81 results about "Total rna" patented technology

The invention provides a macro virus group sequencing library construction method for removing host ribosome RNA through an enzyme method and a kit, and belongs to the technical field of biology. The method comprises the following steps: hybridizing total RNA (Ribonucleic Acid) of a sample with a DNA (Deoxyribose Nucleic Acid) probe aiming at host ribosome RNA to form a DNA / RNA hybrid chain; rNase H is used for digesting RNA in the hybrid chain, and DNase I is used for digesting the remaining DNA probe in the system; purifying to obtain virus RNA (Ribonucleic Acid); carrying out fragmentation treatment on the virus RNA; carrying out reverse transcription on the fragmented RNA to synthesize a first chain cDNA, then synthesizing a second chain cDNA, and carrying out terminal repair and dA tail addition to obtain double-chain cDNA; connecting the linker with the dT tail with the double-chain cDNA; and carrying out PCR amplification and purification on the connection product to obtain the macro virus group sequencing library. According to the construction method and the kit, the problem that the full-length sequence of the virus cannot be obtained from a low-load sample can be solved.

The invention discloses a rapid sterile detection method and kit based on a 16S / 18S rRNA-cDNA digital PCR technology, and the method comprises the following steps: S1, extracting total RNA in a to-be-detected sample to obtain bacteria 16S rRNA and fungi 18S rRNA; s2, carrying out reverse transcription on the bacterium 16S rRNA and the fungus 18S rRNA to obtain a stable cDNA (complementary deoxyribonucleic acid) fragment; and S3, carrying out digital PCR amplification by taking the cDNA fragment as a template, and judging whether bacterial contamination exists or not according to an amplification result so as to realize rapid sterile detection. According to the characteristics of high conservative and high copy of bacteria 16S rRNA / fungi 18S rRNA, 30 sets of universal primer probe combinations are verified, and gt can be covered by selecting the optimal two sets of primer probe combinations; 99.99% of common pollution bacteria / fungi in clinic and pharmacy, and no cross amplification with mitochondrial rRNA of mammals such as human, hamster, cattle and the like.

The invention belongs to the technical field of plant genetic engineering, and particularly discloses a nitrate ion transport channel gene PbrSLAH3b and application of the nitrate ion transport channel gene PbrSLAH3b in promoting growth of pear pollen tubules. The method comprises the following steps: extracting total RNA (Ribonucleic Acid) of Dangshan pears from pollen of Dangshan pears; carrying out reverse transcription on the RNA to obtain cDNA (complementary deoxyribonucleic acid); and by taking the cDNA as a template, carrying out amplification to obtain the pear pollen tube nitrate transport channel gene PbrSLAH3b, the PbrSLAH3b gene provided by the invention belongs to a slow anion channel SLAC / SLAH family member, the PbrSLAH3b is remarkably up-regulated in expression in the early growth stage of a pollen tube, and the PbrSLAH3b is positioned in a plasma membrane. The invention verifies that PbrSLAH3b can form a functional anion channel with NO3 <-> selectivity under the condition of not depending on proton gradient or phosphorylation state, and the PbrSLAH3b has relatively strong selectivity on NO3 <->. Therefore, the PbrSLAH3b gene can mediate the absorption and transport of the pollen tube to NO3 <-> and maintain the polarity growth directivity of the pollen tube, and has important application value for revealing the ion regulation mechanism of the growth of the fruit tree pollen tube and improving the reproductive development of the fruit tree.

The invention provides a fish creatine kinase sensitization and immune cross reactivity analysis method which comprises the following steps: preparing a fish muscle water-soluble protein crude extract, separating 40-45kDa protein components in the fish muscle crude extract through SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and screening a target sensitization protein combined with IgE / IgG through immunoblotting; the method comprises the following steps: extracting total RNA of grass carp and synthesizing cDNA, designing a creatine kinase specific primer for amplification to obtain a creatine kinase gene sequence, and carrying out bioinformatics analysis after sequencing verification; the method comprises the following steps: cloning a creatine kinase gene to an expression vector, transforming escherichia coli BL21, performing IPTG induced expression, and performing urea gradient dialysis renaturation on an inclusion body to obtain the recombinant grass carp creatine kinase. Detecting the IgE binding activity of the recombinant grass carp creatine kinase and the serum of the allergic patient through dot hybridization; the IgG cross reactivity of the recombinant grass carp creatine kinase and the blue crab arginine kinase is verified through indirect ELISA; the IgE cross reaction degree of the recombined grass carp creatine kinase and the blue crab arginine kinase is quantified through inhibitory dot hybridization.

