88results about How to "High amplification efficiency" patented technology

The invention discloses a hormone-treated osmanthus fragrans miRNA reference gene as well as a primer and application thereof. According to the present invention, the optimal miRNA internal reference gene under hormone treatment is obtained, the miRNA internal reference under ethephon treatment is novel33 and ofr-miR159b-3p, the miRNA internal reference under ABA treatment is novel3 and ofr-miR159b-3p, the miRNA internal reference under MeJA treatment is ofr-miR159b-3p, novel8, novel2 and novel3, the miRNA internal reference under co-treatment of the three hormones is ofr-miR159b-3p, novel8 and novel3, and the current situation that the osmanthus fragrans miRNA fluorescent quantitative internal reference does not exist under the treatment of different hormones is filled with the PCR primer; powerful support is provided for expression quantification of related miRNA under treatment of different hormones of osmanthus fragrans.

The invention provides a primer probe group for simultaneously detecting American strains and European strains of porcine reproductive and respiratory syndrome viruses, a detection method and application, and particularly belongs to the technical field of porcine pathogen detection products. The invention provides a primer probe group for simultaneously detecting American strains and European strains of porcine reproductive and respiratory syndrome viruses. The primer probe group comprises universal forward primers PRRSV1-F and PRRSV2-F for detection, reverse primers PRRSV1-R and PRRSV2-R for detection and a universal probe PRRSV-P. A target gene detected by the primer probe group is a 3'non-coding region gene (3 'UTR) of a porcine reproductive and respiratory syndrome virus genome, and the primer probe group can realize efficient detection of American strains and European strains of the porcine reproductive and respiratory syndrome virus, and is simple and convenient in detection, high in sensitivity, good in specificity, high in speed, low in false positive and capable of realizing high-throughput detection.

The invention provides a miRNA reference gene in the opening and senescence process of sweet osmanthus petals and a primer and application of the miRNA reference gene, and belongs to the field of plant molecular biology. The petal opening and senescence process comprises S1 (the pedicel period), S2 (the initial flowering period), S3 (the initial stage of full-bloom), S4 (the full-bloom period), S5 (the later stage of full-bloom) and S6 (the petal falling period). According to the invention, 14 candidate reference genes are screened, and the stability of the candidate genes is evaluated through five algorithms (delta-CT, geNorm, NormFinder, BestKeeper and RefFinder), so that miRNA internal references, namely novel33 and ofr-miR395e, which are suitable for the opening and senescence processes of the sweet-scented osmanthus petals are obtained. According to the invention, the real-time fluorescent quantitative PCR primer of the internal reference is designed, the primer has strong specificity and high amplification efficiency, the detection efficiency of real-time fluorescent quantitative detection of sweet osmanthus miRNA can be greatly improved, and the credibility of the detection result is improved.

The invention belongs to the technical field of biology, and particularly relates to an SSR molecular marker primer group for identifying dracaena dracaena varieties and application of the SSR molecular marker primer group. The SSR molecular marker primer group totally contains 16 pairs of primers, the polymorphic information indexes (PIC) of the 16 pairs of primers are all greater than 0.6, the number of repetitive units is 3-5, and the number of repetitive times is 5-7. The SSR primer provided by the invention has the characteristics of high amplification efficiency, stable amplification result, good polymorphism, strong specificity and the like. By utilizing the SSR molecular marker primer group, not only can different dragon tree varieties and dragon tree species be identified, but also the dragon's blood raw material can be subjected to traceability detection. The method disclosed by the invention has important technical guidance significance for development and utilization of the dragon's blood medicinal material and breeding of fine dragon tree varieties.

The invention belongs to the technical field of molecular biology, and particularly relates to a pseudo-ginseng component rapid detection method based on a Proofman-LMTIA technology. According to the method, a pseudo-ginseng ITS2 sequence is taken as a specific target, and high-specificity and high-sensitivity detection of pseudo-ginseng components is realized through a designed LMTIA primer and a Proofman probe. Compared with the traditional detection technology, the invention has the advantages of simplified primer design, low-cost equipment requirement, rapid detection (only about 30 minutes) and wide application range. The method is not only suitable for detection of raw materials such as pseudo-ginseng seeds, seedlings, roots and rhizomes, decoction pieces and the like, but also can be used for authenticity identification of terminal products such as food, medicines, health care products and the like containing pseudo-ginseng components. Through innovative application of the Proofman-LMTIA technology, an effective solution is provided for rapid and accurate identification of pseudo-ginseng and derivative products thereof, and the method has important market application prospects and social benefits.

