78 results about "Viral isolation" patented technology

A method of predicting the virulence of a new or uncharacterized PRRS virus isolate is provided wherein the isolate is injected into swine and allowed to replicate for a period of from about 3-15 days. During this period, the rate of virus growth and / or the magnitude of viremia is determined, and this data is compared with a corresponding growth rate and / or viremia magnitude of a PRRS virus isolate of known virulence, as a measure of the virulence of the new or uncharacterized isolate. Additionally, a method of selecting an isolate for inclusion in an immunogenic composition based on the predicted virulence is also provided, together with compositions incorporating attenuated forms of viruses predicted to be virulent.

The invention provides arrays and probes for resequencing a SARS virus using an array of probes that are complementary to a SARS reference sequence and to each possible single nucleotide substitution of the reference sequence. Methods of identifying mutations in viral sequences and methods of characterizing viral isolates are also provided. The invention also provides high throughput methods to monitor epidemics and pandemics caused by pathogens such as viruses.

The invention is related to a method for selecting an influenza virus for growth on tissue culture cells to produce a tissue-culture adapted viral isolate. The invention also includes vaccines produced from the isolate.

The invention discloses a multiplex RT-PCR detection kit for a porcine reproductive and respiratory syndrome virus. The kit comprises two pairs of primers used for amplification of genes of an American porcine reproductive and respiratory syndrome virus and a European porcine reproductive and respiratory syndrome virus; and the invention provides an application method for the detection kit. The invention has the following beneficial effects: the multiplex RT-PCR detection kit for the porcine reproductive and respiratory syndrome virus provided by the invention is fast compared with classical virus isolation, single RT-PCR and serum typing methods and can conveniently identify and distinguish any one belonging to American and European porcine reproductive and respiratory syndrome viruses in one shot so as to facilitate timely and fast responses to epidemic disease situations, regions and environments and implementation of correct treatment in shortest time; meanwhile, the Trizol extraction method for extraction of the whole genome RNA in the invention has the characteristics of rapidness, simpleness, low cost and usage of a small amount of desired reagents, is especially applicable to extraction of small samples and has wide market application prospects.

The invention discloses a method for detecting infectious bovine rhinotracheitis virus. Internal amplification control (IAC) markers indicating the false negative are added to a polymerase chain reaction (PCR) system; an IAC fragment is built through the rearrangement of the base, the design of the primers and the PCR gene amplification with the bypass method; the added concentration of an internal maker template is 10<2> MuL in each reaction process, the added concentration of the Mg2+ is 2.0 mmol / L, the added concentration of the each dNTP is 0.3 mmol / L, the added concentration of each primer is 0.5 mmol / L, and the added concentration of a probe is 0.2 mmol / L; the primers are amplified at 50 DEG C for 2min in 1 cycle, 95 DEG C for 5min in 1 cycle, 95 DEG C for 15s in 45 cycles and 60 DEG C for 60s in 45 cycles. Therefore, the existence of inhibitory components or the occurrence of false negative indication can be accurately showed. The method is 10-100 times more sensitive than the conventional PCR method and the virus isolation test method and can be widely used for detecting the infectious bovine rhinotracheitis virus.

The invention discloses a double antibody sandwich ELISA kit used for Seneca viral antigen detection and application thereof. The kit comprises a Seneca viral guinea pig anti-IgG enveloped elisa plateand an HRP marked Seneca viral rabbit anti-IgG. The HRP marked rabbit anti-IgG is used for replacing primary antibodies and secondary antibodies in traditional ELISA, so that the operating steps aresimplified. Simultaneously, the SVV guinea pig anti-IgG is enveloped to the surface of a solid-phase carrier by changing an enveloping stabilization technology, the preparation technology of an enzyme-labeled antibody diluent is changed, an enzyme-labeled antibody working solution can be stored stably without changing the activity and valence, and a double antibody sandwich ELISA kit for SVV antigen specific detection and a detection method thereof are established. The vacancy of making up SVV ELISA antigen detection is proposed, the problem that existing virus separation and detection for SVVantigens are low in repeatability and sensitivity and complex in operational program is overcome, and effective technical means is provided for SVV antigen detection.

