165 results about "Total rna" patented technology

The invention discloses a gastric signet-ring cell carcinoma biomarker and a detection kit thereof, belongs to the technical field of biological medicines, and particularly relates to the detection kit of the gastric signet-ring cell carcinoma biomarker, which comprises a total RNA (Ribonucleic Acid) extraction reagent, an RNA purification reagent, a cDNA (Complementary Deoxyribonucleic Acid) first strand synthesis premixing reagent, a rapid fluorescent quantitative premixing reagent (SYBR Green), nuclease-free double distilled water and a specific primer. The RNA purification reagent at least comprises isopropanol, chloroform and absolute ethyl alcohol; the specific primer comprises an upstream specific primer and a downstream specific primer. The detection kit for the gastric signet-ring cell carcinoma biomarker is high in RNA purification and recovery efficiency and excellent in specificity.

The invention relates to the technical field of molecular biology, in particular to a fluorescent quantitative PCR reference gene screening method in different tissues and stress treatment of perilla frutescens, which specifically comprises the following steps: step 1, respectively selecting perilla frutescens leaves, stalks, roots, fruits at different development stages after flowering and perilla frutescens leaves treated by 20% PEG6000 for 0H, 3H, 6H, 9H, 12H and 24H as samples; extracting total RNA (Ribonucleic Acid) of each sample, and carrying out reverse transcription on the RNA to synthesize cDNA (Complementary Deoxyribose Nucleic Acid) as And step 2, screening out candidate reference genes of the purple perilla by using the sequencing data of the early-stage purple perilla transcriptome, namely PfActin, Pf18S, PfEF-1alpha, PfCYP, PfTUA, PfTUB and PfGAPDH respectively, and screening out the candidate reference genes of the purple perilla, namely PfActin, Pf18S, PfEF-1alpha, PfCYP, PfTUA, PfTUB and PfGAPDH. According to the method, the most stable reference genes in perilla leaves, stems, roots, fruits in three development stages and perilla leaves treated by 20% PEG6000 for 0H, 3H, 6H, 9H, 12H and 24H are screened from seven candidate reference genes, and a foundation is laid for deep excavation of functional genes in the later period.

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 provides a method for eliminating ribosomal RNA in a mouse total RNA sample, which comprises the following steps: carrying out reverse transcription on the total RNA in the mouse sample into cDNA, and amplifying the cDNA by adopting a primer sequence group to obtain a DNA fragment covering all sequences of the mouse ribosomal RNA; carrying out amplification by taking the DNA fragment as a template and adopting a single primer in the primer sequence group to obtain a single-stranded DNA probe; hybridizing the total RNA of the mouse sample by adopting a single-stranded DNA probe, and performing RNase H digestion to obtain a digestion product; sequentially carrying out DNase I digestion and RNA purification on the digestion product to obtain a target product; wherein the primer sequence group comprises upstream primers from the first to the nineteenth and downstream primers from the first to the nineteenth, and the primer sequence group has the characteristics of low cost, stable performance and high reduction efficiency.

The present invention discloses a kit for detecting residual total RNA of Escherichia coli, a detection method and an application thereof. By designing a pre-treatment system, a plasmid sample can be directly used for quantitative detection of residual total RNA of Escherichia coli by RT-qPCR after digestion. The detection method eliminates the influence of the enzyme digestion process on the detection result by setting a digestion quality control product, ensures the accuracy of the entire detection result, and has excellent specificity, accuracy, sensitivity and precision.

The invention relates to the technical field of biological research data analysis, and discloses a computational biological research data analysis display method, which comprises the following steps of: extracting total RNA (Ribonucleic Acid) from biological tissues or cells, reversely transcribing the RNA into cDNA (Complementary Deoxyribonucleic Acid), constructing a library, and sequencing the library by utilizing a high-throughput sequencing platform to obtain a large number of short sequences. According to the method, the number of the short sequences mapped to the same genome coordinate interval is compared and the threshold value is judged, so that the abnormal repeated area can be effectively identified, whether the gene expression difference result is credible or not is clearly judged, and when the number of the abnormal sequences exceeds the preset repeated sequence threshold value, the influence operation of the repeated sequences is eliminated, and the accuracy of the gene expression difference result is improved. That is to say, completely identical and overlapped short sequences are removed under the condition that the FPKM value is relatively high, and the updating result is compared again, so that the problem of expression quantity distortion caused by repeated reckoning can be corrected.