The invention relates to the field of crayfish evaluation, and discloses a method and a system for evaluating the WSSV resistance of procambarus clarkii, and the method comprises the following steps: sampling the procambarus clarkii, extracting target tissues from a sample, and extracting total RNA (Ribonucleic Acid) for accurate quantification of concentration and synthesis of cDNA (Complementary Deoxyribonucleic Acid); then TRIM23 gene quantitative detection is carried out on procambarus clarkii through a probe method, the purpose of calculating the Ct value is achieved, and finally the WSSV resistance of the procambarus clarkii is evaluated in combination with a relative quantitative mathematical model. According to the method, high pathogenicity or virus operation is avoided, the safety of the operation process is improved, the monitoring process is greatly shortened, and the accuracy is improved.

The application relates to the field of biomedical technology, in particular to a heavy chain and light chain variable region gene sequence of a LSDV ORF123 monoclonal antibody, an expression immunoglobulin full gene sequence and application thereof, the application extracts total RNA of hybridoma cells (1G1-1G12) by hybridoma sequencing, takes mRNA in the total RNA as a template, carries out reverse transcription to obtain cDNA of corresponding antibody genes, and then uses specific PCR to obtain a heavy chain variable region (Variable region of heavy chain, V H ) and a light chain variable region (Variable region of light chain, V L ) of the corresponding antibody, and carries out cloning and sequencing analysis. Finally, the heavy chain and light chain variable region gene nucleotide sequences of a monoclonal antibody (1G1-1G12) targeting the LSDV ORF123 protein are obtained, and the application is realized. Sequence analysis of the heavy chain (V H ) and light chain antibody (V L ) of the above-mentioned monoclonal antibody shows that the V H and V L gene subtypes of 1G1-1G12 are as follows: the heavy chain is IgG type, and the light chain is kappa type.

This invention discloses a method for constructing a tomato core membrane-associated protein interaction library based on Gateway technology under Fusarium oxysporum infection conditions and its application, relating to the field of genetic engineering technology. The method includes: using tomato root tissue infected with Fusarium oxysporum as material, extracting total RNA and enriching mRNA; synthesizing first-strand cDNA using Biotin-attB2-Oligo(dT) primers; synthesizing double-stranded cDNA enzymatically; ligating it with a specific double-stranded attB1 adapter; introducing attB1 and attB2 sites at both ends of the cDNA; cloning the cDNA into the donor vector pDONR222 via BP recombination to construct a primary entry library; and recombinating the primary entry library with vectors pGADT7 and pPR3-N via LR recombination to construct a nuclear secondary library and a membrane secondary library, respectively. The constructed libraries have high titers, long insert fragments, and low empty vector rates. This invention also relates to the application of the library in screening the interaction between Fusarium oxysporum effector proteins and tomato nuclear membrane-associated proteins, providing an efficient and specific resource platform for studying plant-pathogen interaction mechanisms.

A method for detecting and treating Type II Diabetes includes the use of hsa_piR_020485, a PIWI-interacting RNA isolated from urinary extracellular vesicles (ECVs), as a biomarker for the diagnosis and treatment of Type 2 diabetes mellitus (T2DM). The method is non-invasive, utilizing the differential expression of hsa_piR_020485 in diabetic versus non-diabetic subjects to identify subjects in need of treatment for T2DM. The method includes obtaining a urine sample from a subject, isolating urinary ECVs from the urine sample, extracting total RNA from the isolated urinary ECVs, quantifying hsa_piR_020485 levels from the total RNA, determining if the hsa_piR_020485 expression level exceeds a threshold, and administering one or more T2DM treatments.