A novel immunogenic HIV-1 Env, particularly gp120, DNA construct is disclosed in which either the V1 / V2 loop and the V4 loop, or all three variable loops, including V3, are replaced with a V3 sequence each of which is from a different viral isolate. Preferably, each replacement V3 loop is a consensus sequence of V3 of a different clade. Such constructs are useful as immunogens as each presents three independent V3 epitopes, so that the immunized subject generates a more broadly reactive neutralizing antibody response than with conventional gp120 or V3 DNA or polypeptide immunogens. Also disclosed are methods of using the DNA construct to immunize a mammal, preferably a human, particularly in a priming regiment in which the DNA immunogen is followed by administration of a V3 fusion protein boosting immunogen.

The invention provides a special kit for detecting highly pathogenic porcine reproductive and syndrome virus mutation strain. The highly pathogenic porcine reproductive and syndrome virus mutation strain is detected by self-designing specific primers PSX1 / PSX2, extracting virus RNA and carrying out reverse transcription into cDNA and utilizing a PCR method; the size of the estimated amplification segment of an amplification mutation strain is 404bp and an amplification traditional PRRSV amplification segment is 494bp. Compared with the prior art, the special kit has strong specificity and sensitivity, and the coincidence rate can achieve more than 90 percent compared with a virus separation and IPMA method. The amplification results of the primers to hogcholera virus, pseudorabies virus and porcine parvovirus are negative, which can distinguish a NSP2 mutation strain and a traditional strain; the method can detect the PRRSV with 12.8ng / L, thus the invention can be widely used for the clinical detection of the highly pathogenic porcine reproductive and syndrome virus mutation strain.

The invention relates to the field of biomedicine, and specifically relates to an enterovirus 71 antigen detection test strip (colloidal gold method) and a preparation method and application thereof. Enterovirus 71 can cause hand-foot-and-mouth disease, which has largegeneration proportion of severe infections (viral encephalitis, meningomyelitis virus and pulmonary edema), and a high death rate reaching 10%-25%. The test strip of the invention is used for rapid diagnosis of EV(enterovirus)71 infection. A virus separation and an RT-PCR (reverse transcription-polymerase chain reaction) are methods first used for EV71 antigen detection, but are not suitable for primary clinic usage due to defects of difficult operation and high costs, etc. The invention overcomes the above insufficiencies and provides a reagent, which is highly demanded in clinic detection, simply operated, suitable for various medical disease control sections, and capable of detecting EV71 antigens in human oropharyngeal swabs, bubble liquid, serum or excrement, and also provides the preparation method and application thereof. A technical scheme is as follows: a specimen is dropped on a sample pad, and the EV71 antigen wherein combines with a gold-labeled EV71 polyclonal antibody in a gold-labeled pad and migrates along a chromatography membrane. A detected line captures colloidal gold particles to form a red line visible to naked eyes, so as to realize detection of the EV71 antigen.

The invention discloses a method for obtaining nucleic acid of influenza virus in an influenza sample and a special primer thereof. A primer pair for amplifying the nucleic acid of the influenza virus consists of a primer as shown in a sequence 9 in a sequence table and a primer as shown in a sequence 10 in the sequence table. The method for obtaining influenza viral genome DNA (deoxyribonucleic acid) comprises the following steps: based on the total RNA (ribonucleic acid) of a sample infected by influenza virus as a template, carrying out RT-PCR (reverse transcription-polymerase chain reaction) amplification with the primer pair so as to obtain a RT-PCR amplification product, namely the nucleic acid of the influenza virus. Through the method in the patent, virus separation is not carried out and the genome of the influenza virus is amplified directly from a nucleic acid extractive of a clinic sample, thereby greatly simplifying the program of influenza virus gene research and saving experiment cost. Based on the method, the primer is amplified through designing the segments of the influenza virus, so that all the segments of the influenza virus can be directly and successfully amplified.

