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Gene chip for detecting porcine respiratory disease complex virus and method for detecting porcine respiratory disease complex virus

A technology for porcine respiratory diseases and syndrome virus, which is applied in the field of gene chip and detection, can solve the problems of difficult and difficult diagnosis, etc., to improve the efficiency of diagnosis, improve the speed and accuracy of diagnosis, and have good repeatability and specificity. Effect

Inactive Publication Date: 2013-01-23
LONGYAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Porcine Respiratory Disease Syndrome is a multifactorial disease, and traditional methods such as serological detection and PCR detection of a single virus are difficult to diagnose accurately and timely. The present invention adopts gene chip technology to establish simultaneous detection Gene chips and detection methods for several different virus infections that cause porcine respiratory disease syndrome can improve the diagnostic efficiency of the disease, thereby solving the difficult problem of its clinical diagnosis

Method used

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  • Gene chip for detecting porcine respiratory disease complex virus and method for detecting porcine respiratory disease complex virus
  • Gene chip for detecting porcine respiratory disease complex virus and method for detecting porcine respiratory disease complex virus
  • Gene chip for detecting porcine respiratory disease complex virus and method for detecting porcine respiratory disease complex virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Design and preparation of probes:

[0041] 1. Sequence acquisition of oligonucleotide probes:

[0042] Download the NPS2 gene of porcine reproductive and respiratory syndrome virus (PRRSV), the gene between 990 and 1500 bp of the genome sequence of circovirus type II (PCV-2), and the gE gene and pK gene of pseudorabies (PRV) from the Genbank public database , HA, NA and M genes of swine influenza virus H1N1, HA and NA gene sequences of swine influenza virus H3N2;

[0043] 2. Probe design for oligonucleotide probes:

[0044] Import the above sequence into Primer Premier 5.0 software, extract the ORF sequences of all viruses, and perform global alignment, obtain the characteristic segment of a specified virus according to the alignment map, design specific probes in the characteristic region, and the specific site should be as close as possible to the In the middle of the probe or slightly close to the 3' end of the probe, set the parameters, try to set the Tm ...

Embodiment 2

[0055] Example 2: Design and preparation of primers

[0056] 1. Sequence acquisition:

[0057] Download the NPS2 gene of porcine reproductive and respiratory syndrome virus (PRRSV), the gene between 990 and 1500 bp of the genome sequence of circovirus type II (PCV-2), and the gE gene and pK gene of pseudorabies (PRV) from the Genbank public database , HA, NA and M genes of swine influenza virus H1N1, HA and NA gene sequences of swine influenza virus H3N2;

[0058] 2. Design primers:

[0059] Import the above sequence into Primer Premier 5.0 software, set the parameter Tm value to 50°C-70°C, length 20bp±2bp, and then run the program.

[0060] 3. Primer selection:

[0061] From the output results, select primers with a Tm value of 50° C. to 70° C., a length of 20 bp±2 bp, and including the probe sequence used in the gene chip. Individual probes are manually adjusted, and the primers are appropriately increased or shortened by a few bases to make them contain probes and meet ...

Embodiment 3

[0066] Example 3: Gene Chip Preparation - Chip Spotting

[0067] 1. Dissolving the probe: Dissolve the probe synthesized in the above Example 1 in 50% DMSO, mix well with a shaker, and then centrifuge quickly to remove the liquid on the tube wall. After standing at room temperature for 1 hour, take 1 μL and dilute it with 50% DMSO to measure OD, measure the nucleic acid concentration of the probe, and then dilute the probe to a final concentration of 40 μM.

[0068] 2. Add plate: Add 5 μL 30-60 μM oligonucleotide aqueous solution to the 384-well plate, add 5 μL 2× gene chip sample solution, and mix thoroughly with a pipette.

[0069] 3. Spotting: use the automatic spotting instrument to spot the probes in the above-mentioned 384-well plate on the glass slide to form a designed microarray. In this embodiment, it is completed by using the crystal core microarray chip spotting system produced by Boao Biotechnology Co., Ltd. The specific operation steps are as follows: use the c...

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Abstract

The invention relates to a gene chip for a detecting porcine respiratory disease complex virus and a method for detecting the porcine respiratory disease complex virus, wherein the gene chip comprises a solid phase carrier, and an oligonucleotide probe and a quality control oligonucleotide probe which are fixed on the carrier. The oligonucleotide probe comprises DNA sequences selected from an NPS2 gene of porcine reproductive and respiratory syndrome virus, gene among circovirus II type genome sequence 990-1500bp, gE gene and pK gene of pseudorabies, HA, NA and M genes of swine influenza virus H1N1, and HA and NA genes of swine influenza virus H3N2. After a to-be-detected sample genomic DNA is amplified and labeled by utilizing a designed primer, the gene chip is used for hybridization, and the porcine respiratory disease complex virus can be detected according to the hybridization signals. According to the gene chip and the detection technology of the gene chip, four samples and five viruses can be simultaneously detected on the same one chip, and the diagnosis efficiency can be greatly improved.

Description

technical field [0001] The invention relates to a gene chip and a detection method, in particular to a gene chip for detecting porcine respiratory disease syndrome virus and a detection method thereof. Background technique [0002] Porcine respiratory disease syndrome (PRDC) is a disease problem with multiple manifestations that has become one of the most prominent diseases in swine. PRDC is the result of synergistic and additive effects of multiple viruses and bacteria infected simultaneously or successively. It is often caused by viruses first, and usually secondary to conditional pathogenic bacteria infection. Since porcine respiratory disease syndrome is a multifactorial disease, the pathogens isolated from different pig farms or even the same pig farm are different. For the diagnosis of this disease, traditional methods include serological detection and PCR detection of a single virus. Some technologies take a long time, some are expensive, and none of them can det...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C40B40/06
Inventor 杨小燕黄翠琴戴爱玲黄其春尹会方
Owner LONGYAN UNIV
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