The present application relates to the technical field of biological research data analysis, and discloses a kind of computing biology research data analysis display method, including extracting total RNA from biological tissue or cell, RNA reverse transcription is cDNA, constructs library, and utilizes high-throughput sequencing platform to sequence library, to obtain a large number of short sequences.The present application can effectively identify abnormal repeat regions by comparing and threshold judging the number of short sequences mapped to the same genomic coordinate interval, so as to determine whether the gene expression difference result is reliable, when the number of abnormal sequences exceeds the preset repeat sequence threshold, by eliminating the influence of repeat sequence operation, i.e. for the case of high FPKM value, remove the completely same and overlapping short sequences, and re-align the updated results, the expression distortion problem caused by repeat counting can be corrected.

The invention discloses a method for identifying germ cells of grass carps and carps by using dazl genes. The method comprises the following steps: cloning dazl cDNA sequences of the grass carps and the carps, and performing comparative analysis by using DNAMAN software; designing a universal non-specific primer, a grass carp specific primer and a carp specific primer according to sequence differences; extracting total RNA (Ribonucleic Acid) of testis of 12-month-old grass carp and carp, and reversely transcribing into cDNA Respectively carrying out PCR (Polymerase Chain Reaction) amplification by using three pairs of primers by taking the cDNA as a template; carrying out 1% agarose gel electrophoresis and imaging on the PCR product, and identifying and distinguishing germ cells according to the size of a strip; the method is simple in structure and reasonable in design, high-specificity primers are designed based on small difference of dazl gene sequences for the first time, precise amplification is achieved by combining with optimized annealing temperature (60 DEG C / 65 DEG C), special instruments are not needed, operation is easy and convenient, sensitivity is high, grass carp germ cells in carp gonads after transplantation can be rapidly identified, development of the'belly borrowing reproduction 'technology is supported, and the method is suitable for large-scale popularization and application. The grass carp breeding period is remarkably shortened, and the important industrialization prospect is achieved.

The application discloses application of a peach fruit hexokinase PpHXK3 gene in preparation of a plant cold-resistant product, and is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; a cloning method of PpHXK3 comprises the following steps: extracting total RNA of peach fruit and reversely transcribing the total RNA into cDNA as a template, and designing upper and lower amplification primers according to a gene sequence of PpHXK3; finally, a PpHXK3 gene amplification product is obtained through PCR amplification; the application of PpHXK3 in preparation of polygalacturonase PpPG21 inhibitors and / or polygalacturonase PpPG23 inhibitors, and the application of PpHXK3 in preparation of peach fruit cold-resistant products and heterologous plant cold-resistant products are also provided; and the advantages are that the decomposition of pectin is reduced, the integrity of a cell wall structure and the hardness of a fruit are maintained, the fruit has higher cold resistance, and cold damage in a storage and transportation process is reduced.

Described herein is a method of selecting allosteric biosensor proteins which bind a target ligand. The method includes providing a library of replicating plasmids each including an expression construct and a reporter, wherein each expression construct includes a gene encoding the allosteric protein variant and the reporter, wherein the reporter includes a barcode sequence for identification of the allosteric protein variant or allosteric domain variant. The method further includes mapping the variants in the library to the barcode sequence or sequences associated with the variant and assigning variant-barcode pairs, growing a population of cells transfected with the library of replicating plasmids in the presence of the target ligand and isolating target ligand total RNA and target ligand library plasmids; performing next generation sequencing to determine a quantity of each barcode in the target ligand total RNA, determining a fold enrichment for each allosteric protein variant or allosteric domain variant in the target ligand total RNA, and selecting a subpopulation of variants with the highest fold enrichment as the selected allosteric biosensors.

The invention provides a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection method for total RNA (Ribonucleic Acid) residual quantity of escherichia coli. A reaction system comprises 3-5 mu L of Taqman RT-PCR Buffer (5 *), 1-3 mu L of TaqMan Enzyme Mix (10 *), 0.3-0.5 mu L of ROX Reference Dye (50 *), 1-1.5 mu L of 8-12 mu M forward specific primer, 1-1.5 mu L of 8-12 mu M reverse specific primer, 0.5-0.8 mu L of 8-12 mu M specific probe, 4-6 mu L of standard substance or to-be-detected sample and RT-PCR-grade water which is supplemented to 20 mu L, and a reaction procedure is as follows: the temperature is 52-54 DEG C for 4-6 minutes; the temperature is 94-96 DEG C for 18-22 s; the detection time is 4-6 seconds at the temperature of 94-96 DEG C, 38-42 seconds at the temperature of 56-58 DEG C and 44-46 cycles, and the detection limit of the method is as low as 2 pg / mL.

