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A gene chip kit for detecting porcine circovirus type 2

A gene chip and kit technology, which is applied in the field of kits for detecting porcine circovirus type 2, can solve problems such as immune function damage, death, and decreased body resistance, and achieve good market prospects, application value, and detection results. Accurate, easy-to-use effects

Active Publication Date: 2015-10-28
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus is persistently infected, and the nucleic acid of PCV2 can be detected in the secretions and excreta of infected pigs; the virus can cause primary infection or even death in pigs, and in severe cases, the immune function of the body is damaged, resulting in body resistance decline, causing concurrent or sending infections, making the condition worse and causing greater economic losses

Method used

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  • A gene chip kit for detecting porcine circovirus type 2
  • A gene chip kit for detecting porcine circovirus type 2
  • A gene chip kit for detecting porcine circovirus type 2

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0037] Example 1 A gene chip kit for detecting porcine type 2 circovirus

[0038] 1. Probe synthesis:

[0039] According to the ORF2 gene encoding the structural protein of porcine circovirus type 2 in GenBank, the DNAStar software was used to compare the sequences to find out the highly conserved sequences. Then use primer3 software to design specific primers. Finally, compared in NCBI, the designed specific primers were compared with the ORF2 gene in the porcine circovirus type 2 sequence, and the coincidence rate was 100%. Primers were synthesized by Shanghai Invitrogen.

[0040] Amino modified capture probe: 5'-H2N-GTATTGACGTCGGCTAC-3';

[0041] Sulfhydryl modification detection probe: 5'-GGGAGCCTAGCAGTGAC-SH-3';

[0042] ORF2 conserved region upstream primer: 5'-TTCTGACTGTGGTTCGCTTG-3';

[0043] Downstream primer of ORF2 conserved region: 5'-ACGTATCCAAGGAGGCGTTA-3'.

[0044] 2. Preparation of nano-gold probes

[0045] 1) 2ml nano gold solution, 4°C, 14000rpm, centr...

Embodiment 2

[0062] The establishment of the assay procedure of embodiment 2 kit

[0063] 1. DNA nucleic acid template extraction and target nucleic acid fragment amplification and identification

[0064] 1) Preparation of viral DNA template

[0065] cell collection

[0066] A. Scrape the cells with a sterile cell scraper, put them in a 15ml centrifuge tube, centrifuge at 4°C, 800rpm / min, 10min; discard the supernatant.

[0067] B. Add 1ml of pre-cooled PBS to each centrifuge tube, gently blow the cell pellet evenly, add it to the pre-cooled EP tube, and centrifuge under the same conditions as in 1. Discard the supernatant.

[0068] C. Add 1ml of pre-cooled PBS to wash again, centrifuge, and discard the supernatant.

[0069] D. Wash with PBS and store at -80°C.

[0070] Total DNA extraction from cells:

[0071] DNA was extracted from PCV2-infected cells using QIAGEN DNeasy Blood & Tissue Kit, and then the PCV2ORF2 fragment was amplified by the designed PCV2ORF2-specific primers. Afte...

Embodiment 3

[0096] The detection of embodiment 3 sample

[0097] Use the visualized gene chip method that embodiment 2 establishes to detect a small amount of PCV2 samples and directly detect, the results are shown in Figure 6 , It can be seen from the results that a total of 24 samples were tested, of which 22 cases were positive results and 2 cases were negative results detected by the gene chip. Both the PCV2 detection point and the positive control detection point have good color development results, which are clearly visible to the naked eye, while the negative control has no obvious color development results, and the background is clear. Utilize the PCR detection method to detect the same 24 samples, see Example 2 for specific operations, see the test results Figure 7 , as can be seen from the figure, the PCR method detected positive results in 22 cases and negative results in 2 cases. The positive results of PCV2 gene chip detection and PCR method were consistent.

[0098] ...

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Abstract

The invention provides a gene chip kit for detecting porcine circorvirus type 2 (PCV2). The method for detecting the porcine circorvirus type 2 by the kit is established by applying a nanogold marking probe to a gene chip, the detecting principle of the kit is as follows: a 5' amino-terminated modified oligonucleotides gene probe, which is connected on the gene chip, is used as a capture probe, oligonucleotides modified by 3' terminated sulfydryl and gold nanoparticles are linked covalently to be used as a detection probe, target nucleic acid to be detected as well as the capture probe and the detection probe are hybridized in a double-probe sandwiched mode, silver developing liquid is added for amplification, the result is analyzed by visual inspection or a flat-bed scanner, and the target sequence hybridization can be quantitatively researched. The gene chip kit is used for detecting PCV2 nucleic acid, is low in cost, and has simplicity in operation, high sensitivity, strong specificity and good application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting porcine type 2 circovirus. Background technique [0002] In 1997, Clark, Harding, etc. reported for the first time a new piglet disease in western Canada at the annual meeting of the American Pig Producers Association, and named it Postweaning multisystemic wasting syndrome (Postweaning multisystemic wasting syndrome) according to its histopathological characteristics. syndrome, PMWS). Since the outbreak of PMWS, the etiological identification proved that it was caused by porcine circovirus type 2 (PCV2), and thus carried out in-depth research on PCV2. PCV2 infection can also cause several other syndromes and diseases, including porcine dermatitis nephrotic syndrome, reproductive failure, porcine respiratory syndrome, granulomatous enteritis, necrotizing lymphadenitis, and exudative dermatitis. The virus is persistently infected, and the nucleic acid of PCV2 can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C12R1/93
Inventor 朱珊珊
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES