51 results about "Genome amplification" patented technology

The invention relates to a micro-fluidic chip for sequencing a single virus genome and a sequencing method. The micro-fluidic chip comprises a first functional area, a delay pipeline, a fusion pipeline, a second functional area and a fusion electrode, the first functional area is used for generating first liquid drops of virus-in-oil and lysate; two ends of the delay pipeline are respectively connected with the first functional area and the fusion pipeline; the second functional area is used for generating second liquid drops of the oil bag amplification reaction liquid, and the second functional area is connected with the fusion pipeline to enable the second liquid drops to enter the fusion pipeline; a fusion point is arranged on the downstream of the joint of the fusion pipeline and the second functional area, and the fusion electrode can act on the fusion point to fuse the first liquid drop and the second liquid drop. According to the micro-fluidic chip disclosed by the invention, single-virus whole genome amplification can be completely completed on a single chip, so that the operation steps are simplified, the complexity of sample treatment is reduced, pollution and sample loss are effectively avoided, and the efficiency, the stability and the reliability are improved.

The invention provides a primer group for detecting a porcine epidemic diarrhea virus based on a whole genome, a library building method and application. The primer group comprises 27 pairs of primers, and sequences of upstream primers and downstream primers of the 27 pairs of primers are shown as SEQ ID NO.1-SEQ ID NO.54. The invention further provides a kit for detecting the porcine epidemic diarrhea virus based on the whole genome. The primer group disclosed by the invention has good specificity, sensitivity and repeatability, and has a good application prospect in amplification of the whole genome of the porcine epidemic diarrhea virus and preparation of reagents or kits required by amplification. The library building method provided by the invention comprises the following steps: carrying out multiple PCR amplification by using the primer group provided by the invention; the library building method is suitable for a next-generation sequencing platform and a nanopore sequencing platform, the automation degree is high, 95% or above of porcine epidemic diarrhea virus sequences can be obtained even in a sample with the virus content as low as 1 copies / microliter, and the sequencing depth can reach 1000 X or above.

The invention discloses a method for identifying Yushan black pigs by adopting an SNP (Single Nucleotide Polymorphism) molecular marker technology, and particularly relates to the technical field of molecular detection.The method comprises the following steps: S1, sample preparation: collecting any one of ear tissues, tail tips or blood samples of to-be-detected pig individuals, and extracting to obtain genome DNA (Deoxyribose Nucleic Acid); the genome DNA is used as a template, amplification detection is conducted through a first primer pair, a second primer pair, a third primer pair and a fourth primer pair corresponding to the first SNP site, the second SNP site and the fourth SNP site, and the nucleotide sequences of the first primer pair, the second primer pair and the fourth primer pair are sequentially SEQ ID NO.5-SEQ ID NO.12. By detecting the specific single nucleotide polymorphic sites in the Yushan black pig genome and carrying out genetic typing comparison, the Yushan black pigs and the filial generation thereof can be quickly and accurately identified.

The invention belongs to the technical field of biological detection, and relates to a method for detecting lentivirus titer by a double-standard-curve RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) method. Infecting the target cells by adopting a to-be-detected sample containing a recombinant lentivirus, and collecting and extracting genome DNA of the infected target cells; carrying out a viral genome RT-qPCR amplification reaction and an internal reference genome RT-qPCR amplification reaction by respectively taking the original virus standard substance and the internal reference standard substance which are diluted in a gradient manner and the extracted target cell genome DNA as templates; and calculating the lentivirus titer corresponding to the sample according to data obtained by the RT-qPCR amplification reaction. The method can be used for absolute quantitative qPCR detection of lentivirus titer, can accurately determine lentivirus successfully integrated into a cell genome, and reflects the real infection ability of the virus; in addition, the technology has high sensitivity and specificity, is not influenced by the extraction efficiency of the cell genome DNA, and ensures the reliability of the detection result.