The invention discloses an SNP (Single Nucleotide Polymorphism) site, a DNA (Deoxyribose Nucleic Acid) fragment, a molecular marker primer and a method for identifying the color of cerasus humilis flesh, the SNP site is located at the 422nd basic group site of a cerasus humilis DFR gene, the polymorphism is T / G, the SNP site of red flesh is T, and the SNP site of yellow flesh is G. The nucleotide sequence of the DNA fragment is as shown in SEQ ID NO.1 or SEQ ID NO.2, and the molecular marker primer comprises a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.4. The molecular marker primer has the advantages that the nucleotide sequence of the DNA fragment is as shown in SEQ ID NO.1 or SEQ ID NO.2; the method comprises the following steps: (1) collecting a to-be-detected cerasus humilis plant tissue sample and extracting total RNA (Ribonucleic Acid); (2) carrying out reverse transcription on the total RNA to obtain cDNA (complementary deoxyribonucleic acid); step (3), amplifying the cerasus humilis DFR gene by using the molecular marker primer by taking the cDNA as a template; (4) purifying and sequencing an amplification product; and (5) judging whether the pulp of the to-be-detected cerasus humilis sample is red or yellow according to a sequencing result. The technical problems that a traditional cerasus humilis pulp color identification method is long in period and not beneficial to popularization can be solved.

The invention belongs to the technical field of toxic substance recognition, and particularly relates to a toxic substance recognition method oriented to a dose-dependent omics effect, which comprises the following steps: storing surface water in a container, washing the container with tap water, methanol and deionized water in sequence, rinsing the container with surface water to be sampled, and loading a sample; the method comprises the following steps: coarsely filtering a water sample by using a 0.45 mu m filter membrane, enriching organic micropollutants by using a hydrophilic-oleophylic equilibrium small column activated by ultrapure water and HPLC (High Performance Liquid Chromatography)-grade methanol, and controlling the flow velocity of the small column to be 3-5mL / min; after extraction, freeze-drying an HLB small column for 24 hours to remove moisture, eluting the adsorbed organic micropollutants by using LC-MS grade methanol, and concentrating the eluent by using a nitrogen instrument; the method comprises the following steps: selecting total RNA (Ribonucleic Acid) of which the RINnumber is greater than 7.0, purifying mRNA (Messenger Ribonucleic Acid), segmenting into short fragments, carrying out reverse transcription to synthesize cDNA (Complementary Deoxyribose Nucleic Acid), preparing U-labeled second-chain DNA, and carrying out PCR (Polymerase Chain Reaction) amplification after heat-sensitive The method comprises the following steps: sequencing by adopting an Illumina paired terminal RNA-seq method, and filtering the reading by using Cutadapt software.

The invention discloses application of a peach fruit hexokinase PpHXK3 gene in preparation of a plant cold-resistant product. The peach fruit hexokinase PpHXK3 gene is characterized in that a nucleotide sequence is shown as SEQ ID NO.1, and an amino acid sequence is shown as SEQ ID NO.2; the cloning method of the PpHXK3 comprises the following steps: extracting total RNA (Ribonucleic Acid) of peach fruits, reversely transcribing the total RNA into cDNA (Complementary Deoxyribonucleic Acid) as a template, designing upstream and downstream amplification primers according to a PpHXK3 gene sequence, and finally, carrying out PCR (Polymerase Chain Reaction) amplification to obtain a PpHXK3 gene amplification product. The invention further provides application of the PpHXK3 in preparation of a polygalacturonase PpPG21 inhibitor and / or a polygalacturonase PpPG23 inhibitor and application of the PpHXK3 in preparation of peach fruit cold-resistant products and heterologous plant cold-resistant products, and the PpHXK3 has the advantages that decomposition of pectin is reduced, integrity of a cell wall structure and hardness of fruits are maintained, so that the fruits have high cold resistance, and the yield of the fruits is increased. And the cold damage in the storage and transportation process is reduced.

