26 results about "Genomic rna" patented technology

The present invention discloses a kit for direct amplification detection of goat pox virus based on single-domain antibody 9‑4‑2 immunomagnetic beads and its application. The kit includes single-domain antibody 9‑4‑2 immunomagnetic beads obtained by coupling the goat pox virus single-domain antibody 9‑4‑2 shown in SEQ ID NO.1 with streptavidin‑biotin and magnetic beads. The present invention uses the single-domain antibody 9‑4‑2 protein purified by gel cutting to couple with magnetic beads to obtain immunomagnetic beads. After the immunomagnetic beads are enriched with goat pox virus, the genomic RNA does not need to be extracted and can be directly amplified. The proposal of the present invention overcomes the phenomenon that goat pox virus is not easy to be found at the peak of virus replication due to the long time of forming the skin, the virus content of the collected diseased material is low, so the detection rate is low, and it is easy to miss the detection, etc.; as well as a series of problems such as time-consuming, costly, and laborious when extracting genomic DNA, and polluting the environment during operation, and it is efficient, sensitive, specific, stable, and practical. The proposal of the present invention provides an efficient technical means for the detection of goat pox virus disease.

This invention discloses a quantitative genomic RNA standard for the BCR-ABL1 fusion gene and its preparation method. The method involves extracting genomic RNA containing two mutant forms of the BCR-ABL1P210 fusion gene, b2a2 and b3a2. RNA storage buffer and quantitative standard solutions are prepared, and corresponding primers and probes are designed. A one-step reverse transcription digital PCR method is used, with the BCR-ABL1P210 fusion mutant genomic RNA as a template for PCR amplification. Fluorescence signals are collected to detect the expression of the BCR-ABL1P210 fusion genes b2a2 and b3a2, thereby obtaining the copy number content of the BCR-ABL1P210 fusion genes b2a2 and b3a2 and the abundance of the mutant gene in ABL-WT as quantitative values.

The invention discloses application of neochlorogenic acid in preparation of a medicine for preventing and / or treating hand-foot-and-mouth disease caused by A16 type coxsackie virus (CVA16) infection, and belongs to the technical field of biological medicine. Researches prove that the neochlorogenic acid has the effect of resisting CVA16 infection on HEK293T cells and H4 cells, after the two cells treated by the neochlorogenic acid are infected by the CVA16, the viral genome RNA abundance is obviously reduced, and the cell lesion death condition is obviously relieved; meanwhile, a cytotoxicity test shows that the neochlorogenic acid has no obvious cytotoxicity to HEK293T cells and only has weak cytotoxicity to H4 cells. Therefore, in a reasonable dosage range, the neochlorogenic acid, which is a low-toxicity natural small molecule compound, can be used for preparing the anti-CVA16 medicine for preventing and / or treating the hand-foot-and-mouth disease caused by CVA16 infection.

[Problem] To provide an antiviral oligonucleotide that acts on cells infected by viruses having multiple RNA genomes, exhibiting an antiviral effect, and does not tend to the emergence of resistant viruses, and a pharmacological composition thereof. [Solution] An oligonucleotide that takes on a G-quadruplex structure that inhibits the binding of the genomic RNA and nucleocapsid (N) protein of a virus having an RNA genome, the antiviral oligonucleotide suppressing proliferation of the virus by contact with virus-infected cells, and a pharmacological composition thereof are used.

Type-C endogenous retroviruses (ERVs) embedded in Chinese hamster ovary (CHO) cells were altered to modify the release of retroviral and / or retroviral-like particles in the culture supernatant. Although evidence for the infectivity of these particles is missing. their presence has raised safety concerns. 173 type-C ERV sequences that clustered into functionally conserved groups were identified. Transcripts from one type-C ERV group were identified to be full-length with intact open reading frames, and to have corresponding viral RNA genomes that were loaded into retroviral-like particles. Also, sequence analysis of the genomic RNA from viral particles indicated that they may result from few expressed ERV sequences. Disclosed herein is the disruption / alteration of the gag gene of the expressed ERV group using CRISPR-Cas9 genome editing. Comparison of CRISPR-derived mutations at the DNA and mRNA level led to the identification of a single ERV locus responsible for the release of viral RNA-loaded particles from CHO cells. Clones bearing a Gag loss-of-function mutation in this particular ERV locus showed a reduction of viral RNA-containing particles in the cell supernatant by over 250-fold. Notably, ERV mutagenesis did not compromise cell growth, cell size or recombinant protein production. Provided herein is a new strategy and cells, in particular engineered CHO cells, to mitigate potential contaminations from CHO endogenous retroviruses during biopharmaceutical manufacturing.

