93results about How to "Amplification realized" patented technology

The invention belongs to the technical field of PCR (polymerase chain reaction), and particularly relates to a digital PCR chip with silicon substrate arrays and micro-reaction pools and a method for manufacturing the digital PCR chip. The digital PCR chip mainly comprises an upper cover and a chip. Micro-pores which are arrayed to form honeycombs are formed in the chip, and the upper cover is fixed onto a chip groove. The method includes particular steps of selecting a silicon chip with a single polished surface; cleaning the silicon chip; coating a layer of uniform photoresist on the polished surface of the silicon chip in a spinning manner; forming circular graphic arrays by means of exposure; etching silicon by the aid of a dry process in masks, namely, the circular graphic arrays to form micro-pore structures; removing the photoresist and scribing the chip to completely manufacture the digital PCR chip.

The invention discloses a method and a primer composition for detecting soybean fusarium oxysporum based on an LAMP (loop-mediated isothermal amplification) technology. The primer composition comprises four specific primers FIP, BIP, F3 and B3 for detecting the LAMP molecules of the soybean fusarium oxysporum, and two ring primers LF and LB used for increasing the reaction speed. The method is used for performing LAMP on DNAs to be detected by the primer composition provided by the invention; a detection result is observed by naked eyes or under the irradiation of ultraviolet light with the wavelength of 245 nm; the color change and the fluorescence intensity of SYBR Green I are taken as result judgment standards. The invention provides the new molecular detection method and the primer composition for detecting the soybean fusarium oxysporum, the soybean fusarium oxysporum is subjected to LAMP detection, the detection cycle is short, the accuracy is high, the sensitivity is high, and the detection result is observed by the naked eyes.

The invention belongs to the technology field of the cellular immunology, and particularly relates to a method for the dominant amplification of in vitro peripheral blood nature killer cells with a large amount. The method comprises the steps as follows: (1) separating single nucleus cell from human or animal peripheral blood; (2) suspending the single nucleus cell in an RPMI 1640 culture solution containing 5%-15% autologous serum and pretreating with methyl-Beta-cyclodextrin with an effective concentration of 1-4mmol / L for 36-60 hours; and (3) adding recombined interleukin 2, and amplification-culturing in an RPMI 1640 culture solution containing 5% of new-born calf serum and 5% of autologous serum for above 10 days. The method has the advantages that the blood sample amount is small; the cost is low; the operation is convenient; and the amplification multiple is high, and the nature killer cells can be amplified by 392-1752 times in a short term.

The invention discloses LAMP (loop-mediated isothermal amplification) primer composition for detecting colletotrichum gloeosporioides and an application of the LAMP primer composition. A GS (glutamine synthetase) gene is used as a target gene, primers which have high specificity and high sensitivity and can be used for LAMP detection of the colletotrichum gloeosporioides are designed and screened out, and the LAMP primer composition consists of a forward outer primer GS-F3 shown in SEQ ID NO.2, a reverse outer primer GS-B3 shown in SEQ ID NO.3, a forward inner primer GS-FIP shown in SEQ ID NO.4, a reverse inner primer GS-BIP shown in SEQ ID NO.5, a loop primer GS-LF shown in SEQ ID NO.6 and a loop primer GS-LB shown in SEQ ID NO.7. The primer composition is mainly used for quickly detecting the colletotrichum gloeosporioides, and only 2 hours are spent on detection each time.

The invention discloses a loop-mediated isothermal amplification (LAMP) method for detecting pathogenic bacteria of canker disease of carya cathayensis. An LAMP primer composition is composed of four primers. The invention further discloses an LAMP kit used for detecting pathogenic bacteria of canker disease of carya cathayensis. The kit comprises the previous LAMP primer composition and further comprises 10*ThermoPol Buffer, dNTPs, MgCl2, glycine betaine, hydroxynaphthol blue, 8 U / muL Bst DNA polymerase and ddH2O. According to the method disclosed by the invention, the LAMP primers are designed according to a conserved region fragment sequence of beta-tubulin genes, the fast molecular detection technology system and reaction conditions are optimized, the pathogenic bacteria of the canker disease of carya cathayensis can be subjected to LAMP detection, the method is simple and convenient in reaction process, short in detection period, high in specificity and high in sensitivity, and the detection results can be observed by naked eyes.