The invention discloses a kit and a detection method for detecting an internally-running sheep virus epidemic strain, the kit comprises a crRNA sequence and an RT-RPA primer, the detection method comprises an RT-RPA reaction system and a CRISPR / cas12a reaction system, RT-RPA nucleic acid amplification and CRISPR / cas12a nucleic acid detection are combined, a two-step detection method is adopted, firstly, RT-RPA nucleic acid amplification is carried out, secondly, RT-RPA nucleic acid amplification is carried out, thirdly, RT-RPA nucleic acid amplification is carried out, secondly, CRISPR / cas12a nucleic acid detection is carried out, and thirdly, RT-RPA nucleic acid detection is carried out. Then, the RT-RPA nucleic acid product is added into a CRISPR / cas12a nucleic acid detection system, and the Chinese epidemic strain of the internally-running sheep virus can be detected. The detection method is simple, efficient, low in dependence on instruments and equipment, good in sensitivity, specificity and stability and good in social application prospect.

The invention discloses a universal identification primer, kit and method for hippocampus. The universal identification primer for the hippocampus comprises HipF: 5 '-GGCCATACCCAAAAATG-3' and HipR: 5 '-TTTAGCTTATTAAAGTGTCTGTTTTGCA-3'. The universal identification primer for the hippocampus can be used for identifying 10 types of hippocampus, and the amplification efficiency of the universal identification primer is higher than that of the identification primer in the prior art, so that conditions are provided for developing an efficient, accurate and practical hippocampus variety identification technical method and effectively popularizing and improving the hippocampus variety identification technical capability.

The invention discloses a primer and probe combination for detecting a bulbus fritillariae cirrhosae medicinal material based on Taqman fluorescent probe double-marking linear correction quantitative PCR (Polymerase Chain Reaction). The invention further discloses a method for detecting the bulbus fritillariae cirrhosae medicinal material based on Taqman fluorescent probe double-marking linear correction quantitative PCR, the bulbus fritillariae cirrhosae specific primers and probes designed based on six bulbus fritillariae cirrhosae-based original plant conserved gene sequences and the 18sRNA universal reference gene primers and probes are adopted, a sample to be tested and a standard reference substance are amplified at the same time in a PCR reaction system, and the specific primers and probes are used for detecting the specific genes of the bulbus fritillariae cirrhosae medicinal material based on Taqman fluorescent probe double-marking linear correction quantitative PCR. The method comprises the following steps: qualitatively confirming that a test sample contains the bulbus fritillariae cirrhosae component according to amplification results of a bulbus fritillariae cirrhosae specific primer and a probe, then taking a standard reference substance as a template to obtain two standard curves of a bulbus fritillariae cirrhosae specific gene and a 18sRNA gene, and finally quantitatively analyzing the bulbus fritillariae cirrhosae content in the test sample by adopting a double-marking linear correction method. The method can be used for efficiently, specifically and accurately detecting the ingredients and content of the bulbus fritillariae cirrhosae through PCR.

The invention belongs to the technical field of biological and genetic engineering, and particularly relates to a nucleic acid amplification efficiency optimization control method and a nucleic acid quantitative detection system. According to the nucleic acid amplification efficiency optimization control method disclosed by the invention, the nucleic acid amplification efficiency is optimized by combining control modes such as temperature and fluorescence real-time regulation and control, and the nucleic acid amplification efficiency is optimized by monitoring the real-time state of the nucleic acid amplification reaction and dynamically and adaptively adjusting the temperature condition of the nucleic acid amplification reaction; the nucleic acid amplification efficiency is improved, the quantitative detection repeatability is optimized, the nucleic acid amplification efficiency is improved, the amplification consistency is optimized, and the quantitative detection repeatability of the nucleic acid is effectively improved.