The invention discloses a molecular marker of resistance of chlamydomonas reinhardtii to diflufenican and application of the molecular marker to classification of sensitive populations and resistant populations of the chlamydomonas reinhardtii to the diflufenican, and the molecular marker is CrPDSA113V mutation or CrPDSAF269Y mutation, namely Ala-1113-Val mutation or Ph-269-Tyr mutation. The detection method comprises the following steps: firstly, extracting total DNA and total RNA of chlamydomonas reinhardtii to be detected, taking the total DNA and CDNA after reverse transcription as templates, carrying out PCR (Polymerase Chain Reaction) amplification by using the specific primer pair to obtain PDS gene segments of an amplification product, analyzing whether the PCR amplification product contains the molecular marker or not, and if the PCR amplification product contains the molecular marker, judging whether the PCR amplification product contains the molecular marker or not. Whether the chlamydomonas reinhardtii to be detected is a diflufenican sensitive population or a diflufenican resistant population can be judged. The molecular marker of the resistance of the chlamydomonas reinhardtii to the diflufenican is determined for the first time, and a rapid molecular detection means is provided for the resistance of the chlamydomonas reinhardtii to the diflufenican.

The present invention relates to an RT-qPCR detection method and application for the distribution of human mesenchymal stem cells in mice, belonging to the field of biomedicine technology. The RT-qPCR detection method comprises the following steps: sampling: taking organ tissues of mice injected with human mesenchymal stem cells, extracting their total RNA for testing; detection: using real-time fluorescence quantitative PCR to detect the human mesenchymal stem cell-specific protein gene sequence in the above-mentioned total RNA to obtain the distribution of human mesenchymal stem cells in mice. The above-mentioned detection method can achieve the purpose of quickly and accurately detecting the in vivo distribution of multiple tissues at different time points, providing data support for the selection of stem cell products, and being used in the evaluation of the distribution of stem cells in the body.

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.

The invention relates to the field of plant extracts, in particular to a torreya grandis total RNA extract which at least comprises mRNA, tRNA, miRNA and tsRNA. The torreya grandis total RNA extract can be used as a natural active matter for an external preparation for the skin, and the effects of resisting oxidation, resisting aging or resisting wrinkles and tightening are achieved, so that the skin aging is delayed.

The invention belongs to the technical field of gene interference, and discloses a coral larva RNAi gene editing method and application thereof, and the coral larva RNAi gene editing method comprises the following steps: S1, soaking coral larvae in filtered seawater; s2, an RNAi reagent is added, and siRNA transfection is carried out; and S3, extracting total RNA from the transfected coral larvae, carrying out reverse transcription to obtain cDNA, and carrying out qPCR detection on the cDNA. The method comprises a double-stranded siRNA agent formed by a sense strand and an antisense strand aiming at coral calcavity protein, a transfection method and a qPCR (quantitative polymerase chain reaction) detection method. According to the successfully constructed RNAi-based coral cornu cervi larva gene editing method, functional verification of a target gene can be realized, and a powerful tool is provided for analyzing a coral biological mechanism.

The present invention discloses an AfGF14i gene of Aechmea fasciata, a cloning method, an expression vector and applications thereof, belonging to the field of biotechnology. Specifically, total RNA of Aechmea fasciata is extracted, reverse transcribed into cDNA, and taking this as a template, specific primers are designed, and the full-length CDS of the AfGF14i gene is obtained by PCR method, ligated with a vector, and transformed into Escherichia coli Trans-T1 competent cells, and positive clones are picked to obtain the AfGF14i gene of Aechmea fasciata. The full-length CDS of this gene is 729 bp, encoding a protein containing 243 amino acid residues. The present invention uses transient expression and fluorescence microscopy to detect that the subcellular localization of the protein encoded by the AfGF14i gene is the cell membrane. Through transgenic technology, it is found that the overexpression of the AfGF14i gene promotes the early flowering of Arabidopsis thaliana. The acquisition of the AfGF14i gene lays a foundation for studying the flowering mechanism of Aechmea fasciata, provides a theoretical reference for overcoming the high-temperature obstacle of ethylene-induced flowering of pineapple in production, and has important theoretical and practical significance.