The invention discloses a loop-mediated isothermal amplification primer composition for detecting benzimidazole fungicide E198A genotype-resistant botrytis cinerea Pers.. The LAMP primer composition consists of the following four primers: an F3, a B3, an FIP and a BIP. The primer composition consists of primer groups designed by mismatching a basic group in a mutation region containing a 198 locus of a botrytis cinerea Pers. beta-tubulin gene; loop-mediated isothermal amplification reaction is used for detecting the benzimidazole fungicide E198A genotype-resistant botrytis cinerea Pers.. The invention further provides a loop-mediated isothermal amplification method for detecting the benzimidazole fungicide E198A genotype-resistant botrytis cinerea Pers. and a kit used thereby.

The invention discloses a loop-mediated isothermal amplification (LAMP) technology-based method and primer composition for detection of fusarium graminearum, and the LAMP primer composition for detection of soybean fusarium graminearum comprises a forward inner primer (FIP) as shown by SEQ ID NO.2, a backward inner primer (BIP) as shown by SEQ ID NO.3, a forward outer primer (F3) as shown by SEQ ID NO.4, a backward outer primer (B3) as shown by SEQ ID NO.53, and a loop primer (LB) as shown by SEQ ID NO.6. The primer composition is used for detection of soybean fusarium graminearum. The detection system can be used for fast, convenient, high efficiency, high specificity and high sensitive detection of soybean fusarium graminearum without complex instruments, and can well satisfy the field detection of soybean fusarium graminearum.

The invention discloses a loop-mediated isothermal amplification primer composition capable of detecting colletotrichum truncatum and application thereof. The primer composition comprises a forward outer primer Rpb1-F3 as shown in SEQ ID NO.2, a reverse outer primer Rpb1-B3 as shown in SEQ ID NO.3, a forward inner primer Rpb1-FIP as shown in SEQ ID NO.4, a reverse inner primer Rpb1-BIP as shown in SEQ ID NO.5 and a loop primer Rpb1-LF as shown in SEQ ID NO.6. By the primer composition, the problems that the biological detection method requires long period, wastes time and energy and is tedious and poor in specificity and a thermal cycling instrument is needed and colletotrichum truncatum cannot be rapidly detected by virtue of the PCR detection technology in the prior art are solved and the primer composition has the advantages of short detection period (only 70 minutes), strong specificity and high sensitivity, and the detection results can be visually observed.

The invention provides a movable liquefied natural gas (LNG) pump-free filling device. The movable LNG pump-free filling device comprises an LNG low-temperature storage tank, an LNG filling machine and a storage tank booster. The LNG low-temperature storage tank is divided into two ways from a storage tank liquid outlet valve, one way is connected with the LNG filling machine, and the other way is connected with the storage tank booster through a boosting adjusting valve. The LNG low-temperature storage tank is further provided with a lower liquid inlet valve and an upper liquid inlet valve which are gathered, and the LNG low-temperature storage tank is connected with a gas phage opening of the storage tank booster and a gas return opening of the LNG filling machine through storage tank gas phase opening root valves. The movable LNG pump-free filling device has the advantages and positive effects that the structure is compact and simple; low-temperature parts in the device are all of a vacuum cold insulation structure; the thermal insulation property is good, and the amount of generated boil off gas (BOG) is small; fuel of a pry car is gas phase natural gas in the storage tank, excessive BOG can be consumed and prevented from being discharged, and other fuels are not needed.

The invention discloses a carbendazim resistant genotype F200Y sclerotinia sclerotiorum LAMP detection primer composition and a LAMP test kit and a LAMP test method. The LAMP detection primer composition comprises a forward inner primer FIP, a backward inner primer BIP, a forward outer primer F3 and a backward outer primer B3. The test method is simple and easy, good in practicability, high in sensitivity, strong in specificity, and high in accuracy, and can achieve the isothermal amplification, a new technology platform is provided for testing carbendazim resistant genotype F200Y sclerotinia sclerotiorum, and the method can be used for rapid high sensitivity testing of the carbendazim resistant genotype F200Y sclerotinia sclerotiorum, monitoring of sclerotinia sclerotiorum carbendazim resistant group, and timely understanding of the development trend of the resistance group, and the detection primer composition has great significance to crop sclerotiniose resistance management and scientific guiding drug use, production cost reduction and deduction of pesticide pollution of the environment.

