Establishment of PCR-HRM analysis method for rapid differential diagnosis of different serotypes of avian leukemia viruses
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A kind of avian leukosis virus and virus technology, applied in the field of virus research, can solve the problems of inability to achieve high-throughput detection tasks, expensive subgroup-specific probes, and limitations on the practical application of fluorescent quantitative PCR detection methods, and achieve good expansion Increased, reduced time, good specific effect
Active Publication Date: 2013-05-01
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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[0013] PCR detection technology has high specificity, but this method is to analyze different subgroups according to the size of the electrophoretic bands of PCR amplification products, so this method is not yet available. high-throughput detection tasks, and the high price of subgroup-specific probes also limits the practical application of fluorescent quantitative PCR detection methods
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[0060] Below in conjunction with embodiment, further illustrate the present invention.
[0061] Extraction of Avian Leukosis Virus RNA:
[0062] The kit MiniBEST Viral RNA / DNA Extraction Kit Ver.4.0 was used to extract the avian leukosis virus RNA in the disease samples. Disease samples can be whole blood, plasma, egg white, cloacal swabs and other samples that are easy to obtain and have no serious harm to the animal body. Disease samples can also be tissue samples, such as spleen and thymus.
[0063] Reverse transcription of avian leukosis virus RNA:
[0064] Use the M-MLV RTase cDNA Synthesis Kit reverse transcription kit to reverse transcribe the avian leukosis virus RNA (in order to further ensure the subsequent HRM analysis and simplify the comparative analysis of the results, the concentration of the cDNA obtained by the reverse transcription can be measured with a nucleic acid protein analyzer. Make the PCR starting template concentration relatively consistent). ...
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Abstract
The invention discloses establishment of a PCR-HRM analysis method for rapid differential diagnosis of different serotypes of avian leukemia viruses. According to the invention, universal primers are good in degeneracy, have very good amplification performance to the A, B, C, D and E subtypes of avian leukemia viruses, is conductive to improving the efficiency of PCR (polymerase chain reaction), and reduces the time of identification and typing of viruses; and specific primers provided by the invention are good in specificity, and the J subtype can be amplified in a specific way. HRM analysis is performed on the amplification product obtained by using the primers, so that the typing of avian leukemia viruses can be performed accurately and rapidly in a high-throughput way by being compared with the standard HRM curve of a known subtype, particularly the endogenous type of avian leukemia viruses and the exogenous type of avian leukemia viruses can be rapidly and accurately distinguished.
Description
technical field [0001] The invention relates to a virus research method, in particular to a research method of avian leukosis virus. Background technique [0002] Avian leukosis (Avian Leukosis, AL) is a general term for a variety of tumor diseases in poultry caused by avian leukosis / sarcoma virus (Avian Leukosis / sarcoma virus, AL / SV). According to clinical symptoms, it can be divided into lymphocytic leukemia, erythroblastic leukemia, myeloblastic leukemia, myeloid leukemia, connective tissue tumor and osteosclerosis, among which lymphoid leukemia is the most common. Avian leukosis virus can be divided into six subtypes: A, B, C, D, E and J. Among them, subtypes A, B, C, D, and J are exogenous viruses, while subtypes C and D rarely exist in nature, and subtype E is a very ubiquitous endogenous virus. In my country, exogenous avian leukosis virus is mainly subtype J, and subtypes A and B are rarely reported. [0003] So far, human beings still have almost no way to preven...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 郭鹏举张建峰张春红嘎利兵嘎
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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