This invention relates to the field of biomedical technology, specifically to a snake-derived nanobody against KLH and its preparation and application. The invention involves immunizing the black-browed rat snake with KLH, extracting total RNA from its splenic lymphocytes, constructing a phage display VH gene library, and, after multiple rounds of screening, obtaining nanobody sequences that specifically bind to KLH, such as KLH-1 and KLH-2. This nanobody can be efficiently and solublely expressed and purified in *E. coli*, exhibiting characteristics of small molecular weight and high affinity. This invention further provides applications for this nanobody, particularly in the preparation of KLH-targeting detection reagents or drug delivery systems. This nanobody provides a core tool molecule for developing highly sensitive KLH-targeting detection reagents, efficient drug delivery systems, and novel vaccines.

The invention relates to the field of sequencing, in particular to an oligonucleotide connector containing an RNA bar code and application of the oligonucleotide connector. By using the oligonucleotide linker provided by the invention, multiple biological samples can be simultaneously sequenced in one chip, and extremely-low initial amount total RNA (Ribonucleic Acid) and even single cell level DRS (Dead Respiratory Syndrome) sequencing of a single sample can also be realized; the requirement on the initial quantity of RNA samples is reduced, and a powerful tool is provided for detecting rare samples or micro / trace RNA.

A method for treating primary hyperoxaluria 1 (PH1), comprising administering to a subject in need thereof an effective amount of a gene editing composition, which comprises: (a) a messenger RNA (mRNA) encoding a Cas12i2 polypeptide, (b) a guide RNA (gRNA) comprising a spacer sequence, which is specific to a target sequence in the HAO1 gene; and (c) and lipid excipients, which comprise a cationic lipid, a zwitterionic phospholipid, cholesterol, a pegylated lipid, or a combination thereof. In some instances, the gene editing composition is administered to the subject an amount of total RNA of about 0.4 mg / kg to about 1 mg / kg.

The invention provides an RNA (Ribonucleic Acid) virus nucleic acid enrichment method which comprises the following steps: selecting an enrichment target viridae, and designing an enrichment primer based on the enrichment target viridae to obtain a target primer group; the method comprises the following steps: acquiring a to-be-detected animal tissue sample, extracting total RNA from the to-be-detected animal tissue sample, and performing reverse transcription to obtain sample cDNA; enriching the RNA virus nucleic acid in the sample cDNA by adopting the target primer group to obtain an enriched product; wherein the target primer group comprises upstream primers from the first to the ninety-sixth and downstream primers from the first to the ninety-sixth, the sequences of the upstream primers from the first to the ninety-sixth are as shown in SEQ ID NO.1-SEQ ID NO.96, and the sequences of the downstream primers from the first to the ninety-sixth are as shown in SEQ ID NO.97-SEQ ID NO.192. The primer group disclosed by the invention has the characteristics of low cost and stable performance.

The application provides a full-length lncRNA absolute quantitative transcriptome library construction and sequencing method based on TSO-UMI marking and composite internal standard correction, and the library construction method steps comprise the following steps: S1, total RNA of a sample is extracted and rRNA is removed; S2, a composite internal standard is added; S3, a reverse transcription adapter primer is used to connect a product; S4, a TSO-UMI fusion primer is added, and full-length cDNA is synthesized through template switching; S5, cDNA is used as a template to perform PCR amplification; and S6, the amplified library is purified; the nucleotide sequence of the TSO-UMI fusion primer is shown in SEQ ID NO. 1 or SEQ ID NO. 2. Through TSO-UMI fusion primer sequence reconstruction, SIRV / ERCC specific composite internal standard ratio, full-process coupling experimental design, and supporting UMI+internal standard joint correction integrated bioinformatics algorithm, the problems such as PCR deviation, relative quantification, batch difference, isomer recognition, and polyA-free quantification are successfully solved, and reliable technical guidance is provided for lncRNA basic research, disease marker screening, and clinical efficacy monitoring.