The invention relates to the technical field of PCR (Polymerase Chain Reaction) detection, and discloses a high-risk human papilloma virus DNA (Deoxyribose Nucleic Acid) multiplex PCR detection kit, which adopts a primer probe group and comprises a PCR mixed solution, a negative reference substance and a positive reference substance, wherein the PCR mixed solution comprises a primer probe group. Female first-stage urine is used as a detection sample, and compared with a cervix uteri sample, sampling is more convenient, the experience feeling is better, and the willingness of female cervical cancer screening can be provided; meanwhile, the content of cervical exfoliated cells in the urine is also increased by using the first-stage urine, and the clinical sensitivity of clinical HPV detection can be remarkably improved by matching with a provided cell enrichment method.

The invention discloses a performance-improved phi29 DNA polymerase mutant as well as a preparation method and application thereof, and relates to the technical field of biology. The invention specifically discloses a phi29 DNA polymerase mutant which contains mutation sites of Y163K, F181K, Y224K, W277K / W277P, Y298K, Y369K, Y439K and F526R relative to a wild type phi29 DNA polymerase, and a preparation method of the phi29 DNA polymerase mutant. The amino acid sequence of the wild type phi29 DNA polymerase is as shown in SEQ ID NO: 1. Compared with wild type phi29 DNA polymerase, the DNA polymerase has higher specific enzyme activity, stability and amplification efficiency, and has a good application prospect in the fields of nucleic acid amplification, sequencing and the like.

The invention relates to the field of biotechnology primer design, in particular to a target species specific primer design system, method and device based on information entropy. The system comprises a data acquisition module, a data screening module and a data optimization module, the data screening module is used for acquiring short sequence fragment data with identity and coverage greater than or equal to 80%, and the data optimization module is used for processing the short sequence fragment data with identity and coverage greater than or equal to 80%. According to the method, short sequence fragments covering 90% of genomes are screened out, the entropy value of each basic group in the short sequence fragments is obtained, quantitative scoring is conducted on the short sequence fragments according to the entropy values and coverage degree information, the short sequence fragments with the top scores are screened out, the basic groups are replaced with the most conservative basic groups, and the optimized short sequence fragments are obtained. And designing primers based on the short sequence fragments. According to the system, entropy analysis is introduced in the primer design process, and the design quality and application efficiency of primers are remarkably improved.

The invention relates to a picosecond laser with high beam quality, comprising: a pumping source for providing pumping energy to an amplification crystal; the laser oscillator is used for providing signal light with high light beam quality for the amplification crystal; the amplification crystal is used for amplifying the signal light power; the pump light shaping system comprises a first lens group and a third plano-convex lens, the first lens group is arranged between the pump source and the amplification crystal and formed by combining a first plano-convex lens and a second plano-convex lens, the third plano-convex lens and the first lens group are arranged at intervals, and the pump light is subjected to quasi-collimation processing through the first lens group; third plano-convex lenses which are arranged at intervals are used for focusing, so that the Rayleigh length of the pump light after focusing completely falls into the amplification crystal; the signal light shaping system comprises a fourth plano-convex lens arranged on the upstream of the light path of the amplification crystal and a second lens group formed by combining a fifth plano-convex lens and a first plano-concave lens, and the signal light is focused through the fourth plano-convex lens. After beam expansion and collimation are carried out through the second lens group, the Rayleigh length of the signal light completely falls into the amplification crystal, the pump light and the signal light are in a uniform distribution state in the amplification crystal, and the amplification crystal generates uniform heat distribution to eliminate the thermal lens effect. The generation probability of a thermally induced birefringence effect and a thermally induced teratosis effect is reduced, and the amplification efficiency of the light beam and the quality of the output light beam are greatly improved.

The invention discloses a method for detecting the copy number of mRNA (messenger ribonucleic acid) of a novel crown RSV (respiratory syncytial virus) combined vaccine. A primer probe combination adopted by the detection method consists of primer pairs as shown in SEQ ID NO.2 and SEQ ID NO.3 and a probe as shown in SEQ ID NO.4, the 5'end of the probe is marked with a fluorescence reporter group, and the 3 'end of the probe is marked with a fluorescence quenching group. According to the detection method, correlation coefficients of equations between the Cq value and the template copy number are both larger than 0.99, amplification efficiency is both 90%-110%, and specificity and accuracy are good; the method can be used for quality control processes such as release detection in the combined vaccine development process and toxicological experiments, and has important significance and value for research and development and quality control of combined vaccines.