The invention relates to a nucleic acid detection kit for RNA isothermal amplification avian influenza virus H7N9 (2013). The kit includes a virus preservation solution, a nucleic acid extraction solution, a washing liquid, an H7N9 reaction liquid (2013), an H7 detection liquid, an N9 detection liquid, an SAT enzyme liquid, an H7N9 (2013) positive control reagent, and an H7N9 (2013) negative control reagent. The detection kit provided by the invention has the characteristics of high specificity, high sensitivity, low pollution and rapid detection, and plays an important role in clinical diagnosis of early infection of avian influenza virus H7N9 (2013).

The invention belongs to the technical field of benthic animal identification, and discloses a method utilizing environmental sediment samples to monitor freshwater benthic animals. The method includes the following steps: 1) collecting an environmental sediment sample to preserve after cleaning; 2) adding anhydrous ethanol in the preserved environmental sediment sample to perform extraction treatment, taking uniformly mixed extract liquid after treating for a period of time, performing vacuum centrifugation on the extract liquid, and discarding supernatant to obtain dried tissue residues so as to perform DNA extraction; 3) utilizing the extracted DNA barcode fragment to perform amplification so as to obtain an amplified product; and 4) sequencing and analyzing the amplified product. The method performs extraction on the sample by adopting the anhydrous ethanol so as to make the DNA of a benthic animal uniformly released in the solution, so that a homogeneous state can be achieved; andthrough the collection of the ethanol solution containing the DNA sample and the extraction of the DNA after centrifugal concentration, the accurate monitoring of a wide range of environmental samples can be realized.

The invention discloses a multi-liquid hydrogen storage tank parallel-connection injection method for hydrogen-oxygen rocket tests. A multi-storage tank parallel-connection system consists of a precooling injection storage tank group 1, a small-flow injection storage tank group 2, a large-flow injection storage tank group 3, a liquid hydrogen storage tank 4, branch pipelines 5, a main pipeline 6, precooling isolating valves7, small-flow isolating valves 8, a main liquid discharge valve 9, liquid hydrogen storage tank supply and discharge valves 10, a liquid hydrogen storage tank self-pressurizing system 11, and a hydrogen-oxygen rocket 12. Due to function grouping of the storage tanks and switching of the isolating valves, isolation of different flow procedures of precooling, small-flow injection and large-flow injection can be achieved; due to adoption of the liquid hydrogen storage tank self-pressurizing system, effective control on flow, temperature and liquid level in the injection process is achieved; due to arrangement and switching of pipeline components, and an abutting structure of branch / main pipelines, fluctuation impact of the injection process can be eliminated. The multi-liquid hydrogen storage tank parallel-connection injection method disclosed by the invention is simple and reliable, and is already successfully applied.

The present invention is one optimized long segment nucleic acid PCR amplification system based on nanometer metal particle, and belongs to the field of molecular biology technology. One Long-PCR system is configured on the basis of available PCR amplification process, and the Long-PCR system has at least 10 vol% of 0.01 wt% concentration nanometer metal colloid solution or nanometer metal particles in the ultimate concentration not lower than 0.001 wt% added into the PCR system. The present invention has simple operation, obvious optimizing effect, low cost and wide application.

The invention discloses an RDA method and kit for rapidly detecting respiratory syncytial viruses. The kit comprise a specific primer pair and an RDA fluorescence labeling probe to realize safe, specific, sensitive, simple and convenient detection of the respiratory syncytial viruses (RSV) and overcome the defects of the existing traditional detection technology. According to the kit provided by the invention, a nucleic acid extraction step can be omitted, the respiratory syncytial viruses can be detected at the constant temperature of 37-42 DEG C within 20 minutes, the specificity is 100%, and the kit is very suitable for on-site rapid detection. Compared with a common PCR method, the RDA fluorescence method has the advantages that the reaction is performed at constant temperature, the temperature change is not needed, complex instruments are not needed, and the reaction time is short. Therefore, the method and the kit have the characteristics of simplicity and quickness in operation,good specificity, high sensitivity, low cost and the like, provide an effective technical means for on-site quick detection and screening of the respiratory syncytial viruses, and have a wide application prospect.