The present invention embraces a RNA replicon that can be replicated by a replicase of alphavirus origin. The RNA replicon comprises sequence elements required for replication by the replicase, but these sequence elements do not encode any protein or fragment thereof, such as an alphavirus non-structural protein or fragment thereof. Thus, in the RNA replicon according to the invention, sequence elements required for replication by the replicase and protein-coding region(s) are uncoupled. According to the present invention the uncoupling is achieved by the removal of at least one initiation codon compared to a native alphavirus genomic RNA. In particular, the RNA replicon comprises a 5′ replication recognition sequence, wherein the 5′ replication recognition sequence is characterized in that it comprises the removal of at least one initiation codon compared to a native alphavirus 5′ replication recognition sequence. The replicase of alphavirus origin may be encoded by an open reading frame on the RNA replicon or on a separate RNA molecule. The present invention enables efficient and safe expression of a protein of interest in a cell or organism, but is not associated with undesired production of fragments of alphavirus non-structural protein. Methods of protein production in vitro and in vivo, as well as medical uses, are provided herein.

A method of detecting severe disease-associated mutations in an enterovirus 71 (EV-A71), comprising: performing an assay on a test sample containing an EV-A71 genomic RNA, a fragment thereof or an amplicon thereof, or an EV-A71 VP1 protein or fragment thereof to detect one or more severe disease-associated mutations in the EV-A71 genomic RNA, the fragment thereof or the amplicon thereof, or the EV-A71 protein or fragment thereof, wherein the one or more mutations are selected from mutations at positions corresponding to 5′ UTR nucleotide positions C580, A707, and C709 in an EV-A71 5′ UTR nucleic acid sequence and at residues corresponding to A280 and E145 in an EV-A71 VP1 protein sequence.

The invention is in the field of polynucleotides. The invention relates to a method for transcription using a nucleic acid construct that is characterized by the presence of a promoter that has been modified to disable the TATA box. This mutated promoter is followed by a second promoter. The invention further relates to that nucleic acid construct, to an expression vector or a cell comprising that construct, and to its use for improved transcription. Production of long RNA sequences was found to be improved by this construct.

The disclosure relates to methods and systems for the detection of low-quantity (e.g., 1×102 copies / μL) target nucleic acids (e.g., genomic RNA and / or genomic DNA).

To provide a method for producing negative-strand RNA viral vector.SOLUTION: The present invention provides a method for producing a negative-strand RNA virus or viral vector, comprising: expressing a factor from a gene encoding a protein kinase R (PKR)-inhibitory factor operably linked to a control sequence, and supplying the factor to a packaging cell; allowing the packaging cell to express the genomic RNA of the negative-strand RNA virus or viral vector, thereby forming a negative-strand RNA virus or viral vector in the presence of the factor; and recovering the formed negative-strand RNA virus or viral vector; the PKR-inhibitory factor being a PKR-inhibitory viral factor, or nc886 or p58IPK, and the relationship between the virus or viral vector and the PKR-inhibitory factor being heterologous, and / or the relationship between the control sequence and the PKR-inhibitory factor being heterologous.SELECTED DRAWING: None

The application discloses application of MG-132 in preparation of a drug for resisting nerve necrosis virus. Researches of the application show that MG-132 can significantly reduce the cytopathic effect caused by nerve necrosis virus infection, reduce the number of positive cells expressing RGNNV capsid protein, and also can significantly reduce the mRNA transcription level of a key gene of RGNNV, the protein expression level and the copy number of virus genomic RNA; and with the extension of RGNNV infection time, MG-132 can also significantly inhibit virus gene transcription, protein synthesis and genome replication, and reduce virus titer. Therefore, MG-132 treatment can significantly reduce the virulence of RGNNV to cells, has the efficacy of resisting nerve necrosis virus, and has low cytotoxicity and good safety. The application provides a new drug intervention strategy for prevention and treatment of nerve necrosis virus infection, and has important application value for prevention and control of NNV related diseases in aquaculture.