The invention provides a special prime designed according to conservative M genes of influenza virus. By the aid of the principle of specific binding of biotin and streptavidin, biotin-labeled PCR (polymerase chain reaction) products are bound on a microtiter plate, anti-digoxin peroxidase is added, and finally TMB (tetramethylbenzidine) color development solution is added, so that positive products can develop colors. On the basis, a special kit is successfully prepared. The method combines PCR with a high-sensitivity digoxin detection system, the sensitivity of the method is 100 times higher than that of a conventional agarose gel electrophoresis detection method, and the method is high in specificity and overcomes difficulty in detecting AIV (avian influenza virus) of a low content in a tissue sample. Compared with a virus isolation method, the method has the advantages that coincidence rate can reach more than 95%, pollution of chemical agents (such as ethidium bromide) is eliminated, automation is benefited, the method is suitable for detecting a large number of samples, and a new approach is provided for early diagnosis of avian influenza and molecular epidemiological investigation.

The invention provides a building method of a transposable element carrier expressing pig telomerase reverse transcriptase, and application in building a pig immortalization cell line, and belongs to the field of biotechnology research. The building method includes the steps of cloning of a promoter of a pig protein translation elongation factor 1a and detection of transcriptional activity, building of an SB transposable element expression vector and exogenous gene integration, pig telomerase reverse transcriptase cDNA cloning, activity detection and building of the pig fibroblast immortalization cell line. Compared with other methods, the pig immortalization cell line built through the transposable element carrier comprises non-transformed normal cells, does not contain resistance genes, virogenes or oncogenes, and is suitable for cell physiological function researching, pig virus separation cultivation and vaccine production.

The invention relates to a primer group and a high-sensitivity detection kit for carrying out spot quick detection on shrimp covert mortality nodavirus. The primer group comprises six primers. The high-sensitivity detection kit comprises a sampling tube, a rinsing tube internally filled with distilled water, a nucleic acid denaturation tube internally filled with a TE buffer solution, an amplification detection tube internally filled with an amplification reaction liquid and a dye, a negative control tube, a positive control tube, an FTA membrane and a quick drying fluid thereof. The primer group can be used for specifically distinguishing and efficiently amplifying target nucleic acid of detected CMNV (covert mortality nodavirus). The detecting method adopting the high-sensitivity detection kit has specificity, sensitivity and convenience which are higher than those of an electron microscope detecting method, a histopathology detecting method, a virus isolation detecting method and the like, is low in cost, convenient to use, relatively accurate and quick in detection, and extremely high in sensitivity.

The invention relates to an extraction method for envelope protein of grouper iridovirus. According to the method, virus particles are purified by sucrose density gradient centrifugation; purified viruses are treated by 1% sodium dodecyl sulfonate (SDS); and virus envelope proteins are separated. The invention is mainly directed at virus envelope proteins, is favorable for promoting development of research on virus envelope proteomics and for further research on functions of grouper iridovirus envelope proteins in the process of virus infecting of hosts and for understanding of the interacting mechanism between viruses and hosts at molecular and cellular level, and provides a convincing scientific basis and application basis for controlling iridovirus diseases and developing viral vaccines.

The invention patent relates to a diagnosis technology in the technical field of agriculture. Specific primers L1, L2 and L3 are designed automatically, and virus DNAs are extracted; on the basis, the PCR method is used for discriminating and detecting different serotypes of the porcine circovirus, the size of an estimated amplified fragment of the amplified porcine circovirus II type (PCV2) is 404bp, and the size of an estimated amplified fragment of the amplified porcine circovirus I type (PCV1) is 198bp; on the basis, a special-purpose kit is successfully developed. Compared with the prior art, the diagnosis method and the special-purpose kit have stronger specificity and sensibility, and compared with methods of virus separation, IFA and the like, the coincidence rate of the method can reach more than 90%. The amplified results of the primers for a porcine reproductive and respiratory syndrome virus, a hog cholera virus, a porcine pseudorabies virus, porcine parvovirus are all feminine, and the primers can be widely used for detecting the porcine circovirus in clinical applications and laboratories.