The invention discloses a loop-mediated isothermal amplification (LAMP) detection method for detecting strawberry anthracnose in the seedling stage and an LAMP primer used in the method; and the method can be used for quickly detecting whether strawberry seedlings carry germs or not on the site. The invention also discloses an LAMP detection method for detecting anti-QoI germicide G143A genotype strawberry colletotrichum gloeosporioides and an LAMP primer used in the method; and the method can be used for specifically detecting strawberry colletotrichum gloeosporioides with high resistance toQoI germicides. The method disclosed by the invention has the characteristics of simplicity, quickness, low cost, high sensitivity and the like, can greatly improve the detection efficiency, and has important practical significance for seedling stage detection and drug resistance epidemic early warning of strawberry anthracnose.

The invention discloses a method for molecular detection of Phytophthora cambivora(Petri)Buisman with the loop-mediated isothermal amplification (LAMP) technique based on color differentiation, and primers. The sequences of the primers are shown from SEQ ID NO.1 to SEQ ID NO.6. According to the detection system, under the isothermal condition of 62 DEG C, Phytophthora cambivora(Petri)Buisman can be detected quickly, conveniently and efficiently with high specificity and sensitivity, no complicated instrument is needed, a new technical platform is provided for detection of Phytophthora cambivora(Petri)Buisman, and the requirement for field detection of Phytophthora cambivora(Petri)Buisman can be well met. The detection system is suitable for inspection and quarantine of entry / exit plants and plant products, and investigation, rapid diagnosis and monitoring of diseases, and is of great importance in preventing Phytophthora cambivora(Petri)Buisman from entering China. Meanwhile, the establishment of the system provides technical guidance and theoretical basis for detection of other pathogenic bacteria.

The invention discloses a carbon fiber tow layered conveying device which comprises a tow collecting device and a tow expanding device. The tow collecting device is provided with a machine frame, a first rotating guide roller and a second transmission guide roller are arranged on the upper portion of one side of the machine frame, a first guide roller is fixed below a second rotating guide roller on the machine frame, a second guide roller is fixed below the first guide roller on the machine frame, a first supporting roller and a second supporting roller are fixed on the upper portion of the other side of the machine frame, a third guide roller is fixed below the first supporting roller on the machine frame, a fourth guide roller is fixed below the third guide roller on the machine frame, and the tow collecting device and the tow expanding device are of the same symmetrical structure. The tow collecting device and the tow expanding device are respectively placed on the front of a carbide furnace and on the back of the carbide furnace, the tow collecting device is capable of achieving layering and collecting of tows, and the tow expanding device is capable of achieving expanding of the tows.

The invention discloses an RDA method and a kit for rapidly detecting influenza A virus. The kit comprises a specific primer group and an RDA fluorescence labeling probe so as to realize safe, specific, sensitive, simple and convenient detection of the influenza A virus (FluA), and to make up for the defects of existing traditional detection technologies. According to the kit provided by the invention, a nucleic acid extraction step can be omitted, the influenza A virus can be detected within 20 minutes at a constant temperature of 37-42 DEG C, the specificity is 100%, and the kit is very suitable for on-site rapid detection. Compared with common PCR methods, the RDA fluorescence method has the advantages that: the reaction is carried out at constant temperature, the temperature change isnot needed, complex instruments are not needed, and the reaction time is short. Therefore, the method and the kit provided by the invention have the characteristics of simple and rapid operation, goodspecificity, high sensitivity, low cost and the like, provide an effective a technical means for on-site rapid detection and screening of the influenza A virus, and have a wide application prospect.