This invention belongs to the field of hybrid identification technology of *Fraxinus pubescens*, specifically relating to an InDel marker and its application for identifying the authenticity of hybrids between *Fraxinus pubescens* and *Astragalus membranaceus*. The InDel marker described in this invention is located at positions 187 to 240 of the sequence shown in SEQ ID NO:1, and the sequence of the InDel marker is shown in SEQ ID NO:2. Furthermore, based on this InDel marker, this invention has obtained a set of primers with stable polymorphism, high amplification efficiency, and clear bands, establishing a technical system that can identify the authenticity of hybrid offspring at the seedling stage without relying on expensive equipment. This provides an efficient and low-cost molecular tool for the breeding of new varieties among *Fraxinus pubescens* species, the protection of variety rights, and market supervision, and has significant application value.

This invention discloses a primer set for amplifying a novel HCV subtype 6xp and a primer set for detecting drug resistance mutations. The primer set for amplifying the novel HCV subtype 6xp includes nested PCR primers for amplifying the full length of the novel HCV subtype 6xp, while the primer set for detecting drug resistance mutations is a set of primers targeting drug resistance mutation sites in the three functional regions of NS3, NS5A, and NS5B. The primer set provided by this invention has high specificity and high amplification efficiency, enabling specific amplification of the entire genome of the novel HCV subtype 6xp and accurate detection of drug resistance mutation sites in the three functional regions. It is suitable for clinical diagnosis, antiviral treatment guidance, epidemiological surveys, and viral evolution research of this subtype, and has significant clinical application value and scientific research significance.

The invention provides a preparation method of a terahertz traveling wave tube based on an electron beam gasification chambering technology and the terahertz traveling wave tube, and the preparation method comprises the steps: machining an initial electron channel in a high-frequency interaction assembly, and enabling the diameter of the initial electron channel to be smaller than the design diameter of an electron beam; centering and welding the high-frequency interaction assembly and the electronic gun; carrying out vacuumizing operation on the terahertz traveling wave tube through a vacuum exhaust furnace; the electron gun is started, the anode is used for accelerating the electrons for the first time, high voltage is applied to the tube body of the high-frequency interaction assembly for accelerating the electrons for the second time, and the high voltage is larger than the voltage of the anode; wherein the material on the peripheral side of the initial electron channel is gasified and extracted by using the electrons subjected to secondary acceleration.

The present invention discloses a primer, crRNA and application thereof for broad-spectrum detection of mycoplasma based on LAMP-CRISPR / Cas12a, and relates to the technical field of mycoplasma detection. The primer and crRNA combination provided by the present invention includes 2 outer primers, 5 inner primers, 4 loop primers and crRNA. Based on this, the present invention also provides a LAMP-CRISPR / Cas12a system and product for broad-spectrum detection of mycoplasma, as well as a method for detecting mycoplasma that is not for the purpose of disease diagnosis and treatment. The primer and crRNA combination provided by the present invention and the corresponding detection method can simultaneously meet the requirements of specificity, specificity, detection coverage (broad-spectrum detection) and sensitivity stipulated by various countries and the European Pharmacopoeia; the operation is simple, the cost is low, the time consumption is short, the detection efficiency is high, the dependence on expensive instruments is low, and the instant detection of mycoplasma can be realized.

The invention belongs to the field of molecular biology of forestry insects, and particularly relates to a fluorescent quantitative reference gene of different gender adults of yellow shield elephant, as well as a primer and application of the fluorescent quantitative reference gene. According to the method, 8 candidate genes commonly used as reference genes are referenced and selected according to transcriptome sequencing data of the hedychium aureum, gene sequences of the 8 reference genes are identified, the stability of the candidate reference genes is evaluated through five algorithms, and the fluorescent quantitative reference genes suitable for the hedychium aureum adults with different genders are obtained. And a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primer of the candidate reference gene is designed. The detection primer provided by the invention is strong in specificity and high in amplification efficiency, fills up the current situation that no reference gene exists under adults with different genders of the hedychium aureum, provides a reliable analysis basis for expression quantification of related functional genes under the adults with different genders of the hedychium aureum, and has a good application prospect. A necessary earlier-stage foundation is laid for analysis of subsequent molecular mechanisms and mining of key functional genes, and repeatability, stability and reliability of functional gene research can be improved at the same time.

