Porcine circovirus PCV1 and PCV2 identifying and detecting test paper card

A technology of differential detection and test strip card, applied in the biological field, can solve the problems of inability to realize the differential diagnosis of PCV2 antigen subtypes, difficulty in popularization and promotion, complicated test operation, etc., and achieve simple and fast operation, vivid test results, and high sensitivity Effect

Active Publication Date: 2009-04-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the above technologies and methods can detect PCV2 virus and have achieved certain results in practice, they cannot realize the differential diagnosis of PCV2 antigen subtypes, and the test op

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  • Porcine circovirus PCV1 and PCV2 identifying and detecting test paper card

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation of embodiment 1 capsid protein recombinant protein

[0022] High-efficiency expression of PCV1 and PCV2 capsid proteins by genetic engineering technology to prepare recombinant capsid proteins. According to PCV1 isolates (GenBank No.AY193712) and 1767 PCV2 isolate HZ0201 (GenBank No. AY188355) complete gene sequence, designed PCV1 and PCV2 capsid protein-specific primers respectively, PCV1 upstream primer is PV1up: 5'-GCGGATCCACGGGTATCTTCAATTC-3', containing BamHI restriction site, PCV1 downstream primer For PV1down: 5'-CCGCTCGAGTTATTTTATTTAGAGGGTC-3', containing XhoI restriction site, PCV2 upstream primer is pG1: 5'-GC GGATCC AATGGCATCTTCAACAC-3', containing BamHI restriction site; PCV2 downstream primer pG2: 5'-CCG CTCGAG TTAAGGGTTAAGTGGG-3', containing XhoI restriction site. Using PCV1 and PCV2 viral DNA as templates, amplify the 573bp and 579bp enucleation localization signal capsid protein genes, the reaction conditions are: 95°C for 30s, 58 or ...

Embodiment 2

[0025] Example 2 Preparation and screening of anti-capsid protein monoclonal antibody

[0026] Mix the recombinant PCV1 and PCV2 capsid proteins with Freund's adjuvant in equal amounts, fully emulsify, and immunize BALB / c mice with 50g-100g / mouse for 3 times with an interval of 15-30 days; the third booster immunization After 3-4 days, bleed the eyeballs of the immunized mice, pull the neck to death, soak in 75% alcohol for 5-10 minutes, and aseptically collect the splenocytes; cut them into pieces and filter them through a 100-mesh nylon mesh, centrifuge at 1000r / min for 10 minutes, and collect splenocytes; mix 1×10 8 splenocytes with 2-5 x 10 7 Mix SP2 / 0 myeloma cells, centrifuge at 1000r / min for 10min, discard the supernatant, slowly add 0.7-1ml of 40%-50% PEG 4000 (pH8.5-9.0) to the cells in a water bath at 37°C, and warm After incubating for 1 min, slowly add 15 ml of serum-free 1640 medium to terminate the effect of PEG, bathe in 37°C for 5-10 min, centrifuge at 1000 r...

Embodiment 3

[0027] Example 3 Screening of anti-capsid protein monoclonal antibodies

[0028] The 25 anti-capsid monoclonal antibodies were screened and identified by indirect immunofluorescence (IFA). Separate PCV1 isolates, 1768 PCV2 isolates, 1767 PCV2 isolates and 1766 PCV2 isolates were inoculated with trypsinized PK-15 cells without PCV contamination at a ratio of 1:10, and the virus cell mixture was added to a 96-well cell culture plate, 100 μL per well, at 37°C in 5% CO 2 After culturing for 96 h, add 100 μL of methanol-acetone fixative mixed at 1:1, and fix at -20°C for 20 min. Discard the fixative and let it dry naturally. Block with 5% skimmed milk powder for 1 hour, add 100 μL of hybridoma supernatant to each well, incubate at 37°C for 1 hour, wash 5 times with PBST, add 50 μL of FITC-labeled goat anti-mouse IgG diluted 1:400, and incubate at 37°C for 40 minutes , washed 5 times with PBST. Add 50 μL of PBS, observe positive cells with an inverted fluorescent microscope, a...

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Abstract

The invention relates to a PCV1 and PCV2 differential detection paper card which can be used for rapid identification and detection of pig PCV2 infection. The reaction reagent carrier adsorption layer comprises a fiber layer, a gold-labeled fiber layer, a cellulose film and an absorbent-material layer; the gold-labeled fiber layer is a gold-labeled glass wool adsorbing colloidal gold-labeled anti-capsid protein monoclonal antibodies which can identify PCV1 and PCV2 common epitope, and the cellulose film is a nitrocellulose film which is orderly printed with a detection trace T1, a detection trace T2 and a control trace C; the detection trace T1 is a strip-shaped PVC1 detection trace printed and produced by monoclonal antibody solution which can identify PCV1-typed specific epitope; and T2is a strip-shaped PVC detection trace printed and produced by anti-capsid protein monoclonal antibody solution which can identify PCV1 and PCV2 common epitope, and the control trace C is a strip-shaped control trace which is printed and produced by anti-mouse IgG polyclonal antibody solution. The paper card has the advantages of strong specificity, high sensitivity, convenience and speediness andintuitive test result, and can be easily promoted for application in the production practice.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a test paper card for the identification and detection of porcine circovirus PCV1 and PCV2 and the identification and detection of PCV2 subtypes. Background technique [0002] Porcine circovirus (PCV) is the smallest animal virus found so far. The diameter of the virus particle is about 17nm. It is a covalently closed, circular, single-stranded DNA virus with two genotypes: porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2). PCV1 is non-pathogenic, but widely exists in pigs and pig-derived cell lines. Its full-length genome is 1759nt or 1758nt. PCV2 is pathogenic to pigs and causes a series of porcine circovirus diseases (PCVD) , especially the main pathogen of post-weaning multisystemic wasting syndrome (PMWS), leading to immunosuppression in pig herds and causing huge economic losses to the pig industry. The full length of the PCV2 genome is 1768nt or 1767nt. Cur...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/558
Inventor 周继勇金玉兰颜焰商绍彬
Owner ZHEJIANG UNIV
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