The invention discloses a novel technology for producing recombinant adeno associated viruses by means of 293-series cell microcarrier cultivation and virus packaging by the aid of WAVE type bioreactors in a pilot test manner. The novel technology includes (1), cultivating cells, to be more specific, inoculating the 293-series cells onto microcarriers in cell cultivation bags, adding cultivation media into the cell cultivation bags and shaking the cultivation bags; (2), packaging viruses, to be more specific, settling the microcarriers, adding fresh cultivation media, transfection reagents and three-plasmid systems for packaging the viruses to the cultivation bags, continuing to cultivate the cells after co-transfection is carried out and packaging the viruses; (3), separating and purifying the viruses, to be more specific, collecting supernatant by means of centrifuging or suction filtration or filtration after the cells are completely cultivated, and separating and purifying the viruses by the aid of AKTA protein and nucleic acid chromatographic systems; (4), determining titer of the viruses by the aid of Q-PCR (quantitative-polymerase chain reaction) instruments. The novel technology has the advantages that the cells can be automatically cultivated on a large scale and can be transfected, the viruses can be packaged, the recombinant viruses can be produced in a pilot test manner, requirements of the majority of scientific research users can be met, supply and demand contradiction on virus and carrier markets can be relieved, and the labor cost input can be reduced.

The invention discloses a primer for detecting porcine sapelo virus, a Taqman-MGB probe and a kit, and belongs to the technical field of animal pathogenic microorganism detection. A nucleotide sequence of the primer for detecting the porcine sapelo virus is represented by SEQ ID NO:1-2; and a DNA sequence of the Taqman-MGB probe is represented by SEQ ID NO:3. The primer and the Taqman-MGB probe are high in specificity, high in stability and high in detection accuracy; the Taqman-MGB diagnosis kit has the characteristics of high detection speed, high sensitivity, high stability, simplicity in quantification and low false positive and is favorable for large-scale and automatic detection analysis. By adopting a real-time fluorescent quantitation RT-PCR method for the Taqman-MGB, the shortcomings of complicated virus separation operation process, difficulty in authentication, low detection speed of the conventional PCR, simplicity in pollution, need of electrophoresis caused by amplification and small number of samples to be detected at each time are overcome.

The invention discloses a micropterus salmoides brain cell line and application thereof, the micropterus salmoides brain cell line has been preserved in the China Center for Type Culture Collection on March 25, 2021, and the preservation number is CCTCC NO: C202166. The micropterus salmoides brain cell line MSBr is stable in passage, has been successfully passed for more than 65 generations at present, is good in growth state, is wide in application range, not only can be used for virus isolation and identification, pathogen function research, environmental pollutant detection, gene screening and function analysis, fish tumor research and the like, but also can be used for preparing the micropterus salmoides brain cell line MSBr. Meanwhile, the method can also be used for monitoring and diagnosing viral diseases of aquatic animals, investigating and researching epidemiology, developing diagnostic kits for related viral diseases, developing vaccines and the like, and has relatively high application value and social and economic value.

Foot and out disease virus (FMDV) is worldwide problem. Rapid isolation, serotyping and vaccine matching of FMDV from infected animals is critical to enable the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Current virus isolation protocols use primary cells, known to be susceptible to FMDV, or baby hamster kidney cells (BHK-21) and other cell lines that are not highly sensitive to some strains of FMDV. The αVβ6 integrin is a principal receptor for FMDV. We therefore transduced the porcine kidney cell line, LFBK, to stably express both the αV and β6 bovine integrin subunits. The LFBK-αVβ6 cell line showed both β6 expression and enhanced susceptibility to FMDV infection for at least 100 cell passages. LFBK-αVβ6 cells are highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophillic strain O / TAW / 97 and are thus a sensitive tool for FMDV isolation.