The present invention discloses a kit for direct amplification detection of peste des petits ruminants virus based on single-domain antibody 9‑4‑① immunomagnetic beads and its application. The kit includes single-domain antibody 9‑4‑① immunomagnetic beads obtained by coupling the peste des petits ruminants virus single-domain antibody 9‑4‑① shown in SEQ ID NO.1 with streptavidin‑biotin and magnetic beads. The present invention uses gel-cutting purified single-domain antibody 9‑4‑① protein to couple with magnetic beads to obtain immunomagnetic beads. After the immunomagnetic beads are enriched with peste des petits ruminants virus, the genomic RNA does not need to be extracted and can be directly amplified. The proposal of the present invention overcomes the problems of low virus content, low detection rate, easy missed detection, etc.; as well as a series of difficulties such as time-consuming, costly, and labor-intensive when extracting genomic RNA, and polluting the environment during operation. The enrichment product is not only time-saving and resource-saving by directly amplifying, but also efficient, sensitive, specific, and stable. The proposal of the present invention provides an efficient technical means for the detection of peste des petits ruminants virus antigens.

The present invention belongs to the technical field of virus detection, and specifically relates to a primer pair, a primer set, a kit and a method for detecting kiwifruit chlorotic ringspot virus. The primer pair RPA‑AcCRaV‑F / RPA‑AcCRaV‑R provided by the present invention can amplify the genomic RNA of kiwifruit chlorotic ringspot virus, has good specificity, and realizes the detection of kiwifruit chlorotic ringspot virus. The primer pair is used in combination with a probe to obtain a primer set for detecting kiwifruit chlorotic ringspot virus, which can directly perform RT‑RPA‑LFD detection on kiwifruit chlorotic ringspot virus, has a short detection time, and the results can be interpreted by the naked eye, which is more convenient, realizes rapid detection of samples to be tested, and has high sensitivity, good specificity, and a minimum detection limit of 10 ‑8 ng / μL.

Modified antisense mucleic acid molecules ending with or without chain terminating bases targeting the HIV-1 viral genomic RNA 3′ LTR region, and uses thereof for inhibiting HIV-1 replication and infection, are disclosed. The antisense mucleic acid molecules more specifically target a sequence corresponding to about nucleotide 9628 to about nucleotide 9642 of HIV-1 clone pNL4-3.

The present invention relates to the adaptation and modification of Coxsackievirus B3 (CVB3) and their use or the use of their genomic RNA for the treatment of cancer, in particular for the treatment of gastrointestinal cancer, and furthermore particularly pancreatic and colorectal carcinomas.

The invention discloses application of CX-5461 in preparation of a medicine for resisting HEV (Hepatitis E Virus) infection, and belongs to the technical field of biological medicine. Bioinformatics prediction finds that a conservative G-quadruplex forming motif exists on HEV genome RNA, and in-vitro tests prove that CX-5461 can reduce the virus load and antigen level of infected cells; furthermore, meriones unguiculatus is taken as an animal model, CX-5461 intervention treatment is performed on gene type 1, type 3 and type 4 HEV infection models, and results show that the medicine can remarkably reduce the fecal virus detoxification amount of experimental animals, reduce the virus RNA load and antigen level of the liver and extrahepatic tissues (kidney and small intestine) and improve pathological injury of the liver. The invention provides a new therapeutic target and a safe and effective therapeutic drug for HEV infection.

A genome RNA extraction kit convenient to use comprises a kit body, first hinges, a kit cover, a built-in partition plate, an arc-shaped pressing rod, a double-torsion spring, a rotating cover plate, a rotating supporting plate and a magnetic sheet, the kit body is connected with the kit cover through the first hinges, the first hinges are distributed between the kit body and the kit cover, the built-in partition plate is matched with the kit body, and the arc-shaped pressing rod is connected with the rotating cover plate through the arc-shaped pressing rod. The rotating cover plates are inserted into the kit body and connected with the side surfaces of the kit body through second hinges, the rotating cover plates are symmetrically arranged in the kit body, the side surfaces of the rotating cover plates are provided with cover plate side plates, the symmetrical cover plate side plates are attached to each other, and the lower surfaces of the rotating cover plates are attached to the built-in partition plates. And the kit body and the kit cover are oppositely combined.

