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Diagnostic gene chip for pig virus disease and its use

A technology of gene chip and porcine virus disease, applied in the field of detection of infectious diseases, can solve the problems of specificity and sensitivity inferior to gene chip, time-consuming, laborious and complicated

Inactive Publication Date: 2005-05-18
山东澳兰生物工程研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, since the livestock to be tested are generally vaccinated against the disease in question, serology to detect antibodies cannot be used for diagnosis
However, the commonly used methods of virus isolation usually take one to two weeks to produce results.
In addition, the ELISA method and fluorescent antibody method for detecting viruses are relatively complicated, and generally take three days, and the specificity and sensitivity are not as good as those of gene chips.
As for the PCR method, it is relatively sensitive, but it is easy to cause false positives due to contamination, especially RT-PCR is difficult to operate and difficult to promote
All of the above methods can only detect one pathogen at a time, which is time-consuming and laborious, and is not conducive to the simultaneous detection of multiple pathogenic pathogens in large quantities.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] The general strategy of embodiment 1 gene chip preparation

[0118] Regarding the preparation of the gene chip of the present invention, the general strategy as detailed below can be adopted

[0119] (1) Gene chips based on glass slides as solid supports:

[0120] 1. Slide treatment: Take a few slides, soak them in 70% ethanol solution of 2mol / L NaOH for 2 hours, take them out, rinse them with water for several times, dry them at 110°C and cool them down to room temperature, put the slides The sheet was soaked in 1% 3-aminopropyltriethoxysilane 95% acetone solution for 2 minutes, taken out and washed 10 times with acetone, baked at 110°C for 30 minutes and cooled to room temperature for later use.

[0121] 2. Spotting: according to the specific type of the gene chip of the present invention that needs to be prepared, take 17 μl each of the characteristic DNA fragments of the pathogenic gene selected in the gene chip, add 20 × SSC buffer solution 3ul to mix, transfer to...

Embodiment 2

[0126] Embodiment 2 The general strategy of the gene chip detection sample to be tested according to the present invention

[0127] (1) Biotin-labeled samples and their detection

[0128] 1. Labeling of samples to be tested

[0129] According to the different detected objects, select the specific sample to be tested, extract the nucleic acid sample according to the following method, and perform biotin labeling on the nucleic acid of the obtained sample to be tested:

[0130] i) According to the RNA extraction method (see Molecular Cloning: Experiment Guide Chinese Third Edition, 2002, J. Sambrook et al. Huang Peitang et al. translation 518-522); the nucleic acid of the sample to be tested is extracted, and the extracted nucleic acid is used as a template, Use the RT-PCR method for amplification and labeling, apply random primers (product number: D3801 company: TaKaRa), dNTP (add 0.5ul less dTTP) to the mixed solution (80ul), and add 0.5ul of 1mmol / L Biotin- dUTP was labeled,...

Embodiment 3

[0168] Embodiment 3 Gene chip detection result of the present invention

[0169] Using the gene chip of the present invention, the samples of 100 cases of sick pigs are detected respectively, and the results are as follows: in 100 samples, there are 17 cases of detecting virus CSFV, 6 cases of FMDV, 4 cases of SIV, and 23 cases of PRV. For example, there were 13 cases in PRRSV, 7 cases in TGEV, 7 cases in PPV4, 6 cases in PRCV, 14 cases in PRTV, and 3 cases in JEV. Among them, there were 3 cases of TGEV and PRTV mixed infection, 2 cases of PRCV and SIV mixed infection, 5 cases of CSFV and PRV mixed infection, and 14 cases of PRV and PPV mixed infection.

[0170] The above detection results are 96% consistent with the prior art virus isolation method and ELISA method.

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Abstract

The present invention relates to pig's viral disease diagnosing gene chip, and the preparation process and the use in detecting infectious disease of the diagnosis gene chip.

Description

[0001] This application is a divisional application of the authorized Chinese patent 02155408.0 submitted on December 12, 2002, and the invention title is "Diagnostic gene chip for poultry and / or livestock disease and its application". field of invention [0002] The invention relates to a diagnostic gene chip for diagnosing porcine viral diseases, a preparation method using the diagnostic gene chip and an application for detecting infectious diseases. Background of the invention [0003] In recent years, my country's animal husbandry has achieved rapid development. For a long time, the main problem that hinders the development of animal husbandry and affects the production of breeding industry is the threat of the poultry and / or livestock suffering from diseases, especially chickens and pigs suffering from infectious diseases. Due to the adoption of intensive feeding methods, the trade and circulation of chickens and pigs has been accelerated, making the infectious diseases...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 吴时友尹燕博
Owner 山东澳兰生物工程研究院
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