79 results about "Peste-des-petits-ruminants virus" patented technology

The invention provides a kit for detecting a peste des petits ruminants virus hemagglutinin protein (H protein) antibody. The kit takes recombinant protein of extracellular region sequence code of peste des petits ruminants virus hemagglutinin protein gene as an envelope antigen. The invention further provides an application method of the kit. The kit can quickly specifically detect the peste des petits ruminants virus H protein antibody in serum, and is good in reaction specificity and high in sensitivity. The operation is simple; the cost is low; and the reaction result is stable and reliable, and is easy to observe. The kit is suitable for monitoring the vaccine immunity effect of import and export immunity, food sanitation, and a livestock farm of peste des petits ruminants.

The invention discloses an ELISA (enzyme linked immunosorbent assay) kit for detecting a PPRV (peste des petits ruminants virus) antibody. The ELISA kit for detecting the PPRV antibody comprises an envelope antigen, wherein the envelope antigen is a recombinant PPRV H-F fusion protein which is a protein of a) or b) as follows: a) protein formed by an amino acid sequence shown in SEQ ID No.2; b) soluble protein obtained from the amino acid sequence shown in SEQ ID No.2 through substitution and / or deletion and / or addition of one or several amino acid residues. The ELISA kit for detecting the PPRV antibody not only can be used for diagnosing PPR (peste des petits ruminants), but also can be used for evaluating a vaccine immunity effect, further can detect PPR rapidly and accurately and is beneficial to clinical monitoring of PPR, thereby playing a positive role in better control of spread of PPR in China.

The invention relates to a peste des petits ruminants virus (PPRV) reverse genetic operating system and an application thereof. The PPRV reverse genetic operating system comprises a transcription plasmid and one or more helper plasmids, wherein the transcription plasmid can express the genome full-length cDNA (complementary deoxyribonucleic acid) sequence of the PPRV; and the helper plasmid(s) can express nucleoprotein (N), phosphoprotein (P) and polymerase large protein (L) of the PPRV, and virus replication-permitting host cells of the PPRV. By using the PPRV reverse genetic operating system, the recombined PPRV is successfully saved. The establishment of the PPRV reverse genetic operating technical platform provides an excellent technical platform for the development of PPRV live vector vaccines and the fundamental research related to PPRV.

The invention relates to the biotechnology field, in particular to an RT-LAMP kit for detecting peste des petits ruminants virus (PPRV). The kit comprises a detecting system composed of LAMP reaction liquid of four LAMP primers. The kit can quickly and sensitively detect PPRV, has simple operation, low cost, easy observation of reaction results and favourable specificity, is suitable for export quarantine, food sanitation and on-site detection of livestock feedlots, and is easy for popularizing and applying in large range.

The invention relates to the field of immune marking vaccines and in particular relates to construction and an application of recombinant peste des petits ruminants virus expressing fused exogenous epitope N protein.

The invention discloses a gene chip and a detection method for detecting FMDV, VSV, SVDV, PPRV and BTV. The detection method comprises the step of detecting foot and mouth disease viruses (type A, Asian type I and type O), vesicular stomatitis virus, swine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus. The method comprises the following specific steps: designing a PCR primer by virtue of sequence analysis of standard strain genome, and performing cloning and sequence analysis on target genes; designing a specific probe, and simultaneously detecting the foot and mouth disease viruses, vesicular stomatitis virus, swine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus. The invention aims at establishing a method for detecting the foot and mouth disease viruses, vesicular stomatitis virus, swine vesicular disease virus, Peste des petits ruminants virus and bluetongue virus by adopting a microarray chip which is high in sensitivity and high in specificity and through which the time and labor are saved and the result is easily observed.

The invention relates to an epitope peptide H362 of an HN protein in PPRV, and determination, a preparation method and application thereof. The amino acid sequence of the epitope peptide is H362: <362>EANWVVPSTDVRDL<375>. The invention detects reactogenicity of a monoclonal antibody and PPRV and specificity of the monoclonal antibody; according to detection results, the monoclonal antibody has good reactogenicity to rPPRV-HN-F protein; immunoinformatic technology is cooperatively used for predicating the B cell epitope of the HN protein; an aminated ELISA plate is employed for detecting candidate epitopes and the monoclonal antibody 10E3, and the epitope peptide H362 corresponding to 10E3 is determined; and determination of the epitope peptide lays a theoretical foundation for preparation of epitope vaccine antigens and diagnostic reagent antigens for PPRV.

