15 results about "Cloning sequencing" patented technology

The application belongs to the field of biotechnology and agricultural pest control, and particularly relates to a membrane protein Sec61 alpha gene and application of dsRNA of the membrane protein Sec61 alpha gene in pest control. After cloning and sequencing of the Sec61 alpha gene of Locusta migratoria, the sequence of the Sec61 alpha gene of SEQ ID NO:1 is obtained; then the gene fragment of SEQ ID NO:2 is selected for synthesis of double-stranded RNA (dsRNA). After the dsRNA of the gene is injected into the body cavity of the Locusta migratoria, specific Sec61 alpha genes can be silenced, and the Locusta migratoria stops feeding and successively dies before molting. The application provides a new specific molecular target for pest control based on RNA interference, and has very important application value.

The application belongs to the field of biotechnology and agricultural pest control, and particularly relates to a membrane protein Sec63 gene and application of dsRNA of the membrane protein Sec63 gene in pest control. After cloning and sequencing of the Sec63 gene of Locusta migratoria, the sequence of the Sec63 gene of SEQ ID NO:1 is obtained; then the gene fragment of SEQ ID NO:2 is selected for synthesis of double-stranded RNA (dsRNA). After the dsRNA of the gene is injected into the body cavity of the Locusta migratoria, specific Sec63 genes can be silenced, so that the Locusta migratoria cannot successfully molt and dies when molting. The application provides a new specific molecular target for pest control based on RNA interference and a new technical approach for pest control.

This invention provides a method for screening highly specific nucleic acid aptamers and specific *Lactobacillus helveticus* aptamers, belonging to the field of nucleic acid aptamers. Specifically, the method for screening highly specific nucleic acid aptamers provided by this invention includes the following steps: S1: A second library is obtained after N rounds of forward screening of a first library; S2: A third library is obtained after M rounds of reverse screening of the second library; S3: The third library is obtained after X rounds of forward screening of the third library to obtain selected random single-stranded oligonucleotides, which are then subjected to PCR amplification, purification, cloning, and sequencing; S4: The sequences cloned and sequenced in step S3 are used for affinity and specificity assays to select sequences with high affinity and specificity, thereby obtaining highly specific nucleic acid aptamers. The aptamers provided by this invention have the advantages of high specificity and strong affinity, and the construction method of the aptamers provided by this invention has the advantage of short screening time.

This invention belongs to the field of biotechnology and discloses a specific primer pair for identifying wheat-specific strains of *Blastomyces oryzae* and its application. Based on the analysis of the genomes of *Blastomyces oryzae* strains specializing in different hosts, this invention identified a conserved fragment in the gene of the wheat-specific strain. Through design, PCR amplification, agarose gel electrophoresis, and cloning sequencing, three pairs of specific primers were ultimately screened to identify the wheat-specific strain of *Blastomyces oryzae*. Using the screened specific primer pairs, the wheat-specific strain of *Blastomyces oryzae* was qualitatively identified by PCR amplification of the test samples. The identification method is simple, efficient, and accurate, and has practical application value. This invention provides molecular markers for screening wheat-specific strains of *Blastomyces oryzae* and offers a convenient and accurate method for observing and studying these strains.

The invention belongs to the technical field of biology, and discloses a specific primer pair for identifying a magnaporthe oryzae wheat specialized strain and application of the specific primer pair. Analysis is carried out according to genomes of different host specialized strains of magnaporthe oryzae, a conserved fragment in a magnaporthe oryzae wheat specialized strain gene is found, and three pairs of specific primers for targeted identification of the magnaporthe oryzae wheat specialized strain are finally screened through design, PCR amplification test, agarose gel electrophoresis and clone sequencing. The specific primer pair obtained through screening is utilized, a sample to be detected is amplified through PCR, the magnaporthe oryzae wheat specialized strain is qualitatively identified, and the identification method is simple, efficient and accurate and has practical application value. The molecular marker is provided for screening of the magnaporthe oryzae specialized wheat strain, and a convenient and accurate method is provided for observation and research of the magnaporthe oryzae specialized wheat strain.

