55 results about "Duck circovirus" patented technology

The invention provides a duplex polymerase chain reaction (PCR) method for rapidly identifying duck circovirus genotype. The method comprises the following steps: comparing sequences DuCV-1 and DuCV-2 released on reference GenBank, selectively and respectively designing a pair of general specific primers SEQ1/SEQ2 aiming at DuCV, a type 1 duck circovirus specific primer SEQ3 and a type 2 duck circovirus specific primer SEQ4 in a conserved region of the DuCV-1 and DuCV-2. According to the method, novel specific primers are designed and screened, various parameters in the reaction process are optimized, the dual PCR sequence-based typing method aiming at the duck circovirus established by utilizing the primers is high in specificity and high in sensitivity, and the sequences DuCV-1 and DuCV-2 can be rapidly and accurately identified.

The invention discloses a duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus, and particularly relates to a primer group for detecting Newcastle disease virus and duck circovirus, which comprises a first primer, a second primer, a third primer and a fourth primer. Nucleotide sequences of the first primer, the second primer, the third primer and the fourth primer are respectively a first sequence, a second sequence, a third sequence and a fourth sequence in a sequence list. Testing shows that the two pairs of primers are designed to establish a duplex PCR detection method for Newcastle disease virus and duck circovirus, and the duplex PCR technique capable of detecting two pathogens, namely the Newcastle disease virus and the duck circovirus has the advantages of simplicity in operation, high sensitivity, high specificity, high repeatability and the like.

The invention discloses a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method for differentiating duck circovirus from goose circovirus. The PCR-RFLP method is used for differentiating by utilizing Rep protein gene sequence enzyme cutting site difference and comprises the following steps of: extracting DNA (Deoxyribonucleic Acid), carrying out PCR amplification to obtain a Rep protein gene segment, and carrying out RFLP analysis after carrying out XhoI digestion. The PCR-RFLP method disclosed by the invention is simple and higher in efficiency and accuracy.

The invention provides a duck circovirus genetic engineering subunit vaccine and a preparation method and application thereof. A nucleic acid molecule of which the sequence is as shown in SEQID NO:1 or a nucleic acid molecule of which the nucleotide sequence is 95% or above same as that as shown in the SEQID NO:1 is used for coding duck circovirus outer capsid structural protein, an immune composition containing the duck circovirus outer capsid structural protein can be used for preparing the duck circovirus genetic engineering subunit vaccine, the antigenicity, the immunogenicity and the function of the vaccine are similar to those of natural protein, and the vaccine is high in expression level, high in immunogenicity and free from pathogenicity to ducks. The vaccine can be in large-scale serum-free suspension culture and preparation through a bioreactor, and the production cost of the vaccine can be greatly reduced.

The invention relates to an indirect ELISA method for detecting a duck circovirus (DuCV) antibody. The method uses a pET-32a(+) protokaryon to express the carrier, constructs a gene engineering bacterium BLpET-32a-Cap which can express the partial capsid protein (Cap) of the DuCV, and coats an ELISA plate with the expressed recombinant protein which is subject to purification and renaturation to detect the antibody level of DuCV in the duck blood serum. The result shows that the method has the characteristics of good repeatability and high specificity, and can be used for serological research on the DuCV.

The invention relates to a duck circovirus and adenovirus bivalent inactivated vaccine and a preparation method of a yolk antibody thereof. A duck circovirus antigen is obtained by infecting 1-day-oldhealthy cherry valley ducklings and collecting infected duck livers at the age of 25 days to obtain antigen tissues, and an adenovirus antigen is obtained by infecting SPF chickens of 20 days old to30 days old and collecting livers of dead chickens; an antigen of the vaccine consists of liver tissues infected with the duck circovirus and liver tissues infected with the adenovirus in a mass ratioof (1-5):1, and an antigen solution for the vaccine is prepared; and a pine pollen polysaccharide with a concentration of 5-40mg/mL is added into the antigen solution to serve as an immunopotentiator, and the antigen solution is emulsified with a conventional white oil adjuvant to obtain the vaccine. The yolk antibody is obtained by immunizing laying hens with the vaccine, and then performing extracting and purifying on egg yolks of high-immunity laying hens, and the yolk antibody simultaneously contains two antibodies against the duck circovirus and the adenovirus. The vaccine and yolk antibody provided by the invention have the advantages of simple preparation process, low cost, good action effect, stable dosage form and easy storage, and has a wide application prospect.