The present invention relates to a method of detecting the presence of exon 8 of ANRIL (Antisense Non-coding RNA in the INK4 Locus) in a subject, comprising the following steps of: 1) Preparation of total RNA from a biological sample of the subject, 2) Conversion of RNA molecules into complementary DNA, 3) Loop-mediated isothermal amplification using trans-exonic oligonucleotides that specifically produces an amplicon containing exon 8 fragment, 4) Addition of Cas12a / RNA guide complexes, wherein RNA guide specifically targets exon 8-containing fragment by base pairing, 5) Addition of a reporter DNA probe, and 6) Detection of cleavage products, Wherein detection of cleavage products reveals the presence of exon 8 of ANRIL. The present invention also relates to a method for in vitro predicting in vitro the efficacy of a therapy targeting exon 8 of ANRIL in a subject, to a kit for implementing the method and to a set of oligonucleotides.

The invention belongs to the technical field of molecular biology, and discloses a saline-alkaline resistant gene MdLAC12 for enhancing apple and application thereof, an extraction method of the saline-alkaline resistant gene MdLAC12 for enhancing apple comprises the following steps: step 1, extracting total RNA (Ribonucleic Acid) from an apple rootstock M9-T337 root system, and taking cDNA (Complementary Deoxyribonucleic Acid) obtained by reverse transcription as a cloning template; 2, designing an MdLAC12 primer according to an apple genome database GDDH13, and carrying out a conventional polymerase chain reaction (PCR); and step 3, carrying out homologous recombination on the obtained PCR product of the MdLAC12 and pMD18-T, transforming a connection product into an escherichia coli DH5 alpha competent cell, and selecting resistant bacterial plaque for sequencing analysis to obtain a CDs sequence of the MdLAC12. The method is helpful for breeding excellent saline-alkaline tolerant apple rootstocks, and can also improve utilization of saline-alkaline land in a prebiotic area. The method has the advantages of simplicity in operation, high saline-alkaline tolerance and low production cost, and can be widely applied to the saline-alkaline tolerance germplasm resource breeding process.

The application provides a KLH-Flag immune phage display antibody library construction method, and steps of the method comprise the following steps: (1) immunizing a New Zealand white rabbit with a KLH-Flag protein, extracting total RNA of lymphocytes, and reversely transcribing the total RNA into cDNA; (2) amplifying heavy chain variable region and light chain variable region fragments and splicing into scFv genes by using a PCR technology; (3) constructing a pCANTAB5E-2sfi1-scFv recombinant plasmid, electrically transforming the recombinant plasmid into TG1 competent cells, and constructing a phage single-chain antibody library; (4) specifically enriching the phage single-chain antibody library, and screening positive clones from the phage single-chain antibody library. And monoclonal antibody amino acid sequences finally obtained for application are disclosed. The application screens a rabbit source single-chain antibody with high affinity by using a phage display antibody library technology, the antibody has high affinity to the Flag protein and high specificity, and the antibody can be used for preparing an antibody of Anti-Flag affinity gel and can be used for WB detection of proteins with a Flag label.

The application discloses a method for detecting viruses and virus replication in media based on Small-RNA high-throughput sequencing. The application belongs to the technical field of virus detection, and the method for identifying virus types in a to-be-detected sample by Small-RNA sequencing developed by the application comprises the following steps: 1) extracting total RNA from a virus-infected to-be-detected sample, constructing a cDNA library, and separating small RNA sequence fragments from the cDNA library; 2) downloading virus sequences and classification databases from GenBank, and comparing the small RNA sequence fragments with virus sequence files; 3) comparing a contigs3 file with a virus nucleic acid database, selecting sequences with the minimum e value in the comparison results in the contigs3 file, calculating the coverage of the contigs3 sequences in corresponding virus genomes, distinguishing virus information with a coverage threshold, and deducing the virus source of the small RNA sequence.

The application provides a screening method for a wheat seed storage-related gene, and belongs to the field of agricultural biotechnology.The screening method comprises the following steps: 1) subjecting seeds of a storage-tolerant variety N38 and a storage-intolerant variety N5 to artificial aging treatment, taking the seed embryos after germination; 2) subjecting the seed embryos to metabolomics analysis to obtain differential metabolites; 3) extracting total RNA of the seed embryos, performing transcriptome sequencing, and screening candidate differential expression genes; and 4) screening the candidate differential expression genes associated with the obtained differential metabolites to obtain final screening differential expression genes.The application subjects known storage-tolerant and storage-intolerant wheat varieties to artificial aging, performs metabolomics detection and transcriptome sequencing, screens differential metabolites and candidate differential expression genes, and further analyzes to obtain a wheat seed storage-related gene, thereby providing key gene resources and technical support for wheat storage-tolerant molecular breeding.

