecessary, and the result can be clearly displayed on a lateral flow chromatography test strip. The primer and probe combination and the kit provided by the invention have the advantages of high sensitivity, strong specificity, simple reaction procedure, short detection time and the like, and are suitable for rapid, accurate and simple detection of foot and mouth disease viruses on a cattle farm.

The invention discloses RPA specific primers and a kit for detecting vibrio parahaemolyticus in food and application and belongs to the technical field of biology. The sequences of the RPA specific primers are shown as SEQ ID No:1 and SEQ ID No:2; the sequences of internal amplification control is shown as SEQ ID No:8. The RPA specific primers disclosed by the invention are low in time cost, i.e., a PCR (Polymerase Chain Reaction) reaction usually takes up 2 to 3 hours, but an RPA technology only needs 20 minutes to complete amplification; amplification can be realized at a normal temperature: constant-temperature reaction at 37DEG C is needed only and no special thermal cycle equipment is needed. The results are easy to judge; unlike a dispersion zone of an LAMP (Loop-Mediated Isothermal Amplification) product, the RPA only needs one pair of primers to complete amplification, and the amplified product has a strip with specific size according to design sites of the primers and also comprises the internal amplification control which can be used for indicating the false negative of the RPA reaction.

The invention discloses a nucleic acid amplification technology and an application thereof in detection of mosquito-borne viruses. The invention provides a method for detecting whether n kinds of target RNA sequences are contained in a sample to be detected or not. The method sequentially comprises the following steps: 1, extracting the total RNA of the sample to be detected; 2, carrying out reverse transcription recombinase polymerase amplification; and 3, carrying out recombinase polymerase amplification, wherein n is a natural number. The invention also provides a primer probe combination. The combination comprises primers and probes represented by sequence 1 to sequence 18 in a sequence table. A micro-fluidic chip, recombinase polymerase amplification and nucleic acid sequence dependent amplification are combined to effectively solve the problem of detection limit reduction of the micro-fluidic chip. The invention further provides a kit used for detecting the mosquito-borne viruses. The kit can simultaneously complete detection of six varieties of the mosquito-borne viruses one time, and has the advantages of high sensitivity, good specificity, fastness, good convenience, and wide application values.

The invention provides a primer, a probe and a kit for detecting brucella based on a recombinase polymerase amplification method and belongs to the technical field of microbiological diagnosis. The primer pair and the probe for detecting brucella based on the recombinase polymerase amplification method are designed and obtained by taking a specific conservative region of a brucella ovis pb 26 genesequence as a template. When the kit is used for detecting the pb 26 gene of the brucella by using the recombinase polymerase amplification method, the detection sensitivity reaches up to 6 CFU; meanwhile, the primer has high specificity in detection of the brucella.

The invention discloses an RPA (recombinase polymerase amplification) primer for detecting a fowl adenovirus serotype 4. The nucleotide sequence of the RPA primer is as shown in SEQ ID No.1 and SEQ ID No.2 in the description. The invention further discloses a method for detecting the RPA of the fowl adenovirus serotype 4, and the method mainly comprises the steps of primer synthesis, extraction of DNA in a sample to be tested, RPA amplification reaction and analysis of an RPA amplification product. According to the RPA primer disclosed by the invention, the specificity is strong, the sensitivity is high, and the detection result is accurate. The detection method disclosed by the invention is simple in operation and good in stability, and a low-cost, rapid and specific on-site diagnosis method for effectively detecting and authenticating chicken cecum hepatitis-hydropericardium syndrome is provided.

The application discloses a reagent for detection of animal influenza virus type A, a detection method and an application thereof. The reagent includes a primer pair and a probe used for recombinase polymerase amplification of the animal influenza virus type A, wherein a forward primer of the primer pair is represented as the SEQ ID No.1, a reverse primer of the primer pair is represented as the SEQ ID No.2, and the probe is represented as the SEQ ID No.3; the 29th basic group in the probe is marked by a FAM group, the 31st basic group is replaced by dSpacer, the 34th basic group is marked by a BHQ1 group, and a 3' terminal is modified by C3Spacer. The reagent provides a novel detection scheme and approach for the detection of animal influenza virus type A. The animal influenza virus type A RPA detection method based on the reagent is quick and accurate, is low in demand on hardware equipment, is suitable for on-site quick detection and is especially suitable for departments such as animal quarantine and the like.