The invention aims at providing a primer and a probe for detecting peste des petits ruminants virus by virtue of a RPA (Recombinase Polymerase Amplification) technique. According to the primer and the probe, a forward primer sequence is represented by SEQ ID NO:1, a reverse primer sequence is represented by SEQ ID NO:2, and a probe sequence is represented by SEQ ID NO:3. According to a method, a large number of primers and probes are designed according to genomic sequences of the peste des petits ruminants virus, and a pair of primer and probe combinations capable of rapidly detecting nucleic acid of the peste des petits ruminants virus are screened out; obvious amplification curves can be obtained by carrying out rapid detection by virtue of the primer and the probe and taking nucleic acid RNA of the peste des petits ruminants virus of domestically separated Tibet strains and Xinjiang strains as a template, and no amplification curve is obtained through a reaction by taking other pathogenic nucleic acids such as foot and mouth disease virus, contagious pustular dermatitis virus, mycoplasma capricolum and bluetongue disease virus as the template.

A multiplex-PCR (polymerase chain reaction) kit for detecting six viruses of sheep and goats simultaneously comprises six pairs of specific primers, wherein the primers used for detection of the bluetongue virus are BTV-F and BTV-R respectively; the primers for detection of the foot and mouth disease virus are FMDV-F and FMDV-R respectively; the primers for detection of the peste des petits ruminants virus are PPRV-F and PPRV-R respectively; the primers for detection of the sheep pox virus are SPPV-F and SPPV-R respectively; the primers for detection of the goat pox virus are GTPV-F and GTPV-R respectively; the primers for detection of the sore mouth disease virus are ORFV-F and ORFV-R respectively. The sequences of the primers are nucleotide sequences shown in SEQ ID NO: 1-12. The multiplex-PCR kit can be used for detecting nucleic acid of the six viruses including the bluetongue virus, the foot and mouth disease virus, the peste des petits ruminants virus, the sheep pox virus, the goat pox virus and the sore mouth disease virus in sheep and goat clinical samples and has the characteristics of high sensitivity, high specificity, simple operation and reduction of detection time.

The invention provides a live vector vaccine for expressing a peste des petits ruminants virus (PPRV) H gene and a preparation method thereof. In the invention, the prepared recombinant virus has good genetic stability so that a goat can be induced to generate a specific antiviral neutralizing antibody after being immunized by the vaccine prepared from the virus. The live vector vaccine has the advantages of good immune protective effect, no toxic side effect, long shelf life and simple production process, and is easy for preservation and convenient for transportation, thus being applicable to industrialized production.

The invention discloses a cell line for stable expression of a peste des petits ruminants virus receptor Nectin-4 and a construction method of the cell line. According to the invention, by virtue of asynthetic Nectin-4 gene and on the basis of a lentiviral vector system, a recombinant plasmid pLOV-eGFP-Nectin-4 is constructed. The recombinant plasmid, together with pSPAX2 and pMD2.G plasmid, is applied to co-transfection of a 293T cell, so that retrovirus-like particles are obtained. A supernatant-infected MDBK cell is cultivated by virtue of cells containing the retrovirus-like particles, and puromycin screening and purifying are conducted, so that the MDBK cell for stable expression of Nectin-4 protein is obtained, and the MDBK cell is named as MDBK-Nectin-4. Through the construction ofthe cell line, an experimental material is provided for deep research of an interaction between PPRV and the receptor Nectin-4 and for the clinical rapid isolation of the PPRV is provided.

The invention provides a genetic marker, real-time fluorescence quantitative PCR (polymerase chain reaction) primers and a probe used for detecting peste des petits ruminants through the N gene sequencing and comparison of the peste des petits ruminants, and the nucleotide sequences of the genetic marker, the primers and the probe are respectively disclosed in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. The invention also provides a quantitive detection method and a detection kit for the peste des petits ruminants virus. The detection method and the detection kit disclosed by the invention have the advantages of accuracy in detection, high flexibility, strong specificity, easiness and rapidness, and the specimen detection capacity is good.

