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

A pathogen and gene technology, applied in the field of biochips, can solve the problems that cannot fully meet the needs of rapid, accurate and high-throughput detection of imported and exported animal diseases, there is no detection method that can detect seven pathogens at the same time, and increase the difficulty of pathogen detection. , to achieve the effects of stability, time saving, good specificity and high sensitivity

Inactive Publication Date: 2015-11-18
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional pathogen isolation and identification and serological methods are time-consuming and require professional operators, which cannot fully meet the rapid, accurate and high-throughput detection of imported and exported animal diseases.
With the development of molecular biology, polymerase chain reaction (PCR) is widely used in the detection of animal diseases, but there is no detection method that can simultaneously detect seven pathogens
In addition, the culture conditions of APP, HPS, and Mhp are relatively harsh, which is not conducive to isolation and culture; TGEV, PRRSV, and CSFV are all RNA viruses, and RNA is easily degraded and unstable, which increases the difficulty of pathogen detection to a certain extent

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the present invention respectively cultures Porcine Infectious Actinobacillus pleuropneumoniae, Haemophilus parasuis and Mycoplasma hyopneumoniae, and extracts pathogen genome DNA; Porcine circovirus type 2, porcine reproductive and respiratory syndrome virus , classical swine fever virus and porcine transmissible gastroenteritis virus cell proliferation and extraction of viral genomes. Specific conserved sequences for Actinobacillus pleuropneumoniae, Haemophilus parasuis, Mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine transmissible gastroenteritis virus Carry out cloning and sequencing analysis, and design 7 oligonucleotide probes according to the sequencing results, which can specifically bind to the PCR products of corresponding pathogens. When PCR is used for genome amplification, the 5′ end of the upstream universal primer is amplified with Cy3 fluorescen...

Embodiment 2

[0088] Example 2, see figure 1 , the gene chip used for the detection of seven important swine disease pathogens, adopts the micro-spotting technology of the gene chip, immobilizes the probes of the samples to be tested and various quality control probes on the chemically modified substrate, forming 8 rows × 7 columns microarray. The distribution of probes on the microarray from top to bottom is as follows: spotting position 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 o'clock, porcine infectious Actinobacillus pleuropneumoniae detection probe division 1: 1 row × 4 dots, Haemophilus parasuis detection probe division 2: 1 row × 4 dots, Mycoplasma hyopneumoniae detection probe division 3: 1 Row × 4 dots, porcine circovirus type 2 detection probe division 4: 1 row × 4 dots, porcine reproductive and respiratory syndrome virus detection probe divi...

Embodiment 3

[0091] Embodiment 3, reagent preparation

[0092] 1) Chip washing solution

[0093] According to needs and actual conditions, prepare washing solution I and washing solution II according to the following proportions.

[0094] Washing solution Ⅰ: the final concentration of SSC is 2×, and the final concentration of SDS is 0.2%. Such as 600mL lotion Ⅰ = 528mL distilled water + 60mL 20 × SSC + 12mL 10% SDS, or according to the need to prepare in proportion.

[0095] Washing solution Ⅱ: The final concentration of SSC is 0.2×. Such as 600mL lotion Ⅱ = 594mL distilled water + 6mL 20 × SSC, or according to the need to prepare in proportion.

[0096] If 10% SDS produces white flocculent precipitate, please put it in a 42°C water bath to dissolve and mix well before preparing the lotion.

[0097] 2) Absolute ethanol.

[0098] 3) Ice-water mixture.

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Abstract

The invention discloses a gene chip for identification of seven swine disease pathogens and a detection method thereof. The gene chip can be used for detection of porcine actinobacillus pleuropneumoniae, haemophilus parasuis, mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine transmissible gastroenteritis 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 construct the gene chip for detection, and optimization is performed to obtain a detection system. The invention aims to establish a method with the advantages of high sensitivity, strong specificity, time saving and labor saving, and easy observation of results to detect the seven important swine disease pathogens.

Description

technical field [0001] The invention belongs to the field of biological chips. Specifically, the present invention relates to a porcine infectious actinobacillus pleuropneumoniae, haemophilus parasuis, mycoplasma hyopneumoniae, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine infectious gastric A chip device for the identification and detection of 7 pathogens including enteritis virus. Background technique [0002] Mycoplasma pneumonia (Mycoplasma neumoniaeofswine, MPS), porcine contagious pleuropneumonia (PCP), porcine polyserositis and arthritis (Swinepolyserositisandarthrithis), porcinecircovirus associated disease (porcinecircovirus associateddisease, PCVAD), porcine reproductive and respiratory syndrome Porcinereproductiveandrespiratorysyndrome (PRRS), classical swine fever (CSF) and porcine transmissible gastroenteritis (transmissible gastroenteritisofswine, TGE) are respectively caused by Mycoplasm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C12Q1/70C12Q1/68C12Q1/04
CPCC12Q1/701
Inventor 聂福平王昱杨俊李应国王国民保雨艾军李贤良张强
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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