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Preparation method and detection method for gene chip

A gene chip and matrix membrane technology, applied in the field of detection, can solve the problems of shortening the detection process, tedious and time-consuming operation, and prone to false positives, etc., and achieve the effect of improving accuracy, specific identification, and high accuracy

Inactive Publication Date: 2014-01-29
珠海出入境检验检疫局检验检疫技术中心
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  • Summary
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AI Technical Summary

Problems solved by technology

The serological method has good specificity and is a common method for identifying subtypes of avian influenza and Newcastle disease and egg drop syndrome. Antiserum cannot detect emerging subtypes, so the method cannot meet the requirements of rapid and accurate detection
PCR technology is highly sensitive and fast, which greatly shortens the detection process compared with traditional methods. The fluorescent PCR and fluorescent RT-PCR developed on the basis of this method further improve the sensitivity, but the disadvantage is that it is prone to false positives and one-time detection. The reaction can only identify a few pathogens, so it is impossible to quickly identify the subtype of avian influenza through a single reaction

Method used

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  • Preparation method and detection method for gene chip
  • Preparation method and detection method for gene chip
  • Preparation method and detection method for gene chip

Examples

Experimental program
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preparation example Construction

[0032] The concrete steps of the preparation method of gene chip of the present invention are as follows:

[0033] S1. Design specific probes for detecting pathogens: select M1, H4, H5, H6, H7, H9, N1, N2, N9 and NDV, EDS-76 gene sequences of avian influenza virus on GenBank, and use bioinformatics Software Primer Express2.0 design specific probe;

[0034] S2. Specific probe synthesis: synthesized by Shanghai Chaoshi Biotechnology Co., Ltd.;

[0035] S3, specific probe processing: label 15-30Poly T at the 5 ' end of probe, so that specific probe is well fixed on the matrix membrane; The material of matrix membrane can be silicon wafer, glass or polymer;

[0036] S4. Spotting: use the DR.Fast Spot chip spotting instrument system to spot the specific probes according to the pre-designed matrix, and dry at room temperature for 10 minutes; each gene chip contains at least one positive control and one negative control; among them, the positive The control is poly PolyT, the negat...

Embodiment

[0074] The Animal Inspection Laboratory of Zhuhai Entry-Exit Inspection and Quarantine Bureau Technology Center received a total of 28 batches of pharyngeal and anal swabs from a chicken farm in Zhuhai City for Australian chickens. The pre-inspection item was avian influenza virus. Select 10 batches of them as objects to test the gene chip diagnosis method established in the present invention, and use pathogen isolation and fluorescent quantitative PCR method for comparison. The specific implementation steps are as follows:

[0075] 1. Sample pretreatment: Cut the pharyngeal and anal swabs of poultry and put them into a 10ml clean centrifuge tube after autoclaving, add 6-8ml of 4000-6000 units / ml of penicillin and streptomycin double antibody solution, 4 ℃ Soak overnight (or a few hours, as appropriate).

[0076] 2. Extraction of nucleic acid: Take 200 μL of the above pharyngeal and anal swab soaking solution and add it to a 1.5ml EP tube, add 4°C pre-cooled lysis solution TR...

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Abstract

The invention discloses a gene chip and a detection method thereof. The gene chip provided in the invention can simultaneously carry out universal screening of Avian influenza viruses in one chip chamber, discriminate subtypes of Avian influenza viruses like H4, H5, H6, H7, H9, N1, N2, N9 and the like, and detect Newcastle disease virus and EDS-76.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a gene chip capable of rapidly detecting and identifying main subtypes of avian influenza virus and simultaneously detecting Newcastle disease virus and chicken egg drop syndrome virus. Background technique [0002] At present, serological and PCR methods are mainly used to detect subtypes of avian influenza (Avian influenza, AI), Newcastle disease virus (NDV), and egg drop syndrome virus (EDS-76). The serological method has good specificity and is a common method for identifying subtypes of avian influenza and Newcastle disease and egg drop syndrome. Antiserum cannot detect emerging subtypes, so this method cannot meet the requirements of rapid and accurate detection. PCR technology is highly sensitive and fast, which greatly shortens the detection process compared with traditional methods. The fluorescent PCR and fluorescent RT-PCR developed on the basis of this method furth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 薄清如罗宝正徐海聂廖秀云沙才华王振全陈伟生
Owner 珠海出入境检验检疫局检验检疫技术中心
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