The invention relates to a primer pair for detecting caprine parainfluenza virus type 3 (CPIV3) and peste des petits ruminants virus (PPRV), and belongs to the field of biotechnology. The invention provides a special primer for detecting viruses, wherein the special primer comprises a primer pair A and a primer pair B, the primer pair A comprises a primer 1 and a primer 2, and the primer pair B comprises a primer 3 and a primer 4; the nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are respectively a sequence 1, a sequence 2, a sequence 3 and a sequence 4 in a sequence table. The primer special for CPIV3 and PPRV duplicate RT-PCR detection, provided by the invention is high in sensitivity, can detect the content of RNA as low as about multiplying 1 by 10<-3>ng / micro-liter, and the sensitivity of which is much higher than 1ng / micro-liter. The duplicate RT-PCR detection method provided by the invention is good in specificity, simple and quick to operate, low in requirement on the quality of a detecting material, and convenient to use clinically.

The invention relates to linear epitope minimum motif peptides of PPRV (peste des petits ruminants virus) N (nucleocapsid) protein and an application of the linear epitope minimum motif peptides. Aiming at a Tibetan isolate PPRV N protein with high immunogenicity, an improved short peptide biosynthesis method and an anti-recombinant protein polyclonal antibody are adopted, a complete-sequence fine linear B cell epitope map is provided and comprises 20 minimum epitope motif contained epitope peptides which can be used for detecting the infection serum of the PPRV independently or in a combined manner. And further, an epitope map with broad spectrum applicable to various PPRVs is provided.

The invention relates to a preparation method of sensitive cell subcloning Vero / Slam / V for enhancing PPRV (Peste Des Petits Ruminants Virus) duplication. The preparation method comprises the steps of designing a goat signaling lymphocyte activation molecule Slam / V primer sequence; constructing a slam / V lentiviral expression vector; acquiring a slam / V replication-defective slow virus; screening and verifying slam / V slow virus infection target object cell Vero, and sensitive cell subcloning Vero / Slam / V; strengthening verification of PPRV susceptibility and the like. The preparation method provided by the invention comprises positive cell subcloning with the capability of enhancing PPRV duplication, the positive cell subcloning has a favorable biological property and hereditary stability, and the stable expressed Slam / V has an effect on remarkably enhancing PPRV duplication.

The invention belongs to the technical field of biological immunoassay, and more specifically relates to an anti-peste des petits ruminants virus N protein monoclonal antibody and an application thereof. The invention discloses the anti-peste des petits ruminants virus N protein monoclonal antibody, which is secreted by a hybridoma cell strain with a preservation number being CCTCC C2016135. The invention also discloses a preparation method and a purpose of the monoclonal antibody. The monoclonal antibody has strong specificity and good biological binding activity, can be reacted with Vero cells infected by peste des petits ruminants virus, and cannot be reacted with the normal Vero cells. The monoclonal antibody can be used for preparing a kit for detecting the peste des petits ruminantsvirus, is used for detecting peste des petits ruminants virus, and has the advantages of high detection specificity and high reaction sensitivity.

The invention provides a recombinant peste des petits ruminants virus N protein and an expression method and an application thereof. The recombinant peste des petits ruminants virus N protein has the amino acid sequence shown in SEQ ID NO:1. The invention also provides a CHO-PPRV-N cell line for stably expressing the recombinant peste des petits ruminants virus N protein, wherein the CHO-PPRV-N cell line has the preservation number of CGMCC No.12281. The invention further provides a kit used for detecting peste des petits ruminants and containing the recombinant peste des petits ruminant virus N protein.

The invention provides a kit for detecting peste des petits ruminants virus by a pyrophosphoric-acid sequencing technology. The kit comprises a general primer for detecting peste des petits ruminants and a pyrophosphoric-acid sequencing primer, and the nucleotide sequences are respectively shown in SEQ ID NO.2-4. The kit adopts the following steps of by extracting the RNA of a sample to be detected, carrying out RT-PCR amplification by the general primer; if the length of the amplified fragment of the primer is 78bp, carrying out pyrophosphoric-acid sequencing reaction; if the sequencing fragment is completely the same as N gene target fragments (SEQ ID NO.1), determining to be the peste des petits ruminants virus; if one or more than one basic groups of the sequencing fragment are different from those of the target fragment, determining that the sample is negative. The kit provided by the invention can be used for rapidly detecting the peste des petits ruminants virus, has the characteristics of high flux and low cost, and has the advantages that the products can be directly used for sequencing, secondary treatment is not needed, the operation is extremely convenient, and the needed sample amount is less, so that the application prospect is good.