The invention belongs to the technical field of PCR (polymerase chain reaction), and particularly relates to a digital PCR chip with silicon substrate arrays and micro-reaction pools and a method for manufacturing the digital PCR chip. The digital PCR chip mainly comprises an upper cover and a chip. Micro-pores which are arrayed to form honeycombs are formed in the chip, and the upper cover is fixed onto a chip groove. The method includes particular steps of selecting a silicon chip with a single polished surface; cleaning the silicon chip; coating a layer of uniform photoresist on the polished surface of the silicon chip in a spinning manner; forming circular graphic arrays by means of exposure; etching silicon by the aid of a dry process in masks, namely, the circular graphic arrays to form micro-pore structures; removing the photoresist and scribing the chip to completely manufacture the digital PCR chip.

To achieve the above object, the present invention discloses a fusion Taq DNA polymerase, which is formed by fusing a DNA single-strand binding protein Sac7d or a DNA single-strand binding protein sso7d with a wild-type Taq DNA polymerase, respectively. The Taq DNA polymerase provided by the invention enhances the affinity with the template chain in the amplification process so as to improve the amplification efficiency, realizes the amplification of difficult templates, has stable enzyme activity and can be directly amplified by using blood as a template.

The invention discloses an RDA method and a kit for rapidly detecting feline panleukopenia virus (FPV). The kit for rapidly detecting the FPV comprises a specific primer pair and an RDA fluorescent label probe so as to realize safe, specific, sensitive, simple and convenient detection of the FPV, thereby making up the defects of the existing traditional detection technology. The kit provided by the invention can save the step of nucleic acid extraction, and is capable of detecting the FPV within 20 min at a constant temperature; and moreover, the kit has a specificity of 100%. Compared with acommon PCR method, the RDA fluorescence method has the advantages of being capable of carrying out reaction at a constant temperature, free of requirements for temperature change and complex instruments, and short in reaction time. Therefore, the kit is suitable for on-site rapid detection. The method and the kit thereof disclosed by the invention have the characteristics of being simple and rapidin operation, excellent in specificity, high in sensitivity, low in cost and the like; and thus, an effective technical means is provided for on-site rapid detection and screening of the FPV. The method and the kit have a wide application prospect.

The invention relates to the field of submerged arc furnace equipment, in particular to an automatic electrode paste adding device and method for submerged arc furnaces. By means of the device, an electrode paste storage bin, a conveying device capable of achieving long-distance conveying and a servo system single beam crane are combined, so that electrode paste can be injected to multiple sets ofsubmerged arc furnaces automatically, the electrode paste injecting efficiency is improved, in addition, the electrode paste stored remotely can be conveyed to the submerged arc furnace, and the field utilization rate is increased. The invention further provides an automatic electrode paste adding method for the submerged arc furnaces, an electric control system and a monitoring device are used for conducting programming control and real-time detection, the electrode paste injecting process is more intelligent, and the work efficiency is improved.

The invention belongs to the technical field of biomedical materials, and particularly relates to a cell large-scale culture method based on porous nanoscale temperature-sensitive soft colloid. The method comprises the following steps: mixing the porous nanoscale temperature-sensitive soft colloid with a cell or tissue culture to obtain a mixture, conveying the mixture in a liquid state into a hollow column reactor, and performing curing at 32-37 DEG C for culture. According to the culture method, pancreatin digestion is not needed in the cell elution process, cells are not affected at all, and the cells can be conveniently amplified into reactors of more levels. Generally, the density of the cells after first-stage amplification can reach 108 per milliliter, the cells carried by the colloid are digested together through temperature changes, a previous inoculation method is repeated to input the cells into other reactors to realize amplification, or the diameter of a hollow column cavity is increased to realize volume expansion, and process amplification and culture volume amplification are realized by increasing the number of hollow fiber columns.

The invention provides a generation method for general amplification of an intelligent automobile recombination scene, which comprises the following steps: determining key elements, including static elements and dynamic elements, for scene generation according to test requirements, and setting related fields for each kind of information; determining a value domain of each element field and whetherthe element fields have correlation or not by using a statistical analysis method based on historical data of a natural driving database, and randomly selecting and traversing elements without correlation in the value domain; for elements with correlation, obtaining constraint conditions through a statistical analysis method, and carrying out selection in a value domain corresponding to the constraint conditions of the fields. According to the generation method for universal amplification of the intelligent automobile recombination scene, the recombination scene can be rapidly generated, scene amplification is achieved, and the purpose of covering all possible working conditions is achieved.