The application belongs to the technical field of biological detection, and particularly relates to a nucleic acid detection primer probe composition for typhus and an integrated microfluidic chip kit. The primer probe composition is used for detecting typhus, and has good sensitivity and accuracy, high amplification efficiency, and simple operation, rapidness and time saving. The application integrates nucleic acid extraction, purification, amplification and detection by using the microfluidic mode on the integrated chip, solves the problem of rapid detection of pathogens, realizes a POCT detection scheme of "sample in-result out", and directly adds the equipment after sampling, purifies and amplifies, and the result is obtained within 60 minutes.

The invention belongs to the technical field of biology, and particularly discloses a membrane protein imaging method and application thereof.The method comprises the steps that different DNA molecules are used as response molecules to mark cell membranes and target membrane proteins, then the response molecules marked on the target membrane proteins can trigger molecular dynamic recruitment based on entropy driven circulation through a strand displacement reaction, and the dynamic recruitment is obtained. Causing a large number of probes in the solution to be recruited to the cell membrane; signals are expanded to the whole cell membrane from the membrane interface protein, so that ectopic amplification imaging of the membrane protein based on molecular dynamic collection is realized; in addition, UCNPs are selected to replace a traditional fluorescent part to further enhance the signal, and the method can effectively enhance the imaging signal of the low-abundance target membrane protein; meanwhile, the biological significance and the dynamic regulation and control process of the cell membrane protein can be known more deeply, and the method has great application value in the field of biological medicine.

The invention discloses a reagent for detecting African swine fever virus gene type II and application of the reagent. The reagent comprises a primer group, the primer group comprises a forward primer with a sequence as shown in SEQ ID NO: 2 and a reverse primer with a sequence as shown in SEQ ID NO: 3. Compared with other similar kits, the kit for detecting the African swine fever gene type II, provided by the invention, has the advantages that the sensitivity can be remarkably improved through the design of the primers, the specificity is high, the sensitivity is high, the amplification efficiency is high, the detection time is short and the like; according to the kit provided by the invention, a user does not need to carry out reagent split charging, the extracted sample nucleic acid is directly added into the pre-split freeze-dried reagent to be detected in a fluorescent PCR instrument, and the operation is simple and rapid.

The invention discloses a giardia visual detection system based on RPA-PfAgo and application of the giardia visual detection system, by optimizing a reaction system and reaction conditions, the efficiency, sensitivity and specificity of RPA-PfAgo amplification are improved; compared with a CRISPR / Cas9 system, the whole operation process is simpler, the synthesis cost of PfAgo and gDNA is lower, the stability is better, storage and transportation are easy, and the system is more suitable for field detection; the whole reaction only needs RPA amplification at 39 DEG C and PfAgo cutting reaction at 95 DEG C, the reaction can be completed through a water bath or a metal bath, professional temperature control equipment is not needed, whether giardia exists in a sample or not can be directly observed through blue light excitation according to a used probe, the needed time and equipment cost are greatly reduced, the operation method is simple, and the method is suitable for large-scale industrial production. The method has low professional requirements on personnel and is more suitable for on-site diagnosis.

The invention provides an SLAMP primer group and a kit for detecting mycobacterium tuberculosis, and relates to the technical field of biological detection. The invention specifically discloses an SLAMP primer group and a kit for detecting mycobacterium tuberculosis. The SLAMP primer group is a primer group aiming at the amplification of a conserved region fragment of mycobacterium tuberculosis, and is a primer pair MTB-SF / MTB-SR; wherein the nucleotide sequence of the MTB-SF is as shown in SEQ ID NO. 1, and the nucleotide sequence of the MTB-SR is as shown in SEQ ID NO. 2. According to the method or the visual kit provided by the invention, rapid and accurate detection of the mycobacterium tuberculosis can be completed without expensive instruments or complicated operation, so that the method or the visual kit is suitable for on-site rapid detection in a scene with a low professional degree.