The invention provides a primer group for detecting a porcine reproductive and respiratory syndrome virus based on a whole genome, a library building method and application. The primer group comprises 16 primer pairs, and sequences of an upstream primer and a downstream primer of each primer pair are shown as SEQ ID NO.1-SEQ ID NO.32. The invention further provides a kit for detecting the porcine reproductive and respiratory syndrome virus based on the whole genome. The primer group disclosed by the invention has good specificity and sensitivity, and has a good application prospect in amplification of the whole genome of the porcine reproductive and respiratory syndrome virus and preparation of reagents or kits required by amplification. The library building method provided by the invention comprises the following steps: carrying out multiple PCR amplification by using the primer group provided by the invention; the library building method is suitable for a next-generation sequencing platform and a nanopore sequencing platform, the automation degree is high, 95% or above of porcine reproductive and respiratory syndrome virus sequences can be obtained even in a sample with the virus content as low as 1 copies / microliter, and the sequencing depth can reach 1000 X or above.

The application belongs to the field of fish development, and particularly relates to a molecular marker for identifying Nibea albiflora sperm-induced Pseudosciaena heteroclita gynogenesis offspring and hybrid offspring, a primer set thereof, a kit and application. The primer set for identifying the molecular marker of the Nibea albiflora sperm-induced Pseudosciaena heteroclita gynogenesis offspring and hybrid offspring comprises primers F and R, the nucleotide sequence of the primer F is shown as SEQ ID NO. 1, and the nucleotide sequence of the primer R is shown as SEQ ID NO. 2. Based on genomic data analysis, the application develops the molecular marker F / R which can effectively identify the Pseudosciaena heteroclita gynogenesis offspring and hybrid offspring in the juvenile stage. The method comprises extracting genomic DNA of parents and offspring, PCR amplification and agarose gel electrophoresis detection, and finally distinguishing the gynogenesis individuals and hybrid individuals by whether the Nibea albiflora parent marker is contained in the electrophoresis band.

The application discloses a set of general primers for amplifying the whole genome of the mitochondrion of Trionychidae, and belongs to the technical field of molecular biology; the specific steps comprise the following steps: extracting the total DNA of the genome of Trionychidae, designing the general amplification primers of the whole sequence of the mitochondrial genome, and performing PCR amplification and sequencing on the mitochondrial genome DNA. The amplification primers for the whole genome sequence of the mitochondrion of Trionychidae are composed of 11 pairs of single-stranded oligonucleotides. The general amplification primers of the application can be used to quickly and efficiently amplify the whole sequence of the mitochondrial genome of various Trionychidae, and provide a favorable technical means for the identification of unknown germplasm of Trionychidae, the protection of species resources, and the research on the system evolution.

The invention relates to a method of determining somatic allele-specific copy number alterations (CNAs) in the genomes of cells in a test-sample from a subject, the method comprising: i) providing an indexed-DNA library of DNA fragments resulting from whole-genome amplification of genomic DNA from cells of the test-sample, ii) providing whole genome sequencing data of reference non-cancer cells from a reference-sample from the subject; and iii) determining somatic allele-specific copy number alterations in the genome(s) of the cells of the test-sample; and associated methods and uses in cancer therapy.

The invention relates to the technical field of gene engineering, in particular to a method for rapidly and economically carrying out gene function analysis on lepidoptera insects and application. The method comprises the following steps: designing four different sgRNAs by aiming at an exon region of a target gene, and constructing a sgRNAs synthetic primer mixing pool; 4 different sgRNA-DNA templates are synthesized at the same time, 4 different sgRNAs are further synthesized at the same time, and a sgRNAs mixed pool is formed; the sgRNAs-Cas9 protein and Cas9 protein are prepared into an sgRNAs-Cas9 compound, and the sgRNAs-Cas9 compound is microinjected into newly laid eggs; after the injected eggs develop into larvae, genome DNA is extracted, sgRNAs targeted areas are amplified, the mutation rate of target genes is determined through T7E1 enzyme digestion, and phenotype analysis is conducted on the larvae. According to the method, the mutation rate of the target gene is increased through redundant targeting of the four sgRNAs, so that functional analysis of the target gene is quickly realized in the G0 generation, and the research period of the gene function of the lepidoptera insects is greatly shortened. Besides, four different sgRNAs are synthesized at the same time by constructing a primer mixing pool of a plurality of sgRNAs, so that the economic cost for synthesizing the sgRNAs is greatly reduced.

