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Gene chip for identification of six swine disease pathogens and detection method thereof

A gene chip and pathogen technology, applied in the field of biochips, can solve the problem of lack of parallel detection of six pathogens, and achieve the effect of strong and stable hybridization signal, ensuring the safety and stability of animal husbandry.

Active Publication Date: 2015-11-18
重庆海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, gene chip detection technology has been applied to the detection of many diseases, but there is a lack of parallel detection technology for six pathogens

Method used

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  • Gene chip for identification of six swine disease pathogens and detection method thereof
  • Gene chip for identification of six swine disease pathogens and detection method thereof
  • Gene chip for identification of six swine disease pathogens and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, the present invention is to cultivate Actinobacillus pleuropneumoniae, Haemophilus parasuis and Mycoplasma hyopneumoniae respectively, and extract pathogen genomic DNA; porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and swine fever virus cell proliferation to extract the virus genome. Cloning, sequencing and analysis of the specific conserved sequences of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and swine fever virus. Results Six oligonucleotide probes were designed, which can specifically bind to the PCR products of the corresponding pathogens. When PCR is used for genome amplification, the 5' end of the upstream universal primer is labeled with Cy3 fluorescent dye, and the probes can be combined with this product. Hybridization was performed at 42°C. According to the position and intensity of the fluoresc...

Embodiment 2

[0081] Example 2, see figure 1 , a gene chip for the detection of important pathogens of respiratory disease syndrome, using the micro-spotting technology of the gene chip, the sample probes to be tested and various quality control probes are fixed on the chemically modified substrate, forming 8 rows × 7 columns of microarrays. The distribution of probes on the microarray from top to bottom is: spot quality control partition P1: 1 row × 8 points, hybridization positive quality control partition P2: 1 row × 4 points, hybridization negative quality control partition N: 1 row × 4 points, Actinobacillus pleuropneumoniae detection probe partition 1: 1 row × 4 points, Haemophilus parasuis detection probe partition 2: 1 row × 4 points, Mycoplasma hyopneumoniae detection probe partition 3: 1 Row × 4 points, porcine circovirus type 2 detection probe partition 4: 1 row × 4 points, porcine reproductive and respiratory syndrome virus detection probe partition 5: 1 row × 4 points, swine f...

Embodiment 3

[0084] Example 3, reagent preparation

[0085] 1) Chip wash solution

[0086] According to the needs and actual situation, prepare washing liquid I and washing liquid II according to the following proportions.

[0087] Washing solution I: the final concentration of SSC is 2×, and the final concentration of SDS is 0.2%. For example, 600mL of washing solution I = 528mL of distilled water + 60mL of 20×SSC + 12mL of 10% SDS, or prepared in proportion as required.

[0088]Washing solution II: The final concentration of SSC is 0.2×. Such as 600mL lotion II = 594mL distilled water + 6mL 20×SSC, or prepare according to the needs.

[0089] If 10% SDS produces a white flocculent precipitate, please place it in a 42°C water bath to dissolve and mix, and then prepare a lotion.

[0090] 2) Absolute ethanol.

[0091] 3) Ice water mixture.

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Abstract

The invention discloses a gene chip for identification of six swine disease pathogens and a detection method thereof. The gene chip is used for detection of porcine actinobacillus pleuropneumoniae, haemophilus parasuis, mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and classical swine fever virus. A PCR primer is designed by means of standard strain genome sequence analysis, cloning and sequencing analysis are carried out on a target gene, and specific probes are designed to perform identification detection on porcine actinobacillus pleuropneumoniae, haemophilus parasuis, mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and classical swine fever virus 6 pathogens at the same time. The invention aims to establish the microarray chip with the advantages of high sensitivity, strong specificity, time saving and labor saving, and easy observation of results to identify and detect the important pathogens of porcine respiratory disease complex.

Description

technical field [0001] The invention belongs to the field of biological chips. Specifically, the present invention relates to the identification and detection of six pathogens of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and swine fever virus. A chip device and a detection method thereof. Background technique [0002] Mycoplasma swine pneumonia (Mycoplasmapneumoniaeofswine, MPS), porcine contagious pleuropneumonia (porcinecontagious pleuropneumonia, PCP), swine polyserositis and arthritis (Swinepolyserositisandarthrithis), porcine circovirus associated disease (porcinecircovirusassociateddisease, PCVAD), porcine reproductive and respiratory syndrome Signs (Porcine reproductive and respiratory syndrome, PRRS) and swine fever (classicalswinefever, CSF) are caused by Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp), Actinobacillus pleuropneumoniae (Actin...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12R1/21C12R1/35C12R1/93C12R1/01
CPCC12Q1/6837C12Q1/689C12Q1/701C12Q2531/113C12Q2565/501
Inventor 李应国王昱聂福平保雨杨俊艾军张强王国民
Owner 重庆海关技术中心
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