Method for detecting infection of avian anemia viruses in pathologic material by combination of polymerase chain reaction (PCR) and nucleic acid probe and dot blot hybridization technology

A technology of dot hybridization and virus infection, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve problems such as failure to detect

Inactive Publication Date: 2012-07-18
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Method for detecting infection of avian anemia viruses in pathologic material by combination of polymerase chain reaction (PCR) and nucleic acid probe and dot blot hybridization technology
  • Method for detecting infection of avian anemia viruses in pathologic material by combination of polymerase chain reaction (PCR) and nucleic acid probe and dot blot hybridization technology
  • Method for detecting infection of avian anemia viruses in pathologic material by combination of polymerase chain reaction (PCR) and nucleic acid probe and dot blot hybridization technology

Examples

Experimental program
Comparison scheme
Effect test

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 the infection of avian anemia viruses in a pathologic material by the combination of polymerase chain reaction (PCR) and a nucleic acid probe and dot blot hybridization technology. According to the method, deoxyribonucleic acid (DNA) of a tissue sample of an animal (dead animal) which is suspected to be infected with the avian anemia viruses is subjected to PCR amplification by using a specific primer for the avian anemia viruses, and then a PCR product is subjected to dot blot hybridization by using a specific nucleic acid probe for the avian anemia viruses, so that the specificity of the PCR product can be shown, and the detection rate is also greatly improved. The experimental result shows that: the infection of the avian anemia viruses in the pathologic material is detected by the combination of the PCR and the nucleic acid probe and dot blot hybridization technology, so that the sensitivity and specificity of the detection of the infection of the avian anemia viruses can be remarkably improved.

Description

[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 technology for detecting avian anemia virus infection in disease materials, belonging to the field of 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 biol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 崔治中
Owner SHANDONG AGRICULTURAL UNIVERSITY
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