The application discloses a single cell whole genome amplification sequencing method. The application comprises the following steps: 1) separating a single cell, adding a single cell lysis solution to sufficiently lyse the single cell and digest proteins combined on the genomic DNA; 2) using a Tn5 transposome with a specific adapter sequence to fragment the DNA, while adding adapters to both ends of the fragments; 3) after end repair, using a ribonuclease to cut modified ribonucleotide residues on the adapter sequence; 4) using the adapter sequence as a primer to linearly amplify the fragmented DNA by using a single primer PCR method; and 5) adding sequencing adapters to both ends of the DNA fragments by using a chain extension and PCR method to obtain a final sequencing library. The method disclosed by the application can amplify single cell whole genome DNA with high coverage, uniformity and fidelity, and simultaneously and accurately detect chromosomal copy number variation CNV and single nucleotide mutation SNV on a single cell genome.

The present application relates to a primer combination for amplifying whole genome DNA of sheep embryo cells and application thereof, and the primer combination is composed of 20 or 30 primers. The present application designs random primers based on sheep reference genome sequence, optimizes the reaction system based on MDA whole genome amplification technology, so that the whole genome DNA amplification product with high coverage and low mismatch rate can be generated by using 10 sheep embryo cells or as low as pg level genomic DNA, which can be directly adapted to downstream applications such as second-generation sequencing and targeted capture sequencing, and the detection cost is reduced.

The application discloses a single-cell mitochondrial whole-genome efficient and specific amplification method. The application provides a single-cell or multi-cell mitochondrial genome DNA high-fidelity amplification method, which comprises the following steps: taking total DNA obtained by lysing a test single cell as a template, using a chimeric primer composed of a 5' end non-template matching common sequence and a 3' end mitochondrial DNA (mtDNA) specific matching sequence, and a primer corresponding to the 5' end non-template matching common sequence of the chimeric primer, and using a high-fidelity DNA polymerase with strand displacement activity to simultaneously amplify several overlapping amplicons which completely cover the single-cell or multi-cell mitochondrial genome DNA. The application avoids various problems in the current single-cell mtDNA in-vitro amplification approach, and is a very reliable mtDNA whole-genome amplification method.

The invention develops a method for absolute quantification of fungal population based on a Pacbio three-generation long fragment sequencing platform and application thereof, and belongs to the technical field of microbial amplicon detection. According to the method, the synthesized chimeric DNA is used as an internal standard, and a fungal community in a sample is absolutely quantified on the basis of an amplicon high-throughput sequencing platform. The specific method comprises the following steps: directly adding the synthesized chimeric DNA into an environmental sample; extracting genome DNA of the environment sample; carrying out high-throughput sequencing on the ITS full-length sequence of the fungus through PCR amplification; the high-throughput sequencing results calculate the relative abundance and absolute abundance of the amplicon family (e.g., the copy number of fungal ITS per unit mass sample). A highly complex environmental sample is taken as a research object to prove that the method can sensitively and accurately quantify the absolute abundance of a specific group. The method is widely applicable to microbial community analysis of samples from different sources, including environmental samples from soil, samples from plant tissues, samples from human intestinal tracts, and samples from food samples. The method disclosed by the invention is simple to operate, accurate in detection and wide in application, and has a wide market prospect in identification and analysis of microbial communities.

The invention provides a method for carrying out whole genome amplification by taking cell culture fluid as a template, the whole genome amplification is carried out by simulating different cell culture fluids, an optimized whole genome amplification reagent and program are determined, and effective amplification of various culture fluid cells or trace genome templates is realized. Whole genome amplification products are subjected to quality inspection, database establishment and machine loading, and off-machine data are analyzed, so that the effectiveness and the accuracy of whole genome amplification data obtained by the method disclosed by the invention are proved. The problem that an amplification reagent on the current market cannot resist inhibition amplification is solved.