The application discloses a kind of based on single-domain antibody 9-4-1 immunomagnetic beads direct spread detection goat poxvirus kit and its application.The kit includes the single-domain antibody 9-4-1 immunomagnetic beads obtained by the single-domain antibody 9-4-1 shown in SEQ ID NO.1 goat poxvirus is coupled with magnetic bead by streptavidin-biotin.The single-domain antibody 9-4-1 protein purified by gel cutting is coupled with magnetic bead to obtain immunomagnetic beads, and after goat poxvirus is enriched, it can be directly amplified without extracting genomic RNA.The application overcomes the phenomenon that goat poxvirus is not easy to be found at virus replication peak period due to long time of forming sandwich, virus content is low when collecting disease material, detection rate is low, and a series of problems such as easy to miss detection;And it is time-consuming, costly and laborious to extract genomic DNA, and a series of problems such as environmental pollution during operation, and efficient, sensitive, specific, stable and practical, and the application provides an efficient technical means for detection of goat poxvirus disease.

We identified a novel spiralin protein on the membrane of a Spiroplasma sp. isolated from CWD infected deer to which we have made recombinant protein that immune reacts with sera from CWD infected deer and not with controls. This recombinant spiralin protein is being developed into live diagnostic tests for CWD including ELISA and lateral flow assays which are also applicable for diagnosis of scrapie in sheep and Creutzfeldt-jakob disease (CJD) in humans. Monoclonal antibodies developed against the recombinant spiralin protein will be used to identify the microbe in formalin fixed tissues by immune histochemistry. Identification of the entire spiralin gene for this CWD associated Spiroplasma is ongoing using real time PCR since we have the genomic RNA sequence of this microbe and PCR primers from past experiments that are effective in identifying CWD cases. The recombinant novel spiralin protein is being tested as a preventative vaccine.

The present application relates to RNA replicons for multifunctional and efficient gene expression. The present invention includes RNA replicons that can be replicated by a replicase derived from an alphavirus. The RNA replicon contains sequence elements required for replication by the replicase, but these sequence elements do not encode any protein or fragment thereof. In the RNA replicon according to the present invention, the sequence elements required for replication by the replicase and the protein coding region are decoupled. According to the present invention, decoupling is achieved by removing at least one start codon compared to the natural alphavirus genomic RNA. In particular, the RNA replicon contains a 5' replication recognition sequence that contains at least one start codon removed compared to the natural alphavirus 5' replication recognition sequence. The alphavirus-derived replicase can be encoded by an open reading frame on the RNA replicon or on a separate RNA molecule. Methods for in vitro and in vivo protein production and medical uses are provided herein.

Alphaviruses, such as chikungunya virus (CHIKV), are a group of emerging / re-emerging mosquito-borne viruses that represent an ongoing challenge to medicine and public health. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling molecule whose structure and function remain elusive. NsP3 is a component of the plasma membrane-bound viral RNA replication complexes (vRC) and is also found in large cytoplasmic aggregates of unknown functions. Here, the inventors report the cryo-electron microscopy structure of the CHIKV nsP3 at 2.6 Å resolution. NsP3 forms highly organized tubular structures made by a helical arrangement of its alphavirus unique domain (AUD) which acts as an oligomerization module. NsP3 helices organize into a cytoplasmic ring above the nsP1 capping pores at the vRCneck. These helices assemble into cytoplasmic tubular structures organized in a honeycomb network that contains viral genomic RNA and capsid as well as host factors required for productive infection. CHIKV harboring mutations in the AUD disturbing nsP3 helices have a strong defect in viral replication, assembly and release. Altogether, the results reveal a unique nsP3-dependent molecular organization required for alphavirus infection and suggest that blocking nsP3 function may represent a potent antiviral strategy.