The invention relates to a primer and probe for real-time fluorescence quantitative PCR detection of two genotypes of duck circovirus. Sequences of the primer and the probe are as follows: an upstreamprimer DuCV-F: 5'-CAGTTTGTKGCTAARACVTTG-3'; a downstream primer DuCV-R: 5'-AGTTTATTGGRAASGGGAGG-3'; a probe DuCV-1-pobe: 5'-AGAGCGAGTTTGACCTYT-3'; a probe DuCV-2-pobe: 5'-TTTGATTTGTCCGCCTTAT-3', wherein the 5'- end of the DuCV-1-pobe is labelled with a fluorescent reporter group Cy5; and the 5'- end of the DuCV-2-pobe is labelled with a fluorescent reporter group FAM. The method is high in sensitivity and specificity, and can be used for genotype identification of two kinds of duck circovirus.

The invention discloses a duck reovirus and duck circovirus bivalent inactivated vaccine and a preparation method thereof. The bivalent inactivated vaccine comprises an effective amount of inactivated antigen and an immunologic adjuvant in immunization, wherein the inactivated antigen comprises duck reovirus antigen and duck circovirus antigen proteins. The invention further discloses a preparation method of the bivalent inactivated vaccine. The bivalent inactivated vaccine provided by the invention is good in safety, does not generate mutual interference or influence of antigen components, can achieve the protection effect of secondary immunization of each single vaccine through one-time immunization, can prevent duck reovirus and duck circovirus at the same time, and can avoid adverse reaction caused by multiple times of immunization, and the preparation method has the advantages of simplicity, convenience, multiple effects, low cost, high vaccine titer content and the like.

The invention discloses a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primer group for visual detection on infection conditions of duck circovirus (DuCV) and goose circovirus (GoCV) and a method thereof. According to the method, the detection on the infection conditions of DuCV and GoCV is carried out by using dissolution curve temperature Tm value difference caused by the difference between nucleotide GC contents of DuCV and GoCV specific gene fragment regions amplified by primers, and the infection conditions of DuCV and GoCV can be subjected to visual differential diagnosis specifically by only combining the SYBR Green I based real-time fluorescent quantitative PCR primer group to difference of dissolution curve Tm values which are automatically generated after reaction is ended. The method disclosed by the invention is simple and is relatively high in efficiency and accuracy.

The invention provides an in vitro proliferation and culture method for a duck circovirus. The method comprises the following steps: (1) collecting healthy adult Muscovy duck blood for isolated culture of PBMC to prepare a PBMC cell suspension; and (2) adding a disease material which is detected duck circovirus positive into the PBMC cell suspension to be cultured. The invention provides the method of culturing DuCV by in vitro proliferation. The virus titer after virus inoculating culture is improved greatly, isolation and identification of the virus can be finished in a lab, and relatively good help is provided for researching the nosogenesis and damage of the virus.

The invention discloses a triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck virus hepatitis and a preparation method of the triple inactivated vaccine. The triple inactivated vaccine comprises an effective amount of inactivated antigens and immunologic adjuvants in immunization, and the inactivated antigens include a duck circovirus antigen, a novel duck reovirus antigen and a duck hepatitis virus antigen. The invention further discloses a method for preparing the triple inactivated vaccine. The triple inactivated vaccine for the duck circovirus disease, the novel duck reovirus disease and the duck virus hepatitis is good in safety and does not generate mutual interference or influence of antigen components. Immune protection efficacy tests prove that the triple inactivated vaccine can prevent duck reovirus, duck circovirus and duck hepatitis virus at the same time, can avoid adverse reactions caused by multiple inoculation immunization, and has the advantages of simple preparation method, convenience, multiple effects, low cost, high vaccine titer content and the like.

The invention aims to provide a modified duck circovirus Cap protein as well as a preparation method and application thereof, that is, 21-36 peptide fragments rich in arginine at the N-terminal of theduck circovirus Cap protein are deleted, and a universal T cell epitope is introduced into a deletion region to promote immunogenicity; the amino acid sequence of the obtained modified duck circovirus Cap protein is SEQ ID NO: 1, and one nucleotide sequence is SEQ ID NO: 2. The modified duck circovirus Cap protein gene is connected with an expression vector and then transferred into escherichia coli BL21 (DE3), efficient soluble expression of duck circovirus Cap protein is achieved, the expressed protein is spontaneously assembled into virus-like particles, and the particles can be used for development of products such as duck circovirus gene engineering subunit vaccine and yolk antibody.

The invention relates to a primer and a probe for dual real-time fluorescent quantitative PCR detection of duck circovirus type 1 and duck circovirus type 2. The sequences of the primer and the probeare as shown in SEQ. ID. No.1 to SEQ.ID. No.6. A dual real-time fluorescent quantitative PCR detection method established by utilizing the primers and the probe is high in sensitivity, good in stability, strong in specificity and good in repeatability, can be used for duck circovirus typing detection, and lays a foundation for subsequent scientific research on genotype pathogenesis of two duck circoviruses and development of molecular epidemiology.

