243 results about "Porcine epidemic diarrhea virus" patented technology

The invention relates to an application of nucleoside analogs in virus resistance. Specifically, the invention relates to the use of nucleoside analogs and pharmaceutical compositions thereof as (a) inhibitors for inhibiting the replication of coronavirus, influenza virus, respiratory syncytial virus, flaviviridae virus, filoviridae virus and/or porcine epidemic diarrhea virus (PEDV); and/or (b) medicines for treating and/or preventing and relieving related diseases caused by infection of coronavirus, influenza virus, respiratory syncytial virus, flaviviridae virus, filoviridae virus and/or porcine epidemic diarrhea virus (PEDV). The nucleoside analogue disclosed by the invention can be used for treating and/or preventing and relieving related diseases such as respiratory tract infection, pneumonia (COVID-19) and the like caused by 2019 novel coronavirus infection.

The invention discloses a primer, a probe and a detection kit for detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. The detection primers and probes are SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence table, wherein the sequences SEQ ID NO: 1 and SEQ ID NO: 2 are sense primers and antisense primers for detecting porcine transmissible gastroenteritis virus respectively, and the sequence SEQ ID NO: 3 For detecting the fluorescent probe of porcine transmissible gastroenteritis virus, sequence SEQIDNO: 4 and SEQIDNO: 5 are sense primer and antisense primer for detecting porcine epidemic diarrhea virus respectively, and sequence SEQIDNO: 6 is the detection primer of porcine epidemic diarrhea virus fluorescent probe. The invention also provides detection kits for porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. The primers and probes selected by the present invention have very strong specificity. The total viral RNA extracted from porcine diarrhea does not need to be transcribed into cDNA first. The synthesis of the first strand of cDNA and double PCR are completed in one step, and two viruses can be detected at one time. ,Improve efficiency.

The invention discloses an immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as a preparation method and application thereof. The test strip sequentially comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, an absorbent paper and a PVC (poly vinyl chloride) base plate arranged below and used as an assembling platform according to a connection sequence, wherein the colloid gold pad comprises a glass cellulose membrane of a PEDV (porcine epidemic diarrhea virus) monoclonal antibody adsorbed with colloidal gold marks, the nitrocellulose membrane is provided with a goat rat resisting IgG (immunoglobulin G) polyclonal antibody coated quality control line and a rabbit resisting PEDV S protein polyclonal antibody coated detection line, and the PEDV monoclonal antibody is generated in secretion of hybridoma cells with a preservation number CCTCC C201392. Experiments prove that the test strip has the advantages of strong specificity and good stability; the operation is simple, technicists do not need special training, no special equipment is needed, the detection cost is low, the detection speed is fast and the results can be read in 5-10 minutes, and the test strip is applicable to field test.

The invention discloses a colloidal gold immunochromatographic test strip for detecting wild-type classical swine fever virus, which consists of water absorbent paper (1), a cellulose nitrate membrane (2), a colloidal gold pad (3), a sample pad (4) and a support (5), wherein the cellulose nitrate membrane contains a detection line which is formed by coating monoclonal antibody HQ06 of anti-classical swine fever virus E2 protein and a quality control line which is formed by coating rabbit anti-mouse IgG antibody; and the colloidal gold pad is combined with colloidal gold-labeled monoclonal antibody 6E10 of the anti-classical swine fever virus E2 protein. The test strip does not react with C-strain of classical swine fever virus, bovine viral diarrhea virus, porcine reproductive and respiratory syndrome virus, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine rotavirus, pseudorabies virus, porcine parvovirus and porcine circovirus type 2, and can accurately and sensitively identify the wild-type classical swine fever virus, thereby having good specificity, sensitivity and repeatability.

The present invention encompasses porcine epidemic diarrhea virus (PEDV) vaccines or compositions. The vaccine or composition may be a vaccine or composition containing PEDV antigens. The invention also encompasses recombinant vectors encoding and expressing PEDV antigens, epitopes or immunogens which can be used to protect porcine animals against PEDV.

