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Gene chip and method for detecting pathogenic microorganisms of porcine epidemic disease

A pathogenic microorganism and gene chip technology, applied in the field of swine blight pathogenic microorganism detection, can solve the problems of time-consuming, labor-intensive, poor sensitivity and specificity, and achieve the effect of improving detection efficiency, strong specificity and saving time.

Active Publication Date: 2016-03-23
山东宝来利来生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, pathogen isolation and conventional serological methods are mostly used in the diagnosis of pig-derived diseases, but pathogen isolation is not only time-consuming and laborious, but also has poor sensitivity and specificity, and serological diagnostic methods are also different due to different laboratory conditions and diagnostic methods and produce different differences; although PCR diagnostic methods have been applied to virus detection, they need to be diagnosed separately

Method used

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  • Gene chip and method for detecting pathogenic microorganisms of porcine epidemic disease
  • Gene chip and method for detecting pathogenic microorganisms of porcine epidemic disease
  • Gene chip and method for detecting pathogenic microorganisms of porcine epidemic disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: the preparation of gene chip

[0058] Spot the designed oligonucleotide probes corresponding to the pathogenic microorganisms to be detected on positively charged glass slides (AMI Company, U.S.), and the spotted glass slides are exchanged under a long-wave ultraviolet lamp. Integrate for about 10 minutes, and at the same time, 0.2 μL of positive quality control labeled with biotin is also spotted on each glass slide. The distribution of positive quality controls is in the shape of a "T", and the oligonucleotide probes corresponding to the pathogenic microorganisms to be tested are distributed in a microarray. The pattern diagram of the gene chip is as follows: figure 1 shown.

[0059] The sequence of the oligonucleotide probe corresponding to the pathogenic microorganism to be detected is as follows:

[0060] (1) Probe for Actinobacillus pleuropneumoniae

[0061] SEQ ID NO.1: 5'gcaAAGGTAATGATATTCTAAgaggt

[0062] (2) Probe for classical swine fever v...

Embodiment 2

[0080] Example 2: Screening of primers for specific amplification of specific conserved nucleotide sequences of pathogenic microorganisms to be detected

[0081] Effective design of primers for specific amplification is the most critical link in determining the success of the experiment. Although there are some software for designing primers and some basic principles for designing primers, according to these design software and design principles, multiple different primer sequences can be designed, and the use of different primer sequences will produce different effects. The replacement or increase or decrease will have an important impact on the test results. Therefore, the design of amplification primers cannot be obtained by conventional software design. In this example, it is used to specifically amplify Actinobacillus pleuropneumoniae, classical swine fever virus, Haemophilus parasuis, swine influenza virus, Mycoplasma hyopneumoniae, porcine circovirus type Ⅱ, porcine pa...

Embodiment 3

[0163] Example 3: Specific verification of the gene chip

[0164] Sample: Specific virus and bacterial culture fluid (specific viruses and bacteria are respectively: swine fever virus, porcine blue ear virus, Actinobacillus pleuropneumoniae, Haemophilus parasuis and porcine pseudorabies virus).

[0165] 1 sample processing

[0166] Centrifuge 100mL of bacterial culture solution, resuspend in normal saline, add lyoprotectant (5% skim milk powder), freeze-dry and store; take 10mL of virus culture solution, add lyoprotectant (5% skim milk powder), freeze-dry Store dry.

[0167] 2 total RNA extraction

[0168] Freeze-dried samples of bacteria and virus were taken, resuspended in PBS to 50-100 μL, and a total RNA extraction kit (TIANGEN, Germany) was used to operate according to the instructions.

[0169] 3RT-PCR amplification

[0170] The primers optimized in Example 2 were used as specific amplification primers, and the specific amplification primers were biotin-labeled (labe...

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Abstract

The invention discloses a gene chip for detecting pathogenic microorganisms of the porcine epidemic disease. The gene chip comprises an oligonucleotide probe and a positive quality controller, which are fixed on a solid phase carrier and correspond to the pathogenic microorganisms to be detected. The invention further discloses a method for detecting porcine actinobacillus pleuropneumonia, hog cholera virus, haemophilus parasuis, swine influenza virus, mycoplasma hyopneumoniae, porcine circovirus type II, porcine parvovirus, porcine reproductie and respiratousyndrome virus, porcine pseudorabies virus and streptococcus suis by adopting the gene chip. The gene chip realizes efficient detection, aims at 10 kinds of swine pathogenic microorganisms at a time, and greatly improves the detection efficiency. Besides, the gene chip is high in sensibility and strong in specificity.

Description

technical field [0001] The invention relates to the technical field of detecting swine epidemic pathogenic microorganisms, in particular to a gene chip for detecting swine epidemic pathogenic microorganisms and a detection method thereof. Background technique [0002] With the rapid development of large-scale pig farming, the occurrence of pig diseases is increasingly harmful to large-scale pig production, especially infectious diseases, which have become the main epidemic diseases that seriously affect the healthy and stable development of large-scale pig farming in my country. There are more and more types of pig diseases in our country, and there are many new diseases in recent years, the complexity of the diseases is increasing, and even the mixed infection of old diseases and new diseases leads to a significant increase in morbidity and mortality, causing pig farms to suffer economically. huge loss. Clinically, swine diseases are mainly caused by multiple infections or...

Claims

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Application Information

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IPC IPC(8): C40B40/06C12Q1/70C12Q1/68C12Q1/14C12Q1/04
CPCC12Q1/6837C12Q2537/143C12Q2545/113C12Q2545/101C12Q2565/501
Inventor 单宝龙陈雷谷巍程福亮王红聂兆晶王丽荣陈甜甜王春凤
Owner 山东宝来利来生物工程股份有限公司
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