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Method for detecting Marek's tumour

A Marek's tumor and detection method technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as indeterminate tumors, achieve the effects of reducing the probability of onset, accurate results, and sensitive operation

Inactive Publication Date: 2012-05-16
ZHENGZHOU HOUYI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of agar diffusion test is to use the antiserum of Marek's disease to determine whether the virus exists in the feather sac of sick chickens, but this method can only determine whether there is infection, but cannot determine whether tumors occur

Method used

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  • Method for detecting Marek's tumour
  • Method for detecting Marek's tumour
  • Method for detecting Marek's tumour

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Separation of lymphocytes: Select chicken flocks with obvious clinical symptoms, mainly with tumor masses visible to the naked eye, collect 1ml of anticoagulant blood, store in a 4°C refrigerator, add 2ml of lymphatic separation solution to the bottom of the test tube, and then Add 1ml of anticoagulated blood to the wall, centrifuge at 2500g / min for 20min, use a pipette gun to draw the middle layer, that is, lymphocytes, into another clean test tube, and store at -20°C;

[0032] 2. DNA extraction: Extract the DNA of lymphocytes with the cell extraction nucleic acid DAN kit, operate according to the kit instructions, and detect the DNA content and purity. The concentration of the extracted DNA is 100 μg / mL, and the OD260 / 280 is 1.7 ;

[0033] 3. Design primers: The primer sequences are as follows:

[0034] Upstream primer IPCS: 5′-AAT GAG CGA ACT GCC TCA CAC AAC-3′;

[0035] Downstream primer IPCA: 5′-GAT CGC CCA CCA CGA TTA CTA CCT-3′;

[0036] Primers were ...

Embodiment 2

[0050] 1, the separation of lymphocytes: with embodiment 1;

[0051] 2, the extraction of DNA: with embodiment 1;

[0052] 3. Design microsatellite primers: Design primers according to the selected microsatellite locus ABR107. The primer sequences are as follows:

[0053] Upstream primer p1: 5'-CCGTTACTGACTTCTGCTTT-3';

[0054] Downstream primer p2: 5'-TTTGTATTGGCTCCCCTCATC-3';

[0055] Primers were synthesized by Shanghai Yingjun Biological Co., Ltd.;

[0056] 4. PCR amplification of target fragment: PCR amplification system is: template: 1 μL, Taq DNA polymerase: 0.4 μL, upstream and downstream primers 1 μL, dNTP: 2 μL, 10×buffer: 2.5 μL, and finally distilled water to make up 25 μL , the program of PCR amplification is:

[0057] Pre-denaturation at 94°C for 5 minutes;

[0058] Denaturation at 94°C for 30 s;

[0059] Anneal at 55°C for 30 s;

[0060] Anneal at 50°C for 30 s;

[0061] Anneal at 45°C for 30 s;

[0062] 68°C 30s

[0063] 72°C for 7 minutes;

[...

Embodiment 3

[0067] 1, the separation of lymphocytes: with embodiment 1;

[0068] 2, the extraction of DNA: with embodiment 1;

[0069] 3. Design microsatellite primers: Design primers according to the selected microsatellite locus ABR204. The primer sequences are as follows:

[0070] Upstream primer P3: 5'-TAAATAAAGGTGTTGGCAGTT-3';

[0071] Downstream primer P4: 5′-CAGATTGTTAAAATAGTTGGGTT-3′;

[0072] Primers were synthesized by Shanghai Yingjun Biological Co., Ltd.;

[0073] 4. PCR amplification of target fragment: PCR amplification system is: template: 1 μL, Taq DNA polymerase: 0.4 μL, upstream and downstream primers 1 μL, dNTP: 2 μL, 10×buffer: 2.5 μL, and finally distilled water to make up 25 μL , the program of PCR amplification is:

[0074] Pre-denaturation at 94°C for 5 minutes;

[0075] Denaturation at 94°C for 30 s;

[0076] Anneal at 55°C for 30 s;

[0077] Anneal at 50°C for 30 s;

[0078] Anneal at 45°C for 30 s;

[0079] 68°C 30s

[0080] 72°C for 7 minutes; ...

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Abstract

The invention discloses a method for detecting Marek's tumour. The method comprises steps of: separating lymphocytes in an anticoagulant blood sample; extracting a genome DNA in the lymphocytes; designing a microsatellite primer; carrying out PCR amplification on an objective fragment and carrying out denaturing polyacrylamide gel electrophoresis detection on PCR products. The method of the invention has sensitive operation and accurate results; genome detection can be carried out before group generation of Marek's tumour by the method to determine whether Marek's tumour occurs according to genome changing result, so as to reduce morbidity of chicken flocks and increase group safety; and the method can help to discovery cause of disease in order to carry out timely and pertinent treatment.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting Marek's tumor. Background technique [0002] Marek's disease is a lymphoproliferative neoplastic disease, which is currently one of the three major diseases that endanger the healthy development of the chicken industry, causing high morbidity and mortality in chicken flocks, and bringing great economic losses to the breeding. Therefore, early diagnosis can help reduce losses. Usually, a preliminary diagnosis can be made based on clinical symptoms, but those that are difficult to judge clinically need to be sent to the laboratory for virus isolation and identification, serological methods, histological examination, and nucleic acid probes for confirmation. The agar diffusion test method is to use antiserum against Marek's disease to determine whether the virus exists in the feather sac of sick chickens, but this method can only det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02
Inventor 吴红云李建正郭俊清
Owner ZHENGZHOU HOUYI PHARMA
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