The invention belongs to the technical field of gene sequencing, and particularly relates to a library building kit and probe for detecting blood tumor related fusion genes. According to the library building kit, only a trace amount of total RNA is needed to be used for cDNA synthesis, the initial quantity of a template can be as low as 10 ng, so that a cDNA library is built, then the library is subjected to hybrid capture and enrichment by using an autonomously designed probe hematologic tumor fusion gene Panel (RA Panel), the time consumption is short, the efficiency is high, and simultaneous building and on-machine sequencing of 384 libraries can be realized. According to the rapid hybridization system of the library building kit, the shortest hybridization time can reach 30 min, the size of a finally obtained library fragment is about 200-400 bp, and the rapid hybridization system can be compatible with various Illumina sequencing platforms for sequencing. According to the invention, through a simple and easy-to-use solution of NGS sequencing while synthesis, rapid and accurate typing of fusion genes which may affect blood tumor diagnosis is realized.

The invention provides a preparation method and a detection method of a recombinant cat-omega interferon antiviral preparation, and the preparation method comprises the following steps: carrying out reverse transcription on total RNA (Ribonucleic Acid) of cat spleen to obtain cDNA (Complementary Deoxyribose Nucleic Acid); by taking the cDNA as a template, executing a PCR amplification program on the cat IFN-omega gene sequence by using the specific primer to obtain a PCR product; carrying out connection operation on the PCR product and a pGEM-T-Easy vector, and transferring the PCR product and the pGEM-T-Easy vector into DH5alpha competent cells; the method comprises the following steps: extracting positive clone plasmids which are verified to be correct from DH5alpha competent cells by using specific primers to obtain recombinant expression plasmids, carrying out double enzyme digestion on the recombinant expression plasmids and a pET30a vector by using NdeI and XhoI, and transferring obtained recovered target fragments into BL21 (DE3) competent cells to identify positive recombinant plasmids containing target genes; transferring the positive recombinant plasmid containing the target gene into BL21 (DE3) competent cells for bacterial colony culture, and performing induced expression on the obtained culture thalli; and after the induced expression is completed, preparing a preparation corresponding to the cat interferon from the cultured thalli. The preparation efficiency is at least improved.

The present invention relates to a method of detecting and / or quantifying the presence of exons 5 and 6 of ANRIL (Antisense Noncoding RNA in the INK4 Locus) in a subject, comprising the following steps of: 1) Preparation of total RNA from a biological sample of the subject, 2) Conversion of RNA molecules into complementary DNA, 3) Loop-mediated isothermal amplification using trans-exonic oligonucleotides that specifically amplify exons 5 and 6, 4) Addition of Cas12a / RNA guides complexes, wherein RNA guide specifically targets exons 5 and 6 by base pairing, 5) Addition of a reporter DNA probe, 6) Detection of cleavage products, wherein detection of cleavage products reveals the presence of exons 5 and 6 of ANRIL, and 7) possibly quantifying expression of exons 5 and 6 of ANRIL. The present invention also relates to a method for in vitro diagnosis of a cancer in a subject, to a method for in vitro prognosis a cancer in a subject to a kit for implementing the method and to a set of oligonucleotides.

The invention discloses a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) method for detecting capsicum vein mottle virus in tobacco, which comprises the following steps of: firstly, designing and synthesizing a specific primer pair based on a coat protein CP gene conserved sequence of capsicum vein mottle virus isolate in an NCBI (National Center of Biotechnology Information) database; secondly, extracting total RNA of a tobacco sample infected with the pepper vein mottle virus, and performing reverse transcription to obtain cDNA; carrying out real-time fluorescent quantitative PCR (Polymerase Chain Reaction) amplification by using the obtained cDNA as a template and adopting the specific primer pair; and finally, judging whether the sample carries the target virus or not according to the amplification curve, the melting curve and the Ct value, and realizing quantitative analysis of the virus load through the standard curve. The method is high in specificity and sensitivity, compared with a conventional PCR method, the sensitivity is improved by 10 times, and rapid and accurate detection of the pepper vein mottle virus in the tobacco can be achieved.