The invention discloses a primer probe set, kit and detection method for detecting SARS-CoV and MERS-CoV based on Recombinase Polymerase Amplification (RPA) detection. The primer probe set for detecting SARS-CoV and MERS-CoV based on the RPA detection comprises primers for nucleotide sequences as shown in SEQ ID NO.1-4 and probes for nucleotide sequences as shown in SEQ ID NO. 5-6. The invention further provides the kit for detecting SARS-CoV and MERS-CoV based on the RPA detection, wherein the kit comprises the primer probe set. By adopting the technical scheme, the sensitivity, specificity and simplicity for detecting SARS-CoV and MERS-CoV are obviously improved.

The invention discloses a reagent and a detection method for detecting porcine circovirus type 2 and an application. The reagent for detecting the porcine circovirus type consists of a primer pair for conducting amplification detection on recombinant polymerase of the porcine circovirus type 2 and a probe, wherein the sequence of a forward primer of the primer pair is shown as Seq ID No.1, the sequence of a reverse primer of the primer pair is shown as Seq ID No.2 and the sequence of the probe is shown as Seq ID No.3; the 31st base of the probe is marked by BHQ1, the 33rd base is substituted by tetrahydrofuran, the 35th base is marked by FAM and the 3' terminal of the probe is modified by a phosphate group. According to the reagent for detecting the porcine circovirus type 2, a novel detecting scheme and a novel detecting approach are provided for the detection of the porcine circovirus type 2. The porcine circovirus type 2 RPA detection method based on the reagent provided by the invention is rapid and accurate, and low in requirement on hardware equipment; and the method is applicable to on-site rapid detection and is especially suitable for such departments as inspection and quarantine and the like.

The invention aims at providing a primer and a probe for detecting peste des petits ruminants virus by virtue of a RPA (Recombinase Polymerase Amplification) technique. According to the primer and the probe, a forward primer sequence is represented by SEQ ID NO:1, a reverse primer sequence is represented by SEQ ID NO:2, and a probe sequence is represented by SEQ ID NO:3. According to a method, a large number of primers and probes are designed according to genomic sequences of the peste des petits ruminants virus, and a pair of primer and probe combinations capable of rapidly detecting nucleic acid of the peste des petits ruminants virus are screened out; obvious amplification curves can be obtained by carrying out rapid detection by virtue of the primer and the probe and taking nucleic acid RNA of the peste des petits ruminants virus of domestically separated Tibet strains and Xinjiang strains as a template, and no amplification curve is obtained through a reaction by taking other pathogenic nucleic acids such as foot and mouth disease virus, contagious pustular dermatitis virus, mycoplasma capricolum and bluetongue disease virus as the template.

The invention provides a RPA (recombinase polymerase amplification) kit used for detecting Ebola virus, special-purpose primers and probes of the RPA kit, and applications of the RPA kit in detection of Ebola virus. The RPA kit, the special-purpose primers, and the probes are designed based on Ebola virus NP gene conserved sequence, and the Ebola virus NP gene conserved sequence possesses an oligonucleotide sequence represented by SEQ ID NO.1. It is shown by research results that the RPA kit can be used for rapid detecting of Ebola virus in samples with specificity and sensitivity.

The invention belongs to the technical field of biology, and particularly relates to an influenza A virus reverse transcription-recombinase polymerase amplification (RT-RPA) detection kit and a preparation method thereof. The detection kit consists of an RT-RPA reaction system, an RNA enzyme inhibitor, influenza A virus M-segment positive plasmids (Puc-AM), a negative quality control product, an M-segment RPA primer and an exo probe. By using the specificity primer and probe, preparing the RT-RPA reaction system and embedding a Twista real-time fluorescent detector to carry out real-time fluorescent detection, a rapid, sensitive and specific influenza A virus constant-temperature real-time fluorescent nucleic acid detection method is established. The invention relates to an application of a specific RPA primer and an exo probe in the influenza A virus infection clinical identification diagnosis and virus separation strain identification.