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Gene chip and kit for infectious bronchitis viruses and/or infectious laryngotracheitis viruses

A technology for bronchitis and chicken infectivity, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problem that there is no chip specificity and sensitivity test, and it is impossible to explain the specific and sensitive simultaneous detection and other problems, to achieve the effect of good application prospects, fast detection and high sensitivity

Active Publication Date: 2015-01-21
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Patent applications with publication numbers 1616679 and 1616678 respectively disclose gene chips for detecting 10 or 9 viruses including chicken infectious bronchitis virus and chicken infectious laryngotracheitis virus, but there is no chip specificity and sensitivity in the documents It cannot be proved that it can specifically and sensitively detect chicken infectious bronchitis virus and chicken infectious laryngotracheitis virus at the same time

Method used

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  • Gene chip and kit for infectious bronchitis viruses and/or infectious laryngotracheitis viruses
  • Gene chip and kit for infectious bronchitis viruses and/or infectious laryngotracheitis viruses
  • Gene chip and kit for infectious bronchitis viruses and/or infectious laryngotracheitis viruses

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Embodiment 1

[0033] Embodiment 1 Preparation of gene chip of the present invention

[0034] 1. Strains

[0035] ILTV standard strain, IBV H120 strain.

[0036] 2. Experimental method

[0037] 2.1 Preparation of PCR primers and detection probes

[0038] (1) Design of pathogen-specific probes: by comparing and analyzing the nucleic acid sequences of chicken infectious bronchitis virus and chicken infectious laryngotracheitis virus included in GenBnak, selected conserved region sequences: IBV-IBM / IBN, ILTV-TK / gB. Design and detect multiple pairs of probes for conserved sequences, and select the probe sequences with strong specificity.

[0039] (2) Design of specific primers for probe sequences: specific primers were designed for the above conserved probe sequences using bioinformatics software DNAman, Primer5.0, etc. Specific primers were synthesized by Shanghai Bioengineering Company.

[0040] (3) Probe preparation: Extract chicken infectious bronchitis virus and chicken infectious lar...

Embodiment 2

[0165] Embodiment 2 detection method of the present invention

[0166] 1. Nucleic acid extraction

[0167] Extraction of viral RNA: The kit method is used to extract viral RNA, and the test uses a small amount of virus / liquid sample RNA extraction kit. Extract RNA according to the instructions of the kit, the method is as follows:

[0168] a) Take 300 μL of the sample to be tested and place it in a centrifuge tube, add 500 μL of RV solution, shake vigorously for 2 minutes, and then let stand at room temperature for 5 minutes.

[0169] b) Add 750 μL of isopropanol and shake gently.

[0170] c) Pipette 800 μL into the adsorption column and centrifuge at 12000 rpm for 30 s at 4°C.

[0171] d) Discard the liquid in the collection tube, transfer the remaining lysate into an adsorption column, and centrifuge at 12000r for 30s at 4°C.

[0172] e) Add 500 μL RP solution after discarding the collected solution, and centrifuge at 12000 rpm for 30 seconds at 4°C to remove protein.

...

Embodiment 3

[0205] The influence of embodiment 3 colloidal gold on gene chip hybridization efficiency

[0206] 1. Preparation of colloidal gold

[0207] Prepare 1% HAuCl4, 1% trisodium citrate solution. Dissolve 1.0g of HAuCl4 powder in a clean beaker filled with 100mL of sterilized ultrapure water to prepare 1% HAuCl4, and store it in a brown bottle at 4°C in the dark for future use.

[0208] Take 200mL sterilized ultrapure water and 2mL1% HAuCl4 and mix them in a 500ml large beaker, then measure 20mL and divide them into 10 50mL small beakers numbered 1-10, and use a heating magnetic stirrer to mix the divided colloidal gold solutions in turn Heat for 5 minutes, and quickly add 1% trisodium citrate solution, the addition amount is 0.1mL, 0.2mL, 0.24mL, 0.28mL, 0.32mL, 0.36mL, 0.4mL, 0.6mL, 1.0mL, 1.2mL. When the color changes rapidly from light yellow to black and then to red, continue to stir for 10 minutes to stop, wait for the solution to cool to room temperature, and filter throug...

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Abstract

The invention discloses a gene chip and a kit for infectious bronchitis viruses and / or infectious laryngotracheitis viruses. The gene chip and detection kit disclosed by the invention can accurately and effectively detect infectious bronchitis viruses and / or infectious laryngotracheitis viruses, and are strong in specificity, high in sensitivity, short in time consuming and rapid in detection, and has a good application prospect.

Description

technical field [0001] The invention relates to a gene chip and a kit for detecting chicken infectious bronchitis virus and chicken infectious laryngotracheitis virus. Background technique [0002] With the continuous improvement of the degree of intensification of my country's poultry industry, the incidence of various diseases is also increasing year by year. The overall level of poultry disease prevention and control in my country is not high, and the occurrence and harm of poultry diseases are still very serious. According to incomplete statistics, there are more than 80 kinds of diseases that endanger the production of poultry industry in my country, among which infectious diseases account for about 75% of the total number of poultry diseases. , the hazard is serious. At the same time, the occurrence of poultry diseases also has new characteristics, such as the continuous emergence of new infectious diseases; the increase in the types of new poultry diseases; the atypic...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701
Inventor 文心田黄小波曹三杰赵松伍锐文翼平邓静尹人杰张仙常晓霞
Owner SICHUAN AGRI UNIV
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