The invention relates to a method and a system for quality control of single cell whole genome amplification products. The method comprises detection of different chromosome segments. The invention also relates to reagents used in the method.

The invention relates to a mantle cell lymphoma BCL1-IGH rearrangement MRD detection system. The detection system comprises: (1) a sample module, which is used for extracting peripheral blood or marrow genome DNA of a sample to be detected; (2) an amplification sequencing module: performing amplification by taking the genome DNA as a template to obtain an amplification product, and performing high-throughput sequencing on the amplification product; and (3) the bioinformatics analysis module is used for carrying out BCL1-IGH rearrangement cloning identification and cloning tracking on the to-be-detected sample according to the sequencing result. According to the detection system provided by the invention, the detection process is remarkably simplified, the detection cost and the analysis difficulty are reduced, the detection rate of minimal residual focuses of mantle cell lymphoma is high, and the detection system can be applied to the fields of auxiliary clinical diagnosis and the like.

This invention discloses a kit for amplifying the whole genome of bovine embryonic cells and its applications. The invention provides a primer set for amplifying the whole genome of bovine embryonic cells, consisting of 20 primers, each 6 nt in length. This invention can utilize 4-8 bovine embryonic cells or genomic DNA down to the pg level to amplify and generate whole-genome DNA amplification products with high coverage and low mismatch rate. These products can be directly adapted to downstream applications such as next-generation sequencing and targeted capture sequencing, while reducing detection costs. Furthermore, the three-step operation process of this invention (cell lysis-lysis termination-isothermal amplification) can complete efficient amplification within 3.5 hours.

This invention provides a primer set, library construction method, and application for whole-genome detection of porcine reproductive and respiratory syndrome virus (PRRSV). The primer set comprises 16 primer pairs, and the sequences of the upstream and downstream primers for each primer pair are shown in SEQ ID NO. 1 to SEQ ID NO. 32. The primer set of this invention exhibits good specificity and sensitivity, and shows promising application prospects in the amplification of the entire PRSV genome and the preparation of reagents or kits required for amplification. The library construction method of this invention includes multiplex PCR amplification using the primer set described herein. This method is compatible with next-generation sequencing platforms and nanopore sequencing platforms, offering a high degree of automation. Even in samples with viral concentrations as low as 1 copy / μL, it can obtain more than 95% of the PRSV sequence, with a sequencing depth exceeding 1000X.

The invention discloses a pigeon mitochondrial whole genome amplification and assembly method. The method comprises the following steps: (1) extracting genome DNA of a to-be-detected pigeon; (2) carrying out PCR (Polymerase Chain Reaction) amplification through the primers to obtain two sections of pigeon mitochondrial DNA (Deoxyribose Nucleic Acid) fragments, and (3) carrying out high-throughput sequencing; and (4) assembling the mitochondrial genome to obtain the mitochondrial DNA full length.

The invention relates to the technical field of biology, in particular to a single cell methylation detection method based on enriched cast-off cells and application. The invention provides a single cell methylation detection method based on enriched cast-off cells. The single cell methylation detection method comprises the following steps: S1, separating, purifying and enriching cast-off cells to be analyzed; s2, carrying out DNA extraction on cast-off cells to be analyzed; s3, carrying out hydrosulfite conversion on the extracted DN A; s4, carrying out DNA multiple replacement amplification on the transformed DNA sample; and S5, carrying out methylation analysis on the amplified DNA. According to the method, DNA products are increased through a genome amplification technology, the DNA products can be increased to the level suitable for a conventional detection technology, and meanwhile non-specific loss in the capturing process is effectively avoided.

The present invention belongs to the field of gene detection, and specifically relates to a primer set for whole genome amplification of monkeypox virus and its application. The present invention provides a primer set for whole genome amplification of monkeypox virus, the primer set comprising: a first primer set, which comprises at least two selected from the sequences shown in SEQ ID NO:1-178; a second primer set, which comprises at least two selected from the sequences shown in SEQ ID NO:179-358. The primer set provided by the present invention for monkeypox virus has better sensitivity, can achieve whole genome amplification of monkeypox virus, the proportion of fragments with gene coverage of more than 30× is more than 99.9%, and the sequencing depth can reach up to 90000×. The present invention has broad application prospects in monitoring monkeypox virus subtypes.