The invention provides a fast cultivation and identification method of high throughput virus, which comprises the steps of inoculating cells on a culture plate, inoculating a sample to be cultivated and identified to the cells and then centrifuging, then cultivating, and finally dropping the cell sap to be identified obtained by cultivation to a polyporous glass sheet for immunofluorescence assay. The centrifugal rotation speed is 2500-4000 turns per minute, the time is 30-90 minutes and the temperature is 25-37 DEG C; and the polyporous glass sheet comprises a glass sheet and a film with holes, which are tightly connected. The fast cultivation and identification method of high throughput virus has high detection flux, reduces the sample treatment time and virus cultivation time, increases detection sensibility, has less virus reagent usage, and can be widely applied to virus separation culture and identification of related samples of clinical virus laboratories, public health laboratories and sanitary inspection authorities.

The invention discloses a nested RT-PCR detection method for differentiating variant strains and classic strains of PEDV, belonging to the technical field of rapid detection of PEDV virus. According to the nested RT-PCR detection method, the disadvantages that the steps of isolation, identification and gene sequencing of viruses are tedious, the cost is high, and the consumed time is long are overcome, and the problems of low specificity and poor sensitivity of the traditional RT-PCR are solved. The nested RT-PCR detection method has the beneficial effects that the operation is simple and convenient, the use is simple, the accuracy is high, the specificity is strong, and the sensitivity is high; the method is capable of detecting whether PEDV on infected animal engine bodies is the variant strain and qualitatively detecting whether PEDV exists in epidemic materials, so that the detection technique for differentiating whether PEDV is the variant strain in the production is greatly facilitated, and the PEDV research contents in the laboratory are enriched.

The invention discloses a cherry valley duck embryo epithelial cell line and an establishment method thereof, and belongs to the field of cytobiology. The method comprises the steps of taking duck embryo tissue with 9 to 12 age in days in a sterile way, shearing the duck embryo tissue into tissue blocks, adhering the tissue blocks downwards to the bottom of a culture hole, adding nutrient solution into a carbon dioxide incubator, and performing plate adhering culture at 37 DEG C. The high-survivability and high-purity cherry valley duck embryo epithelial cell line is obtained according to the method and conducted to biological preservation, compared with primary cell, the cell line has the advantages that the cellular morphology and the physiological property of the cell line are obvious different from that of the primary cell, continuous and stable passage can be realized, the nutritional requirement is low, the growth cycle is short, and the cell line is suitable for large-scale culture, provides a large amount of high-quality cell material for life sciences, such as gene engineering, cell engineering, immunology, molecular biology and the like, can serve as donor cell for poultry somatic cell clone breeding, and can also serve as a main material for duck virus isolation and vaccine production.

The invention provides a European eel kidney cell line EK and application thereof, belonging to the technical field of animal cell culture. The kidney cell line of Anguilla anguilla is deposited as CCTCC NO: C2018160. The cell line is a fibroblast type cell, which is primary cultured by tissue block adherence method and passaged by trypsin digestion method. The kidney cell line EK in L-15 containing 8% of fetal bovine serum has the optimum state during culture at 26 DEG C.. The typical morphology of fibroblasts can be maintained after 65 passages in vitro. The karyotype of fibroblasts is aneuploid. After cryopreservation and resuscitation, the survival rate of fibroblasts is 85%, and the fibroblasts grew rapidly. The cell line EK can be used to construct the expression model of exogenous gene, can also be used as a host cell for viral isolation and other etiological and immunological research.

The invention discloses a method for separating, enriching and detecting enveloped viruses on the basis of a CL7-CVN and Im7 system. The method for separating, enriching and detecting the enveloped viruses on the basis of the CL7-CVN and Im7 system comprises the following steps: firstly, producing recombinant CL7-CVN protein and Im7 protein; coupling the Im7 protein with beads to obtain beads Im7-Beads; putting the recombinant CL7-CVN protein into a to-be-enriched virus sample, after incubation is conducted, putting the Im7-Beads, conducting enrichment after incubation is conducted, and conducting centrifugation to obtain virus-enriched Im7-Beads; and extracting nucleic acid of the enveloped viruses enriched on the Im7-Beads, conducting RT-PCR detection, and calculating the enrichment efficiency. According to the method for separating, enriching and detecting the enveloped viruses on the basis of the CL7-CVN and Im7 system, CL7-CVN protein is produced through a genetic engineering method, and by means of specific binding of CL7 and Im7 and the performance of CVN of binding the enveloped viruses, surfaces of the Im7-Beads can be enriched with PRV virus particles, so that a brand novel technical measure is provided for separation, enrichment and detection of viruses.

