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Method for detecting infection of Marek's disease virus serotype 1 (MDV-1) in pathological material by combining polymerase chain reaction (PCR) with nucleic acid probe dot hybridization technology
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A technology of Marek's disease and dot hybridization, which is applied to the determination/testing of microorganisms, microorganisms, biochemical equipment and methods, etc., and can solve the problems of inability to detect
Active Publication Date: 2012-07-11
SHANDONG AGRICULTURAL UNIVERSITY
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However, if the amount of virus infection is small, the nucleic acid of the specific virus may be less than 1pg in the total DNA of 1μg of the disease material sample, so it cannot be detected
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Embodiment 1
[0072] Detection of MDV infection in naturally occurring chicken flocks by PCR combined with dot hybridization (take type I chicken Marek's disease virus as an example).
[0073] 1 Collection of disease materials and preparation of sample DNA
[0074] The liver, spleen, heart, kidney, thymus, bone marrow, etc. of chickens and dead chickens suspected to be infected by chicken type I Marek's disease virus (MDV) causing clinical manifestations of different flocks were taken. For tissue samples, each 0.02g was ground, added 0.5mL DNA extraction buffer (100mmol / L NaCl, 10mmol / L Tris-Cl, pH 8.0, 0.25mmol / L EDTA, pH 8.0, 0.5% SDS) and the final concentration was 100 μg / mL proteinase K, digest overnight at 55°C. The next day, extract tissue DNA according to conventional methods (J. Sambrook, E.F. Fritsch, T. Maniartis. Translated by Jin Dongyan, Li Mengfeng, etc. Molecular Cloning Experiment Guide 2nd Edition. Science Press, 1996) , dissolved in an appropriate amount of TE buffer (p...
Embodiment 2
[0098] Detection of REV Infection in Naturally Diseased Chickens by PCR Combined with Dot Hybridization
[0099] 1 Collection of disease materials and preparation of sample DNA
[0100] The liver, spleen, heart, kidney, thymus, bone marrow, etc. of chickens and dead chickens suspected of being infected by avian reticuloendotheliosis virus (REV) causing different clinical manifestations were collected. For tissue samples, each 0.02g was ground, added 0.5mL DNA extraction buffer (100mmol / L NaCl, 10mmol / L Tris-Cl, pH 8.0, 0.25mmol / L EDTA, pH 8.0, 0.5% SDS) and the final concentration was 100 μg / mL proteinase K, digest overnight at 55°C. The next day, extract tissue DNA according to conventional methods (J. Sambrook, E.F. Fritsch, T. Maniartis. Translated by Jin Dongyan, Li Mengfeng, etc. Molecular Cloning Experiment Guide 2nd Edition. Science Press, 1996) , dissolved in an appropriate amount of TE buffer (plus an appropriate amount of ribonuclease), which is the sample DNA. At...
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Abstract
The invention relates to a method for detecting infection of Marek's diseasevirusserotype 1 (MDV-1) in a pathological material by combining polymerasechain reaction (PCR) with a nucleic acid probe dot hybridization technology. After the tissue sample deoxyribonucleic acid (DNA) of a suspected animal infected with MDV-1 (a dead animal) is subjected to PCR amplification by specific primers for MDV-1, a PCR product is subjected to dot hybridization by a virus-specific nucleic acid probe, so that the specificity of the PCR product can be shown, and a detection rate is greatly improved. A test result proves that the sensitivity and specificity of detecting virus infection can be obviously improved by the method for detecting the infection of MDV-1 in the pathological material by combining the PCR with the nucleic acid probe dot hybridization technology.
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[0001] This application is a divisional application with an application date of April 9, 2010, an application number of 201010142266.1, and an invention titled "PCR combined with nucleic acid probe dot hybridization technology for detection of virus infection in disease materials". technical field [0002] The invention relates to a PCR combined with nucleic acid probe dot hybridization technique for detecting type I Marek's disease virus infection in disease materials, which belongs to the molecular biotechnology in the field of veterinary microbiology. Background technique [0003] At present, in large-scale poultry and pig farming, multiple infections of viruses are a common problem and the real cause of clinical morbidity. However, pathogenic detection of multiple viruses is a difficult problem for farms. This is because it is difficult to isolate and cultivate different viruses from different disease materials of multiple individuals at the same time. Modern molecular ...
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