The invention discloses a recombinant porcine epidemic diarrhea virus (PEDV) lactococcus lactis and its construction method and use. The construction method comprises the following steps of carrying out RT-PCR amplification to obtain a major protective antigen COE gene sequence of PEDV, carrying out clone sequencing, designing expression primers according to the sequencing result and expression vector characteristics, carrying out amplification of a PEDV COE coding sequence, connecting the PEDV COE coding sequence to a lactococcus lactis expression vector pNZ8149, and carrying out electrotransformation of the lactococcus lactis expression vector pNZ8149 into a lactococcus lactis NZ3900 cell to obtain the recombinant PEDV lactococcus lactis. The recombinant PEDV lactococcus lactis is induced by 1ng/mL of Nisin and through SDS-PAGE and Western blot analysis, a main s protein of the PEDV is expressed. An expressed recombinant PEDV lactococcus lactis vector oral vaccine can irritate production of humoral immunity and certain cellular immunity of mice. The recombinant PEDV lactococcus lactis has wide application prospects in drugs such as a PEDV vaccine.

The invention provides primers for detecting variants on porcine epidemic diarrhea virus and a detection kit thereof, which belongs to the technical field of biotechnology. The primers include a forward primer PEDV-VF(SEQ ID No.1) which is 5'-TTGGTGAAAACCAGGGTFTC-3' and a reverse primer PEDV-VR(SEQ ID N0.2) which is 5'-CAACACTATGTTCACTCA-3'. The invention further provides the detection kit for detecting the variants on the porcine epidemic diarrhea virus. The detection method comprises the following steps: adopting total RNA as a template, adopting the former primers to carry out inverse transcription PCR(RT-PCR) amplification, and judging according to the positions of the amplified fragments after the reaction is ended. The primer provided by the invention has good specificity. The detection method is simple and quick, has high accuracy, and provides a guarantee for detection of the variants on the porcine epidemic diarrhea virus.

The invention discloses a multiplex RT-PCR detection primer for a porcine delta coronavirus, a porcine epidemic diarrhea virus and a porcine transmissible gastroenteritis virus. The minimum detection capacity of the multiplex RT-PCR for the three viruses is 4.05*10<1> copies/microliter, 4.52*10<3> copies/microliter and 5.47*10<3> copies/microliter respectively. The amplification results for a porcine parvovirus (PPV) and a porcine pseudorabies virus (PRV) are both negative. The multiplex RT-PCR detection results of 57 clinical samples show that one sample is infected with the three viruses at the same time, 11 samples are infected with the PDCoV, 15 samples are infected with the PEDV, one sample is infected with the TGEV, five samples are infected with the PDCoV and the PEDV, and one sample is infected with the PDCoV and the TGEV.

The invention discloses a multiplex PCR primer group for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus simultaneously, and belongs to the field of virus detection. The primer group comprises three pairs of primers, wherein the primer sequences of the first pair of primers used for detecting the porcine epidemic diarrhea virus are respectively SEQ ID NO:1 and SEQ ID NO:2, the primer sequences of the second pair of primers used for detecting the porcine transmissible gastroenteritis virus are respectively SEQ ID NO:3 and SEQ ID NO:4, and the primer sequences of the third pair of primers used for detecting the porcine rotavirus are respectively SEQ ID NO:5 and SEQ ID NO:6. Through the application of the sequences of the primers, different strains of the porcine epidemic diarrhea virus, the porcine transmissible gastroenteritis virus and the porcine rotavirus can be detected simultaneously through the multiplex PCR method, and the detection results of the porcine epidemic diarrhea virus, the porcine transmissible gastroenteritis virus and the porcine rotavirus are masculine, while the defection results of other common pig-derived viruses are feminine, in conclusion, the primer group is strong in specificity, and good in repeatability; the PCR detection is carried out after the virus cDNA is subjected to gradient dilution, which shows that the sensitivity of the primers is high.