The present invention relates to a specific primer and a probe for detecting millet components, wherein the sequences of the specific primers are shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the probe is shown in SEQ ID NO.3. The primers and probes provided by the present invention are used to identify millet components in a sample, which has the advantages of accurate results, high sensitivity, stable and reliable results, low false positive rate, etc., can accurately separate millet components from other closely related species, has high specificity, and its PCR amplification product is only 113bp, has a small molecular weight, and has high amplification efficiency, thereby ensuring the specific identification of millet components in deep-processed foods of different matrices.

The present invention belongs to the field of biotechnology, and discloses an MMLV reverse transcription mutant, which is obtained by replacing the RNaseH domain of a wild-type MMLV reverse transcriptase with a domain that enhances binding to an RNA-DNA hybrid chain. The present invention also performs mutation design on the mutant, wherein the preferred mutation combination is E69K, H126Y, N131M, H204R, and T306K. Compared with the wild type, the MMLV reverse transcription mutant of the present invention has a higher amplification efficiency. The present invention also provides the sequence of the MMLV reverse transcriptase mutant of the present invention and its application in a reverse transcription reaction.

This invention belongs to the field of molecular biology technology, specifically relating to a primer-probe set for RT-RAA detection of goose astrovirus and its application. The primer-probe set includes an upstream primer F1, a downstream primer R2, and a probe P. This primer-probe set can simultaneously detect two genotypes of goose astrovirus, GoAstV-I and GoAstV-II; it exhibits high specificity and amplification efficiency, aiming to provide a rapid and convenient technical means for the early diagnosis, on-site monitoring, and epidemic prevention and control of goose astrovirus. Example results show that using GoAstV-I, GoAstV-II, GPV, GoCV, DTMUV, and NDV nucleic acids as templates for RT-RAA-LFD detection, positive bands were observed for GoAstV-I and GoAstV-II, while all other pathogens were negative, with no cross-reaction, indicating that the specificity of the primer-probe set is 100%.

The invention relates to the technical field of parasite detection, in particular to a kit and a method for detecting toxoplasma gondii based on RPA-CRISPR / Cas12a. The kit comprises a crRNA sequence, wherein the crRNA sequence is as shown in SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.21, SEQ ID NO.23 or SEQ ID NO.30, and the crRNA sequence is as shown in SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.21, SEQ ID Or the difference between the nucleotide sequence and the nucleotide sequence is not more than 2 basic groups. The kit further comprises an RPA primer pair, Cas12a protein and ssDNA, Cas12a carries out DNase activity, the single-stranded DNA marked with fluorophore is cut, and whether a sample to be detected contains toxoplasma gondii DNA or not can be judged by detecting a fluorescence signal. When being used for detecting toxoplasma gondii, the system has the characteristics of short reaction time, strong specificity, high sensitivity, small instrument dependence, simplicity in operation and the like, the effect is outstanding, and the sensitivity is consistent with that of fluorescent quantitative PCR.

The present invention discloses a design method for primers and probes for amplifying low-concentration mutant target sequences. First, the mutation position (mutation point, i.e., position 0) of the target sequence to be amplified is determined; then, a 15-25bp nucleic acid fragment including the 0 base is selected in the negative direction of the mutation point as a forward primer, and a 12-25bp nucleic acid sequence is selected from the ‑1 base or the 0 base in the positive direction of the mutation point as the probe sequence of the amplification system; finally, a reverse primer is designed according to a conventional method at a suitable position downstream of the 3' direction of the probe sequence. The primers and probes designed according to the method of the present invention can effectively (high specificity and high efficiency) amplify the target fragment under the background of a higher content of wild-type template, especially for point mutations, deletion mutations, and insertion mutations in gene mutations, etc., combined with fluorescent real-time PCR technology, can effectively solve the difficulty of insufficient sensitivity in current clinical tumor detection and drug sensitivity detection.

The invention discloses a varicella-zoster virus whole genome capturing method, a primer group and a kit. The primer combination for amplifying the whole genome sequence of the varicella-zoster virus disclosed by the invention can effectively amplify the whole genome sequence of the varicella-zoster virus, is high in coverage rate, and is beneficial to further research on pathogenic mechanisms, molecular epidemiology, etiology and the like of the varicella-zoster virus.