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 provides a primer group for detecting a tomato yellow mottle related virus TYMaV based on RT-RAA-CRISPR / Cas12a. The primer group comprises an RT-RAA amplification primer pair and a CRISPR / Cas12a detection probe. The invention provides a method for visually and rapidly detecting TYMaV. The method comprises the following steps: extracting total RNA (Ribonucleic Acid) of tobacco leaves infected by TYMaV or coarsely extracted virions; the method comprises the following steps: preparing RT-RAA reaction mixed liquid at the bottom of a PCR tube and CRISPR premixed liquid at the cover of the PCR tube, adding total RNA or crude extraction virus particles into the RT-RAA reaction mixed liquid, incubating in a 42 DEG C metal bath for 30 minutes, placing the CRISPR premixed liquid at the bottom of the tube through centrifugation or shaking, mixing with an RT-RAA reaction product, incubating in a 37 DEG C metal bath for 20 minutes, adding 80 mu L of ddH2O, and detecting a target strip by using a flow measurement chromatography test strip. The method can be used for preparing a kit for detecting TYMaV. According to the primer group and the established TYMaV visual rapid detection method based on RT-RAA-CRISPR / Cas12a, a theoretical basis and a technical guarantee are provided for monitoring, predicting, forecasting, preventing and treating the TYMaV in the field.

The present invention relates to the field of marine biology technology, and specifically to a method for analyzing food residues in the gastrointestinal tract of Antarctic krill using molecular sequencing technology. The method comprises the following steps: dissecting Antarctic krill in a low-temperature environment and obtaining total DNA and total RNA using a nucleic acid extraction kit, and amplifying target genes in combination with specific primer design; subsequently, barcode labeling and high-throughput sequencing library construction are performed, paired-end sequencing is performed using an Illumina NovaSeq platform, and data quality control and redundancy removal are completed to obtain high-quality sequence data, accurate species identification is performed based on multi-level sequence alignment and a lowest common ancestor algorithm, phylogenetic tree and network analysis methods are used to analyze the diversity and trophic level distribution of Antarctic krill food residues, and food sources are further traced through environmental DNA comparison. The present invention can significantly improve the accuracy and scope of species identification and is of great significance for understanding the energy flow and trophic level structure of the Antarctic ecosystem.

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 invention relates to the field of gene engineering, and discloses dsRNA of a bombyx mori glucose regulatory protein 78 gene and a preparation method of the dsRNA, and the preparation method comprises the following steps: a) designing and synthesizing an upstream primer and a downstream primer of a BmGRP78 gene; b) extracting bombyx mori total RNA, performing reverse transcription to obtain first chain cDNA, taking the first chain cDNA as a template, performing PCR amplification to obtain a BmGRP78 gene full-length fragment, purifying, cloning to escherichia coli, and performing sequencing verification; c) designing an upstream primer SEQ ID NO: 3 and a downstream primer SEQ ID NO: 4 of dsRNA according to the nucleotide sequence SEQ ID NO: 1 of the BmGRP78 gene, wherein both the upstream primer and the downstream primer carry a T7 promoter sequence; and d) in-vitro transcription synthesis of dsRNA which can specifically inhibit BmGRP78 gene expression and is used for bombyx mori to optimize silk production and improve disease resistance innovation. Compared with the prior art, the dsRNA has the effect of outstanding gene knock-down efficiency, and experimental results prove that the dsRNA can effectively reduce BmGRP78 gene expression in bombyx mori ovary cells (BmN) and silk glands.

The present invention belongs to the field of biotechnology and relates to a pair of cloning primers for the AeC4H gene of okra, a cloning method and its application. The primer pair can efficiently amplify the full-length CDS sequence of the AeC4H gene. The cloning method includes steps such as extracting total RNA from okra tissues, reverse transcription, PCR amplification, product recovery, vector ligation, and positive clone screening. The recombinant expression vector contains the coding sequence of the AeC4H gene and is inserted into the vector through specific restriction enzyme sites. The present invention also provides a method for transforming plants using this vector, as well as a method for increasing the flavonoid content in plants, which is achieved by overexpressing the AeC4H gene. The present invention also provides the application of the AeC4H gene or its encoded protein in regulating the biosynthesis of plant flavonoids. The present invention realizes the overexpression of the AeC4H gene in plants, thereby increasing the flavonoid content in plants, and reveals the application of the AeC4H gene in regulating the biosynthesis of plant flavonoids.

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.