The invention discloses a loop-mediated isothermal amplification primer for detecting botrytis cinerea containing BCbil43 / 144 intron. The invention further simultaneously discloses a loop-mediated isothermal amplification kit for detecting the botrytis cinerea containing the BCbil43 / 144 intron. Besides the LAMP (loop-mediated isothermal amplification) primer composition, the kit further comprises 10*ThermoPol Buffer, dNTPs, MgCl2, betaine, hydroxynaphthol blue, 8U / mu L Bst DNA polymerase and ddH2O. By adopting the loop-mediated isothermal amplification primer and the loop-mediated isothermal amplification kit which are disclosed by the invention, rapid detection on the botrytis cinerea containing the BCbil43 / 144 intron can be implemented in the field, so that scientific medicine application for preventing and treating the botrytis cinerea is timely guided.

The invention provides a multiplex PCR primer group and next-generation sequencing database building method for MLST (multilocus sequence typing) tracing of vibrio parahaemolyticus, and belongs to thetechnical field of microbiological detection. The multiplex PCR primer group for the MLST tracing of the vibrio parahaemolyticus comprises a primer for gyrB gene amplification, a primer for dnaE geneamplification, a primer for dtdS gene amplification, a primer for pyrC gene amplification, a primer for tnaA gene amplification, a primer for pntA gene amplification and one of a primer for recA-1 gene amplification and a primer for recA-2 gene amplification. The next-generation sequencing based database building method is established by multiplex PCR amplification for the seven genes, amplification and sequencing of the two types of RecA genes can be realized, and a sequencing result is relatively consistent with a first-generation sequencing result; and meanwhile, amplification of all genescan be realized by one-time PCR, the method can be directly applied to database building, and the operation is simple.

The invention discloses a method for detecting an African swine fever virus by isothermal fluorescent nucleic acid amplification. According to the method, by designing specific primers and the probe,a to-be-detected sample is amplified under the isothermal condition by utilizing a recombinase mediated chain-shuffling nucleic acid amplification technique, and detection of the African swine fever virus can be completed within 20 min. A kit has high sensitivity, and the lowest detection limit is 10 copies / muL, and the kit has high specificity, and thus, an effect technological means can be provided for on-site quick detecting and screening of the African swine fever virus.

The invention discloses an RDA (Recombinase-dependent amplification) method and a kit for quickly detecting a Coxsachie virus 16 and an enterovirus 71. The kit comprises a specific primer pair and anRDA fluorescence labeling probe to realize a purpose of safely, specifically, sensitively and conveniently detecting the Coxsachie virus 16 (CA 16) and an enterovirus 71 (EV 71) so as to make up for deficiencies in an existing traditional detection technology. The kit provided by the method can omit a nucleic acid extraction step, and the detection of the CA 16 and the EV 71 can be realized in 20min under a constant temperature condition of 37-42 DEG C, specificity is 100%, and the kit is very suitable for field quick detection. Compared with a common PCR (Polymerase Chain Reaction) method, the RDA fluorescence method carries out reaction at a constant temperature, does not require temperature change, does not require a complex instrument and is short in reaction time. Therefore, the method and the kit thereof have the characteristics of being simple and quick in operation, good in specificity, high in sensitivity, low in cost and the like, provide an effective technical means for thefield quick detection and screening of the CA 16 and the EV 71, and have a wide application prospect.

The invention discloses a Mycoplasma pneumoniae detection method based on PCR (polymerase chain reaction)-gold-magnetic nano immunochromatography assay, which comprises the specific steps of designinga specific primer modified with digoxin and biotin according to 16S rRNA nucleotide sequence of Mycoplasma pneumoniae; extracting DNA of a sample under test, and performing PCR amplification by usingthe DNA of the sample under test as a template and the specific primer as a primer to obtain a MP target fragment amplification product of the sample under test; detecting the amplification product with a Mycoplasma pneumoniae detection gold-magnetic immunochromatographic test strip. The Mycoplasma pneumoniae detection method based on PCR-gold-magnetic nano immunochromatography assay is simple toperform and is suitable for detecting Mycoplasma pneumoniae quickly.