The present application relates to a specific probe for detecting HCMV and a kit for isothermal amplification of HCMV, and belongs to the technical field of human cytomegalovirus detection.A specific probe for detecting HCMV, the probe comprises a sequence comprising a T7 promoter SEQ ID NO.21 and a sequence capable of recognizing a Cas13 protein SEQ ID NO.22.The present application uses the genomic RNA of the sample to be tested as a template, uses a specific primer probe set, performs isothermal amplification and CRISPR / Cas13 specific detection, and determines the results according to the fluorescence signal, so as to realize the qualitative detection of the infection state of human cytomegalovirus.Using RNA as a template, the latent state and active state of HCMV can be distinguished.

The invention relates to a genome RNA protective agent and a preparation method thereof, and belongs to the field of biology, the protective agent comprises 0.1%-0.3% of sucrose, 0.01%-0.02% of sodium chloride, 0.01%-0.05% of ammonium sulfate, 0.01%-0.2% of lactoalbumin hydrolysate, 0.01%-0.05% of disodium ethylene diamine tetraacetate, 0.01%-0.1% of triethanolamine and 0.01%-0.05% of procilin300, and a solvent is nuclease-free DEPC water. The genome RNA protective agent provided by the invention is used for protecting genome RNA, has colorless and transparent characters, has no influence on PCR amplification, and can stably preserve the genome RNA for 18 months or more at-20 DEG C or below.

The present specification provides: an antisense oligomer, a pharmaceutically acceptable salt thereof, or a hydrate of the antisense oligomer or the pharmaceutically acceptable salt, which contains a nucleotide sequence complementary to the nucleotide sequence of a target region and has a length of 15 to 30 nucleotides, the target region includes at least 10 contiguous base sequences or complementary sequences thereof in at least one region selected from the group consisting of a 5 'UTR region, an nsp1 region, an nsp10 region, an RNA-dependent RNA polymerase region, an ORF10 region, and a 3' UTR region in genomic RNA of SARS-CoV-2. The antisense oligomer or a pharmaceutically acceptable salt thereof, or a hydrate of the antisense oligomer or the pharmaceutically acceptable salt thereof, has an antiviral effect against a virus selected from the group consisting of SARS-CoV-2, SARS-CoV-1, and MERS-CoV.

Type-C endogenous retroviruses (ERVs) embedded in Chinese hamster ovary (CHO) cells were altered to modify the release of retroviral and / or retroviral-like particles in the culture supernatant. Although evidence for the infectivity of these particles is missing, their presence has raised safety concerns. 173 type-C ERV sequences that clustered into functionally conserved groups were identified. Transcripts from one type-C ERV group were identified to be full-length with intact open reading frames, and to have corresponding viral RNA genomes that were loaded into retroviral-like particles. Also, sequence analysis of the genomic RNA from viral particles indicated that they may result from few expressed ERV sequences. Disclosed herein is the disruption / alteration of the gag gene of the expressed ERV group using CRISPR-Cas9 genome editing. Comparison of CRISPR-derived mutations at the DNA and mRNA level led to the identification of a single ERV locus responsible for the release of viral RNA-loaded particles from CHO cells. Clones bearing a Gag loss-of-function mutation in this particular ERV locus showed a reduction of viral RNA-containing particles in the cell supernatant by over 250-fold. Notably, ERV mutagenesis did not compromise cell growth, cell size or recombinant protein production. Provided herein is a new strategy and cells, in particular engineered CHO cells, to mitigate potential contaminations from CHO endogenous retroviruses during biopharmaceutical manufacturing.

The application discloses a kind of based on single-domain antibody 9-4-1 immunomagnetic bead direct spread detection small ruminant pestivirus kit and its application.The kit includes the single-domain antibody 9-4-1 immunomagnetic bead obtained by the single-domain antibody 9-4-1 shown in SEQ ID NO.1 small ruminant pestivirus through streptavidin-biotin and magnetic bead coupling.The single-domain antibody 9-4-1 protein purified by gel cutting is coupled with magnetic bead to obtain immunomagnetic bead, after small ruminant pestivirus is enriched by immunomagnetic bead, it can be directly amplified without extracting genomic RNA.The application overcomes the problems of low virus content, low detection rate, easy to miss detection, etc.;And a series of problems such as time-consuming, costly, laborious, environmental pollution during operation when extracting genomic RNA, by directly amplifying enrichment product, not only save time, save resources, but also efficient, sensitive, specific, stable, the application provides a kind of efficient technical means for small ruminant pestivirus antigen detection.