The invention is related to a method for selecting an influenza virus for growth on tissue culture cells to produce a tissue-culture adapted viral isolate. The invention also includes vaccines produced from the isolate.

The present invention discloses an HRM primer set for identifying canine parvovirus (CPV) and feline parvovirus (FPV), a kit containing the primer set, and applications of the kit, wherein the primerset comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is represented by SEQ ID NO.1, and the nucleotide sequence of the downstream primer is represented by SEQ ID NO.2. The present invention further provides an HRM analysis kit containing the primer set. According to the present invention, the experiment results prove that the kit has good sensitivity and good specificity in the detection of FPV and CPV, and has high coincidence, high relative specificity and high sensitivity compared to virus isolation, DNA sequencing and other tests; and thekit can provide the effective technical means for the prevention and control of diseases caused by canine parvovirus (CPV) and feline parvovirus (FPV).

The invention provides a virus wastewater treatment process, and relates to the field of virus separation. The wastewater treatment process comprises the following steps: aiming at excrement and otherhigh solid waste flows, a solid-liquid separator is adopted to carry out dry-wet separation on living source pollution discharge. On one hand, the solid-liquid separator adopts screw separation compression, on the other hand, the sewage concentration can be effectively reduced, and on the other hand, the sewage treatment efficiency is improved. On the other hand, the concentrate is pressed into blocks which can be directly sterilized. The high-toxicity wastewater (medical and domestic wastewater) is treated by adopting a pollution-resistant secondary membrane process, so that the method is convenient and has less pollution. The invention has the technical effects and advantages that: 1, wastes generated in the treatment process can be properly treated; 2, fully-sealed forced solid-liquidseparation is adopted in the treatment process, and vent holes and produced liquid are sterilized; 3, the original polluted flow is reduced, and pollutants are not newly added;.

The invention discloses a micro-fluidic cell chip and a virus isolated culture method based on same. The micro-fluidic cell chip comprises a metal frame, a lower cover plate, a chip frame, a core chip and an upper cover plate, wherein a plurality of cell culture chambers are formed in the core chip; a main runner is formed in the surface of the core chip; and the main runner is communicated with the plurality of cell culture chambers. According to the virus isolated culture method based on the micro-fluidic cell chip, a method of co-culturing multiple cell lines is adopted, a to-be-analyzed sample and different cell lines are sequentially incubated, clinical samples can be obviously saved, and the sample utilization rate is improved to the greatest degree. Meanwhile, the consumption of a culture reagent can be reduced, and the separation and identification time is shortened. Because the micro-fluidic cell chip provided by the invention contains the plurality of built-in cell culture chambers, culture of at least ten different cell lines can be simultaneously realized theoretically, the screening flux of virus sensitive host cells is obviously increased, and high-flux, automatic and rapid screening of the virus sensitive cell lines is realized.

The invention discloses a verification virus, especially relates to a detecting method for fowl grippe H5, H9 subtype virus. It uses Trizol kit to extract the sample gene RNA, and then carries on reverse rerecording and synthesizes into cDNA, then disperses several genes special expanded in one-time PCR, finally carries on electrophoresis verification. Because the special gene quantity is small in single suspected case sample, the invention forms large amount of gene replicated segments through rerecording the RNA in sample into cDNA, then expanding the NP, H5A, H9A genes in one time, thus the method is convenient, it can judge if it is fowl grippe or not, and judges the which subtype does it belong to in H5 or H9. The invention is quick and accurate.