The present invention provides universal primers and a method for amplifying the whole mitochondrial genome of mites from the family Acaridae. This invention, for the first time, provides a set of universal primers for amplifying the whole mitochondrial genome of mites from the family Acaridae. By dividing the mitochondrial genome into six overlapping fragments, conventional PCR amplification, Sanger sequencing, and sequence splicing are performed to rapidly obtain the complete mitochondrial genome sequence of mites from the family Acaridae. This method requires relatively low instrumentation and analytical equipment, making it easy to implement. Furthermore, the entire mitochondrial genome sequence can be obtained from the total DNA of a single individual, avoiding interference from the genomic DNA of multiple individuals. This method has been validated in the amplification of the mitochondrial genomes of six species of mites from the family Acaridae. This method is of great significance for obtaining more complete mitochondrial sequences of mites from the family Acaridae, further inferring the phylogenetic relationships between the genera of the family Acaridae, and the phylogenetic relationships between the families of the family Acaridae. It also provides a reference for studying the evolution and phylogenetic relationships of the Acari.

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant for improving long fragment amplification efficiency and application of the DNA polymerase mutant. According to the invention, the DNA polymerase mutants KODL and KODW with enhanced continuous synthesis capability are successfully constructed by carrying out combined mutation of specific amino acid sites on the KOD DNA polymerase, the amplification efficiency of the mutants on a long-fragment DNA template (such as 30kb) is remarkably improved on the basis of retaining the amplification efficiency on fragments with conventional lengths, and the application of the mutants to the preparation of the long-fragment DNA template is facilitated. The technical bottleneck of the existing variable temperature amplification enzyme in long fragment amplification is broken through, and an efficient and reliable enzymology tool is provided for molecular biology application of long fragment gene cloning, whole genome amplification, complex structure analysis and the like.

The present invention relates to a genome amplification module having a branch space adjacent to an inlet through which an extract flows, and provides a genome amplification module in which a branch space that is wider and deeper than the extract flow passage is formed adjacent to the inlet, and after the branch space is filled with extract, the extract can be simultaneously injected along each extract flow passage.

The application belongs to the technical field of biology, and discloses a primer group, a kit and a method for quality control of whole genome amplification of Macaca fascicularis. The quality control method for the whole genome amplification product of Macaca fascicularis is simple and easy to operate, 6 pairs of primers can be used in the same reaction tube, thereby reducing the steps of adding reagent components and sub-packaging, and the whole reaction has low requirements on laboratory conditions, only a conventional PCR amplification laboratory, agarose electrophoresis and a gel analyzer are needed.

The present application relates to the assisted evaluation method of the second biopsy in the pre-implantation genetic testing. The blastocyst first biopsy process collects and cryopreserves the corresponding biopsy droplet; for the blastocyst with CNV detection failure or suspected abnormal CNV detection result in the first biopsy cell, the first biopsy droplet is thawed to complete genome amplification, CNV library construction, high-throughput sequencing and CNV analysis, and the second biopsy suggestion is given according to the detection result. If the biopsy droplet detects the abnormal aneuploidy or chimera clinically interpreted, it is suggested that the corresponding blastocyst has a high probability of chromosomal abnormality, and the second biopsy is not recommended; if the biopsy droplet does not detect the abnormal aneuploidy or chimera clinically interpreted, it is suggested that the corresponding blastocyst has a high probability of chromosomal normality, and the second biopsy is recommended. The present application proves that the first biopsy droplet contains detectable genetic material, and the consistency of the chromosomal ploidy detection result and the biopsy cell is more than 80%, which can assist the second biopsy decision and improve the decision efficiency and scientificity of PGT detection.