The invention provides a duck circovirus tandem repeated sequence and application thereof and belongs to the technical fields of bioengineering and molecular biology. The invention firstly discovers aQTR sequence in which DuCV specifically exists, and the tandem repeated sequence does not appear in other circoviruses. QTR can serve as a gene I type and II type DuCV genetic typing new molecular marker; meanwhile, the invention proves the effect of regulating and controlling mRNA stability by the QTR serving as a DSE component by a dual-luciferase reporter system and a semi-anchored reverse mutation PCR technology, and defines the important effect of regulating and controlling virus gene expression by the QTR. The invention provides important technical and theoretical basis for researchingDuCV genotyping and virus replication regulation and control mechanism.

The invention relates to a triple PCR (Polymerase Chain Reaction) detection primer group for duck circovirus, duck adenovirus and duck tembusu virus. The triple PCR detection primer group comprises a first primer pair, a second primer pair and a third primer pair, wherein the first primer pair has a nucleotide sequence pair as shown in SEQ ID NO.1 and SEQ ID NO.2; the second primer pair has a nucleotide sequence pair as shown in SEQ ID NO.3 and SEQ ID NO.4; the third primer pair has a nucleotide sequence pair as shown in SEQ ID NO. 5 and SEQ ID NO. 6. The invention further provides a triple PCR detection kit for the duck circovirus, the duck adenovirus and the duck tembusu virus, the detection kit disclosed by the invention can be used for simultaneously detecting the mixed infection or single infection condition of the duck circovirus, the duck adenovirus and the duck tembusu virus, the workload is reduced, and the detection time is greatly shortened; and the purpose of rapidly detecting pathogens is achieved.

The invention relates to the technical field of virus detection, and particularly discloses a primer and probe combination for RF-RAA detection of duck circovirus and application thereof. In the primers and the probe for detecting the duck circovirus, the sequence of an upstream primer is shown as SEQ ID NO: 1, the sequence of a downstream primer is shown as SEQ ID NO: 2, and the sequence of the probe is shown as SEQ ID NO: 3. Specific primers and probes are designed according to a conserved motif of DuCV, an RF-RAA method applied to detection of DuCV is established, accumulation of amplification products is positively correlated with the intensity of fluorescence signals, an obvious result can be observed through an instrument after the reaction is performed for 15 min at 41 DEG C, and the method is high in specificity, high in sensitivity, high in accuracy, capable of achieving the lowest detection limit of 1 copy/microliter and capable of being applied to detection of DuCV. Reliable guarantee is provided for early clinical detection and epidemiological investigation of the duck circovirus, and positive significance is achieved on prevention and control of epidemic diseases.

The invention discloses an anti-duck circovirus single-chain antibody and a preparation method and application thereof, and relates to the technical field of bioengineering. The amino acid sequence ofthe anti-duck circovirus single-chain antibody is Seq ID NO.1 or Seq ID NO.3. The invention provides a coding gene of the anti-duck circovirus single-chain antibody. The nucleotide sequence of the coding gene is Seq ID NO.2 or Seq ID NO.4. The anti-duck circovirus single-chain antibody ScFv-DuCV is screened by adopting a phage antibody library technology, antibody structure modification is further carried out on the anti-duck circovirus single-chain antibody ScFv-DuCV so that the anti-duck circovirus single-chain antibody ScFv-mDuCV is obtained, and the anti-duck circovirus single-chain antibody ScFv-DuCV and the anti-duck circovirus single-chain antibody ScFv-mDuCV can be used for duck circovirus detection and diagnosis. The anti-duck circovirus single-chain antibody ScFv-mDuCV and the duck circovirus have good virus recognition specificity, meanwhile, the anti-duck circovirus single-chain antibody ScFv-mDuCV has a good blocking effect on the duck circovirus, and a foundation is laidfor research of novel drugs for treating and inhibiting the duck circovirus.

The invention discloses inverse PCR amplification of a duck circovirus gene. The inverse PCR amplification is characterized in that the inverse PCR amplification includes subjecting a PCR product to electrophoresis on agarose gel having a concentration of 2% and cutting a target fragment under a fluorescent lamp; recovering and purifying; dipping into an LB solution and performing blue/white selection to screen again; subjecting selected bacterial colonies to plasmid extraction, performing PCR again and performing restriction enzyme digestion identification; performing pre-degeneration in an environment having a temperature of 95-100 DEG C for 3-4 min; and then repeating the above step every 3 min for 25 times; agarose gel electrophoresis detection is performed again after the step is repeated for 25 times; and ice bath is performed for 3 min before electrophoresis detection. Beneficial effects of the inverse PCR amplification are that successful extraction of DNA in the duck circovirus genome, high purity, simple and convenient operation, low cost, strong repeatability and capability of meeting experiment standards are achieved.