The invention provides a double-antibody sandwich ELISA quantitative determination kit of a porcine epidemic diarrhea virus and an application and relates to the field of biodetection. A preservation number of a hybridoma cell strain PEDV of an anti-PEDV N protein monoclonal antibody is CCTCC NO: C201410. The double-antibody sandwich ELISA quantitative determination kit of the porcine epidemic diarrhea virus comprises a pre-coated elisa plate and an enzyme labeled antibody. The pre-coated elisa plate is the elisa plate coated by the anti-PEDV N protein monoclonal antibody, and the enzyme labeled antibody is a horse radish peroxidase labeled anti PEDV N protein polyclonal antibody; the anti PEDV N protein polyclonal antibody is obtained by immunizing a rabbit through the protein N of the porcine epidemic diarrhea virus to obtain serum of the rabbit. The kit provided by the invention is good in specificity, high in sensitivity and good in stability, can quickly, simply and efficiently detect the porcine epidemic diarrhea virus quantitatively, and is low in cost and suitable for high throughput detection.

The invention discloses a kit with RT-LAMP nucleic acid test strips for detecting a porcine epidemic diarrhea virus and applications of the kit. The kit comprises a primer group of a nucleic acid represented by SEQ ID No. 1-6 and test strips for detection of nucleic acid. The usage of the kit is as follows: first preparing a RT-LAMP reaction system comprising an AMV retrovirus, a 1* reaction buffer, a strand displacement DNA polymerase, a dNTP mixture, betaine, MgSO4, a FIP primer, a BIP primer, a LoopF primer, a LoopB primer, a F3 primer, a B3 primer and RNA of a sample to be measured; carrying out a reaction at a constant temperature, after testing the obtained products by using the nucleic-acid-detecting test strip, judging and reading directly: the positive result is that two red strips appear, and one strip is in the detection zone while the other strip is in the control zone. The kit has advantages of simple operation, low cost, easy observation of the reaction result, good specificity, easy popularization and application in large scope and being extremely suitable for export quarantine, food hygiene and on-site detection in animal husbandry.

The invention belongs to the technical field of animal virology and animal-borne disease, and particularly relates to a low virulent strain variated from the porcine epidemic diarrhea virus and application thereof. The low virulent strain is a porcine epidemic diarrhea virus AJ1102-R strain having the preservation number of CCTCC NO:V201433, and has stable breeding performance after verification of cell passage and plaque cloning and culturing. The genomic sequence of the low virulent strain has the following variations: the 96th generation has sequence variation on the 71-72 of 3'UTR, and 2 bases TT are lost; and the 96th generation has sequence variation on the 3805-3816 of the S gene, and 12 bases GATGCTATTGTT are added. The immune efficiency of the low virulent strain can be used for inducing an immunized piglet to generate high-level neutralizing antibody and enough attacking protection, and can be used as a vaccine strain for application.

The invention discloses a porcine epidemic diarrhea virus monoclonal antibody, a cell strain thereof and an application. The cell strain PEDV-J11E is collected at the China Center for Type Culture Collection, the collection number of the cell strain is CCTCC NO: C2016145, the monoclonal antibody secreted by the cell strain has high specificity and affinity for PEDV, can be specifically combined with different epitope of the PEDV and can be used for building a double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for quantitative detection of the PEDV. The double-antibody sandwich ELISA method has excellent detection specificity, sensitivity and linear detection range, and a double-antibody sandwich ELISA kit has excellent popularization and application values.

The invention discloses an attenuated strain YN150 of variant porcine epidemic diarrhea virus and applications thereof. The attenuated strain is prepared by consecutively passing strain YN144 (microbial preservation number: CCTCC V201547) for six generations in Vero cells in the presence of pancreatin (10 [mu]g/mL). The PEDV attenuated strain is originated from variant porcine epidemic diarrhea virus, has good safety, and is safe to various pigs. The provided vaccine can stimulate the pigs to generate protective immune response so as to resist variant porcine epidemic diarrhea virus and effectively prevent the infection caused by variant porcine epidemic diarrhea virus.

The invention belongs to the field of animal biomedicine engineering, and in particular discloses protein recombinant lactococcus lactis for secretory expression of a core antigen COE of a PEDV (Porcine Epidemic Diarrhea Virus) as well as a preparation method and an application of the protein recombinant lactococcus lactis. The method comprises the following steps: connecting a signal peptide and other sequences onto a gene of the core antigen COE of the PEDV via an overlap extension PCR method by designing multiple primers, connecting the gene to a lactococcus lactis expression vector pNZ8048 and introducing the vector with the gene into a cell of the lactococcus lactis NZ9000 in an electrotransformation manner so as to obtain recombinant bacteria; and inducing the recombinant bacteria with nisin so as to obtain an expression, and directly taking all induced cultures as oral vaccines capable of stimulating mice and inducing strong cellular immune responses. Thus, the protein recombinant lactococcus lactis can serve as a novel oral vaccine product with a good industrial prospect and has a positive effect for reducing harms of the PEDV to pig industry, thereby playing a great practical significance for promoting the healthy development of the pig industry.