The invention relates to a kit for detecting nocardia seriolae of jewfish based on RPA-CRISPR / Cas12a as well as a detection method and application of the kit. The main component of the kit is a composition for identifying nocardia seriolae, and the composition comprises a specific primer for detecting nocardia seriolae based on RPA-CRISPR / Cas12a, a Cas12a protein, crRNA and a fluorescence reporter probe ssDNA Reporter, wherein the specific primer, the Cas12a protein, the crRNA and the fluorescence reporter probe are used for detecting nocardia seriolae based on RPA-CRISPR / Cas12a. The detection method of the kit comprises the following steps: S1, extracting a sample genome DNA; s2, carrying out RPA (recombinase polymerase amplification); s3, CRISPR (clustered regularly interspaced short palindromic repeats) / And S4, interpreting a detection result. According to the invention, a target gene segment of nocardia seriolae is rapidly amplified at a constant temperature by virtue of an RPA technology, and the target gene segment of nocardia seriolae is specifically targeted by virtue of accurate recognition and trans-cleavage capabilities of CRISPR / Cas12a, so that rapid detection visible to naked eyes is realized. Compared with conventional detection methods such as PCR, qPCR and the like, the kit does not need a professional thermal cycler, has the advantages of high sensitivity, strong specificity, simplicity and convenience in operation, low cost and the like, and is expected to be applied to on-site instant detection of aquaculture sites and basic laboratories.

The invention provides a kit for detecting jewfish aeromonas veronii based on RPA-CRISPR / Cas12a, a detection method and application of the kit. The main component of the kit is a composition for identifying the aeromonas veronii, and the composition comprises a specific primer for detecting the gyrB gene of the aeromonas veronii based on RPA-CRISPR / Cas12a, a Cas12a protein, crRNA and a fluorescence report probe ssDNA Reporter. The detection method of the kit comprises the following steps: S1, extracting a sample genome DNA; s2, carrying out RPA (recombinase polymerase amplification); s3, CRISPR (clustered regularly interspaced short palindromic repeats) / And S4, interpreting a detection result. According to the invention, a target gene segment of the aeromonas veronii is rapidly amplified at a constant temperature by virtue of an RPA technology, and the target gene segment of the aeromonas veronii is specifically targeted by virtue of accurate recognition and trans-cleavage capabilities of CRISPR / Cas12a, so that rapid detection visible to naked eyes is realized. Compared with conventional detection methods such as PCR, qPCR and the like, the kit does not need a professional thermal cycler, has the advantages of high sensitivity, strong specificity, simplicity and convenience in operation, low cost and the like, and is expected to be applied to on-site instant detection of aquaculture sites and basic laboratories.

The present invention provides an isolated peptide of SEQ ID NO: 1 needed for primase active as well as new replicative DNA polymerase enzymes, preferably that of SEQ ID NO: 2, comprising said peptide. Thus, these DNA polymerases are endowed with priming activity and do not require externally provided primers for initiating and performing DNA amplification. These polymerases are able to carry out a faithful and processive de novo DNA synthesis of DNA templates in the absence of pre-synthetized primers. Therefore, these enzymes of the invention act both as primases and DNA polymerases. Furthermore, they show translesion synthesis capacity, so that they may be useful not only for whole-genome amplification but also for the amplification of damaged DNAs. The invention further refers to methods for amplifying templates DNAs using these enzymes.

The invention develops a bacterial population absolute quantification method based on a Pacbio three-generation long fragment sequencing platform and application thereof, and belongs to the technical field of microbial amplicon detection. The synthetic chimeric DNA is used as an internal standard, and the microbial community in the sample is absolutely quantified at the same time. The specific method comprises the following steps: directly adding the synthesized chimeric DNA into an environmental sample; extracting genome DNA of the environment sample; carrying out PCR amplification on the 16S full-length sequence and carrying out high-throughput sequencing; and calculating relative abundance and absolute abundance of the amplicon family according to a high-throughput sequencing result. A highly complex environmental sample is taken as a research object to prove that the method can sensitively and accurately quantify the absolute abundance of a specific group. The method is widely applicable to microbial community analysis of samples from different sources, including environmental samples from soil, samples from plant tissues, samples from human intestinal tracts, and samples from food samples. The method disclosed by the invention is simple to operate, accurate in detection and wide in application, and has a wide market prospect in identification and analysis of microbial communities.