The invention provides a primer composition for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus, a kit and application, and belongs to the field of biotechnology and molecular biology. The primer composition for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus includes a primer pair A and a primer pair B, the primer pair A includes a primer 1 and a primer 2, the primer pair B includes a primer 3 and a primer 4, and the nucleotide sequences of the primers 1-4 are shown as SEQIDNO:1-4. The primer composition can be used for detecting mycoplasma sheep pneumoniae and peste des petits ruminants virus simultaneously or individually, operation is simple, the specificity is high, the sensitivity is high, and the detection efficiency is remarkably improved. The situation is avoided that one pathogen can only be detected at a time, the time is saved, pollution is avoided, rapid detection of clinical respiratory disease samples is facilitated,and the primer composition has broad application prospects.

The invention provides a modified coded nucleic acid of peste des petits ruminants virus N protein which has a sequence of SEQ ID NO:41, and also provides its PCR amplification method and a kit used for the method.

The invention discloses a peste des petits ruminants virus CH / GDDG / 2014 strain whole genome sequence and an amplification primer pair thereof. The virus genome sequence is divided into 11 segments, corresponding cDNA is amplified by using a one-step RT-PCR method, and amplified PCR segments are cloned onto a pGM-T carrier for nucleic acid sequencing. By the virus genome sequence, target segments can be amplified effectively; the whole genome sequence is provided, and studying of genetics, molecular epidemiology and reverse genetics of peste des petits ruminants virus is facilitated.

The invention discloses a reagent kit used for testing Taqman Real-time RT-PCR (reverse transcription-polymerase chain reaction) of the peste des petits ruminants virus. The reagent kit comprises amplification premixed solution, negative control, positive control and a mixture of SEQIDNO.1 to SEQIDN0.2 amplification primers and an SEQIDNO.3 probe primer. The reagent kit has the advantages that variation of each circular amplicon in PCR (polymerase chain reaction) is tested in real time by the aid of variation of fluorescence signals, a starter template is subjected to quantitative analysis through the relation of Ct values and a standard curve, and the test kit used for testing the peste des petits ruminants virus is high in specificity, good in stability and easy to operate; the test kit has higher sensitivity than common PCR and can be used for testing the peste des petits ruminants virus of low and micro-content samples; the test kit is applicable to not only quantitative analysis of research units but also pathogen testing analysis of prevention and control units at all levels, base veterinary stations and large and medium-sized livestock farms and the like, thereby having good application prospect.

The invention provides a nanometer immunogold labeling probe for detecting a peste des petits ruminants virus (PPRV) and a detection kit. Two specific oligonucleotide probes are designed by aiming at a highly conserved region of an N gene of the PPRV. The nucleotide sequence of the 5' modified biotin of one of the probes and the nucleotide sequence of the 3' modified sulfydryl of the other probe are respectively shown by SEQ ID NO.2 and SEQ ID NO.3. The invention also provides a method for detecting the nucleic acid of the PPRV by utilizing the nanometer immunogold labeling oligonucleotide probe. The sulfhydrylated probes are respectively connected onto nanometer gold particles through Au-S bonds; both ends of the targeted nucleic acid are respectively bonded with the two probes to form a hybrid compound, and the targeted nucleic acid is quantitatively detected through bonding the hybrid compound with a solid-phase carrier and performing silver staining on an amplifying signal. According to the detection method provided by the invention, the minimum concentration of the nucleic acid reaches 10fmol / L. The method is high in sensitivity and strong in specificity. A novel method is provided for clinically detecting the PPRV.

The invention relates to the technical field of molecular biology, in particular to a codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine. The codon optimization gene sequencegives consideration to the codon use preference of mammalian cells, and a sequence is shown as SEQ ID NO. 2. According to the peste des petits ruminants virus F gene nucleic acid vaccine, after signal peptide at the 5'end of an F gene is removed, the remaining sequence is optimized through codons, the 5 'end is connected with an encephalitis B virus signal peptide sequence, and finally the sequence is inserted into an eukaryotic expression vector. The nucleic acid vaccine effectively stimulates an immune system after immunizing a mouse, generates a neutralizing antibody and reflects good immunogenicity.

The invention discloses a Taqman fluorescence quantitative PCR kit and method for simultaneously detecting eight common sheep viruses and bacteria, and relates to the technical field of molecular biology. The eight common sheep viruses and bacteria include peste des petits ruminants virus (PPRV), orf virus (ORFV), goatpox virus (GTPV), chlamydophila abortus (C.a), staphylococcus aureus (S.a), escherichia coli (E.coli), streptococcus (Stre.) and salmonella (Salm.). PCR reaction liquid comprises eight groups of primers and eight groups of Taqman probes. The kit can rapidly and effectively detect8 sheep pathogens simultaneously, the detection method is accurate, the specificity and the sensibility are high, the stability is good, and it can be achieved that to-be-detected pathogens is rapidly diagnosed and effectively detected.