The invention discloses dsRNA of a migratory locust nubbin gene and application of the dsRNA in pest control, and belongs to the technical field of biotechnology and agricultural pest control. The invention belongs to the field of biotechnology and agricultural pest control, and particularly relates to application of dsRNA of a nubbin gene in pest control. The method comprises the following steps: cloning and sequencing a nubbin gene of migratory locust to obtain the nubbin gene with a sequence of SEQ ID NO: 1; and then the gene segment with the sequence of SEQ ID NO: 2 is selected and used for synthesis of double-stranded RNA (dsRNA). After the dsRNA of the gene is injected into the body cavity of the migratory locust, the specific nubbin gene can be silenced, so that wing development is abnormal after 100% of migratory locust molts, and migratory flight cannot be realized. A novel specific molecular target is provided for pest control based on RNA interference, and a novel technical approach is provided for pest control.

The present invention relates to the field of biotechnology, and more specifically to specific primers for identifying indole-producing bacteria and their uses. The present invention is designed based on conserved regions of the tryptophanase gene of indole-producing bacteria from different phyla. Through PCR amplification experiments, agarose gel electrophoresis, and cloning sequencing, three pairs of specific primers (a first primer pair, a second primer pair, and / or a third primer pair) for targeted identification of indole-producing bacteria were ultimately screened and obtained. A kit prepared using the screened specific primers can be used to amplify test samples using a PCR instrument to qualitatively identify indole-producing bacteria. The identification method is simple, efficient, and accurate, and has practical application value. The present invention provides molecular markers at the gene level for the screening of indole-producing bacteria, and offers a convenient and accurate method for observing and studying changes in the species diversity of indole-producing bacteria in humans and animals.

The invention discloses a method for confirming the sequence of single-stranded nucleic acid. The method comprises the following steps: (1) preparing double-stranded nucleic acid from the single-stranded nucleic acid; (2) adding A to the tail end of the double-chain nucleic acid, and carrying out TA cloning; and (3) sequencing, such as Sanger sequencing. The method provided by the invention is low in cost, simple to operate, capable of quickly completing confirmation of the sequence of the single-stranded nucleic acid, high in practicability and wide in application prospect.

The application provides a Marek's disease virus meq gene edited deletion vaccine candidate strain SQ01Delta meq and construction and application. The SQ01Delta meq vaccine candidate strain has a preservation number of CGMCC NO.45864. The parent strain of the SQ01Delta meq vaccine candidate strain is MDV super strong Chinese mutant HNSQ01. After in vitro passage of the parent strain HNSQ01, the CRISPR / Cas9 gene editing technology is used to delete a 925bp fragment on each of the two meq alleles in the viral genome. Through PCR identification, gene cloning sequencing, RT-qPCR analysis, IFA identification and SPF chicken challenge experiment, it is ensured that the SQ01Delta meq strain completely loses the pathogenicity and tumorigenicity to the host chicken, and has no immunosuppressive property, and has good biosafety. Animal immune challenge protection evaluation experiment shows that the SQ01Delta meq has good immune protection, and the immune protection index of the parent strain reaches 80.0%. Therefore, the SQ01Delta meq strain constructed by the application can not only be used as a new MD gene deletion vaccine candidate strain, but also be used for the development and utilization of subsequent MD new type gene engineering vaccine.

The application discloses a method for analyzing epigenetic diversity based on a methylation-sensitive amplified polymorphism (MSAP) gene and a biological gene fingerprint table. The method comprises the following steps: obtaining an MSAP amplification electropherogram of a biological sample to be detected, and constructing a 01 matrix of the sample; converting the 01 matrix into an MSAP gene sequence by using a "CCGGN" replacement method; comparing the MSAP gene sequence with a reference genome, and annotating DNA methylation related gene information; based on the annotation result, constructing a gene fingerprint table based on the copy number of the methylation gene and the methylation / hemimethylation mode, and analyzing epigenetic diversity. The method can convert a traditional MSAP band fingerprint map into a gene fingerprint table with annotation function information without performing cloning and sequencing of MSAP differential fragments, can improve MSAP marker analysis to epigenome analysis, solves the problem that MSAP epigenes are difficult to clone, and improves the level of germplasm resource identification and molecular breeding.

This invention discloses a method for sequencing short nucleic acid fragments. The method involves artificially adding a 20-25 bp universal short sequence to both ends of a primer specifically for the target short nucleic acid fragment. After primer synthesis, a first round of PCR amplification is performed to obtain a first-round extended amplification product. Then, a universal long sequence is added to the 5' end of each of the aforementioned 20-25 bp universal short sequences to form universal long primers. After primer synthesis, the first-round extended amplification product is used as a template for a second round of PCR amplification, ultimately obtaining amplification products with 60-70 bp extensions at both ends of the target short nucleic acid fragment. This method eliminates the cumbersome steps of cloning and sequencing, as well as the time required for ligation, transformation, and screening, offering advantages such as high accuracy, shorter time, and lower cost. Furthermore, the two-round PCR amplification method used in this invention to extend the sequencing range is universal and applicable.