The invention relates to the technical field of biological monitoring and specifically discloses a nucleic acid, a real-time fluorescent RT-RPA kit and a method for detecting porcine epidemic diarrhea virus. The real-time fluorescent RT-RPA method comprises the following steps: extracting RNA from a sample; performing isothermal amplification on the obtained RNA; adding a reaction system into a reaction tube filled with a lyophozyme preparation; adding a magnesium acetate solution, and reacting for 20min at a constant temperature of 40 DEG C; when an obvious amplification curve appears in the to-be-detected sample, determining that the result is positive. The real-time fluorescent RT-RPA method provided by the invention has the advantages of easiness in operation, strong specificity and high sensitivity; quick detection of a PEDV positive sample can be realized within 4-15min without using complicated instrument.

The invention discloses a kit for detecting a porcine epidemic diarrhea virus, a porcine transmissible gastroenteritis virus and a porcine rotavirus. The gene detection kit disclosed by the invention can accurately and effectively detect the porcine epidemic diarrhea virus, the porcine transmissible gastroenteritis virus and the porcine rotavirus, and is strong in specificity, high in sensitivity, short in time consumption, fast in detection and good in application prospects.

The invention discloses a DPO (Dual Priming Oligonucleotide) primer group for porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus detection and application of the DPO primer group. Aiming to a TGEV-N gene, a PEDV-N gene and a PRoV-VP7 gene, a pair of DPO primers respectively, and a multiplex DPO real-time RT-PCR detection method of a porcine epidemicdiarrhea virus, a porcine transmissible gastroenteritis virus and a porcine rotavirus is established. Results show that a detection limit of the method is 5.3*100 copies/microlitre, target gene fragments can be efficiently amplified within an annealing temperature range from 40 DEG C to 65 DEG C, and shown that the method is wide in annealing temperature range; and meanwhile, the DPO primers are strong in specificity, and no non-specific amplification is generated in the PCR process. The multiplex DPO real-time RT-PCR detection method provided by the invention is simple to design, strong in specificity and high in sensitivity and provides a new technological means to rapid and accurate detection of three porcine viral diarrhea infective pathogens of TGEV, PEDV and PRoV.

The invention belongs to the field of biological medicine engineering, and particularly discloses a preparation method of an oral subunit vaccine for a porcine epidemic diarrhea virus. The prepared subunit vaccine can be directly used as an oral vaccine to immune a mouse, and the mouse can be stimulated to generate stronger humoral immune response and cellular immune response. The vaccine is not degraded easily in the stomach and intestines, the immune effect is remarkable, the preparation process is simple, and the oral subunit vaccine plays an active role in relieving harm caused by the porcine epidemic diarrhea virus to the pig industry and has important practical significance in promoting healthy development of the pig industry.

Disclosed is a Porcine Epidemic Diarrhea Virus strain PEDV-KB2013-4. The microbial preservation number is CGMCC No.12663. The classification name is Porcine Epidemic Diarrhea Virus, PEDV. The preservation time is August 23, 2016. The preservation organization is China General Microbiological Culture Collection Center which is located in Institute of Microbiology, Chinese Academy of Sciences, No.3, yard 1, Beichen West Road, Chaoyang District, Beijing, China.

The invention provides a specific primer and probe combination for detecting porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV). The specific primer and probe combination comprises two pairs of specific primers and two specific probes used in conjunction with the primers. The invention provides a detection kit or a detection reagent for detecting PEDV and TGEV at the same time. The specific primer and the detection kit or the detection reagent provided by the invention have the advantages of rapidness, sensitivity, specificity and the like, and can lay a foundation for double digital PCR (Polymerase Chain Reaction) absolute quantitative detection of PEDV and TEGV, epidemiological investigation, vaccine usage and the like.