The invention belongs to the technical field of biological genetic engineering, and particularly relates to application of a PRKRA gene as a target spot in inhibition of peste des petits ruminants virus replication. The invention firstly finds that by inhibiting or silencing the host PRKRA gene, the replication of the peste des petits ruminants virus can be inhibited, and the PRKRA gene can be used as a target spot for preparing the medicine for inhibiting the replication of the peste des petits ruminants virus; secondly, by taking the PRKRA gene as a target spot, the small interfering RNA is designed, and the small interfering RNA can interfere with the replication of the peste des petits ruminants virus and can be used for preparing a medicine for inhibiting the replication of the peste des petits ruminants virus; moreover, the invention provides sgRNA for specifically targeting the PRKRA gene, the sgRNA can specifically target the PRKRA gene, complete knockout of the PRKRA gene is realized by combining with a CRISPR-Cas9 technology, and the obtained monoclonal cell line PRKRA-KOs has a resistance phenotype to PPRV, can significantly inhibit duplication of PPRV in cells, and a research tool and a material are provided for further researching a molecular mechanism of the PRKRA gene for regulating and controlling pathogenic microorganism replication in cells.

The invention provides a modified coded nucleic acid of peste des petits ruminants virus N protein which has a sequence of SEQ ID NO:41, and also provides its PCR amplification method and a kit used for the method.

The invention relates to a peste des petits ruminants virus IgG antibody colloidal gold detection card and production and using methods thereof, and belongs to the technical field of immunology. The rapid diagnostic card comprises a shell and test paper, and is characterized in that: the shell is provided with a sample feeding hole and an observation window; the test paper is arranged in the shell; nitrocellulose films are stacked in the middle of a PVC lining plate, and water absorption pads and sample pads are respectively stacked at two ends; the nitrocellulose films are lapped with the water absorption pads and the sample pads; a section of membrane containing a staphylococcal protein A immune colloidal gold compound is additionally arranged in a sample area; and a detection belt containing inactivated peste des petits ruminants virus antigen and a quality control belt containing goat anti-rabbit IgG are formed on the nitrocellulose films. The invention provides a rapid, accurate and sensitive diagnostic card without special instruments, which is easy to operate, and can meet the detection requirements of food safety, slaughter and detection mechanisms.

The present invention relates to a virus clone purification strain for Peste des petits ruminants live vaccine production, and applications thereof. According to the present invention, an introduced Peste des petits ruminants virus attenuated PPRV 75 / 1 strain is cloned and purified to obtain a PPRV 75 / 1Clone9 strain, ie., the virus clone purification strain, wherein the virus titer is increased after the cloning; the virus titer is decreased by not more than 0.3 titer during the storage and the use of the Peste des petits ruminants live vaccine prepared by using the virus clone purification strain; the safety and the immunizing efficacy are higher than the OIE standard; and the immunity protective test results show that the immunizing 10<2>TCID50 protection rate is 96.7%, and the immunizing 10<3>TCID50 protection rate is 100%.

According to the invention, the B cell epitope of the screened peste des petits ruminants virus (PPRV) H protein is used as a basis, four antigen epitope peptides with better reactogenicity are selected and are connected in series to be synthesized into a coating antigen; an iELISA method for the PPRV H protein antibody is established, the optimal coating amount of the multi-epitope antigen peptide is 1.5*10<-6> mu g / hole, the negative and positive critical dilution concentration of to-be-detected serum is 1: 100, and the optimal dilution concentration of the enzyme-labeled secondary antibodyis 1: 80000; the coating solution is a carbonate buffer solution, the serum and the secondary antibody diluent are PBST solutions, and the confining liquid is a 2% BSA solution; a positive and negative critical value is 0.25. Clinical serum detection shows that the variation coefficients of in-batch and inter-batch detection results are both less than 10%; the method has good specificity; comparedwith a cELISA kit produced by the ID-Vet company for 306 parts of serum, the coincidence rate of the cELISA kit and the 306 parts of serum is 92.75%. The PPRV H protein antibody iELISA detection method can be used for clinical peste des petits ruminants antibody detection, and the operation is more time-saving, labor-saving, simple, convenient and rapid than cELISA.