The invention belongs to the technical field of molecular biology, and particularly relates to an anti-CD33 nano antibody as well as a preparation method and application thereof. The anti-CD33 nano antibody provided by the invention comprises a frame region and a complementarity determining region, the complementarity determining region comprises CDR1, CDR2 and CDR3, the sequence of the CDR1 is SEQ ID NO.1, the sequence of the CDR2 is SEQ ID NO.2, and the sequence of the CDR3 is SEQ ID NO.3. The preparation process of the nano antibody comprises the following steps: carrying out phage library construction on immunized alpaca PBMC, carrying out automatic equipment-assisted panning on the phage library, carrying out positive clone sequencing through monoclonal preliminary screening and selecting a correct sequence for construction, expression and purification, combining an overexpression cell line with a to-be-detected antibody for a detection experiment, and screening to obtain the anti-CD33 nano antibody. The nano antibody provided by the invention can recognize and combine with a CD33 antigen, and has strong specificity and high sensitivity; in addition, the development and production cost of the CD33 antibody is remarkably reduced in the preparation process of the antibody, and meanwhile, the expression period of the antibody is greatly shortened.

The invention provides an sgRNA sequence targeting a Rongchang pig CD163 gene and a gene editing method and application of the sgRNA sequence. The nucleotide sequence of a targeting sequence in the sgRNA sequence is as shown in SEQ ID NO. 1. According to the invention, a part of region of the Rongchang pig CD163 gene exon 7 is accurately knocked out through a CRISPR / Cas9 technology, so that the directional regulation and control of the gene function are realized for the first time, and a key foundation is laid for the cultivation of disease-resistant varieties. According to the invention, the susceptibility of the CD163 gene to PRRSV invasion can be reduced only by knocking out a sequence of 1-18 bp, and the whole gene or most SRCR structural domains do not need to be deleted. According to the gene editing method disclosed by the invention, high editing efficiency and high precision are taken into consideration at the same time, editing, cloning and sequencing are performed on 16 randomly selected genes, the Indel mutation rate is up to 93.75%, the target site precise deletion efficiency is up to 75%, the invalid editing proportion is extremely low, and a reliable guarantee is provided for actual industrial application.

The invention discloses a short nucleic acid fragment sequencing method. The method comprises the following steps: artificially adding a section of universal short sequence of 20bp-25bp at two ends of a specific primer of a target short nucleic acid fragment, synthesizing the primer, and carrying out first-round PCR (Polymerase Chain Reaction) amplification to obtain a first-round lengthened amplification product; and adding a section of universal long sequence at the 5'end of each of the 20bp-25bp universal short sequence to form a universal long primer, synthesizing the primer, and carrying out a second round of PCR amplification by using the first round of lengthened amplification product as a template to finally obtain an amplification product obtained by respectively lengthening the two ends of the target short nucleic acid fragment by 60bp-70bp fragments. When the method is used for carrying out first-generation sequencing on the short nucleic acid fragment, tedious operation steps of clone sequencing and connection transformation and screening time are not needed, and the method has the advantages of being high in accuracy, shorter in needed time, lower in needed cost and the like. In addition, the method for lengthening the sequencing range through two rounds of PCR amplification has universality and universality.

The invention relates to the technical field of biology, in particular to a specific primer for identifying indoleproducing bacteria and application. Three pairs of specific primers (a first primer pair, a second primer pair and / or a third primer pair) for targeted identification of the indole-producing flora are finally screened according to the design of tryptophan enzyme gene conserved regions of different bacteriophage indole-producing flora and through PCR amplification test, agarose gel electrophoresis and clone sequencing. The kit prepared by using the specific primer obtained by screening can amplify a sample to be detected through a PCR instrument and qualitatively identify the indoleproducing bacteria, and the identification method is simple, efficient and accurate, and has practical application value. The invention provides a gene-level molecular marker for screening the indoleproducing bacteria, and provides a convenient and accurate method for observing and researching the diversity change of indoleproducing bacteria species in human and animal bodies.