Rapid PCR (Polymerase Chain Reaction) testing method of silkworm densovirus BmDNV

A technology of densovirus and silkworm, which is applied in the field of rapid diagnosis of densovirus and genome cloning and sequencing analysis, can solve problems that need further research, achieve the effect of overcoming sensitivity problems and simple operation

Inactive Publication Date: 2011-07-27
SOUTH CHINA AGRI UNIV
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Problems solved by technology

In recent years, gratifying progress has been made in using PCR technology to diagnose, detect and study silkworm densovirus genes (Kageyasu S, Hayakawa T, Isawa H et al. Detection of the Silkworm-pathogenic virus genomes by PCR. Journal of Sericultural Science of Japan, 1997, 66(6):477-483; Han Xu, Yao Qin, Gao Lu, etc., Replication of Bombyx mori densovirus (Zhenjiang strain, China) in different competent hosts. Chinese Journal of Biotechnology, 2007, 23( 1):145-151; Fu Yanhong, Tang Shunming, Qin Guangxing, etc. Cloning of the genome of Bombyx mori densovirus (Zhenjiang) strain and rescue of silkworms transfected with recombinant plasmids. Sericulture Science, 2008, 34(3 ):453-458; Yu Wei, Yao Qin, Guo Zhongjian, etc. Expression of nonstructural protein 1 (NS1) of the Chinese strain of silkworm densovirus. Acta Microbiology, 2008, 48(2):191-196) Based on the reported silkworm Primers were designed for the sequence of densovirus BmDNV gene fragments, and a PCR diagnostic method for silkworm densovirus BmDNV was established, but the sensitivity of its detection needs to be further studied

Method used

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  • Rapid PCR (Polymerase Chain Reaction) testing method of silkworm densovirus BmDNV
  • Rapid PCR (Polymerase Chain Reaction) testing method of silkworm densovirus BmDNV
  • Rapid PCR (Polymerase Chain Reaction) testing method of silkworm densovirus BmDNV

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Embodiment 1

[0023] In this example, the DNA extracted by boiling precipitation method, boiling method and phenol-chloroform method were respectively used as templates for PCR molecular detection of Bombyx mori densovirus (BmDNV).

[0024] In this embodiment, the silkworm densovirus BmDNV (obtained by the Sericulture Biotechnology Laboratory of South China Agricultural University through conventional mulberry leaf food subculture, can also be used by other laboratories or research institutes in this field. Bombyx mori densovirus BmDNV ) as material, respectively by boiling method, boiling precipitation method, phenol-chloroform method (Chen Dongmei, Lin Ying, Wang Yanxia. A simple method for isolating high-quality silkworm genomic DNA. Silkworm Science Communication, 2007, 27 (2): 5- 9) The BmDNV template DNA was prepared by three methods, and then the molecular detection and identification of the silkworm densokaryophysis DNV was carried out by the PCR method.

[0025] 1. Bombyx mori dens...

Embodiment 2

[0040] (1) Extract the template DNA of the silkworm densovirus BmDNV;

[0041] Biological material: Bombyx mori densovirus, subcultured by the Sericulture Biotechnology Laboratory of South China Agricultural University by adding silkworms to mulberry leaves (according to conventional methods, a certain concentration of virus liquid is applied to fresh mulberry leaves for research purposes in the laboratory The back of the leaves, and then fed silkworms to make silkworms infected with conventional experimental methods); EDTA was purchased from Beijing Dingguo Biotechnology Co., Ltd.; isopropanol was produced by Tianjin Damao Chemical Reagent Factory; absolute ethanol was purchased from Tianjin Damao Chemical Reagents Factory produced.

[0042] Step (1) The extraction of the template DNA of the densovirus BmDNV of the silkworm adopts the boiling precipitation method, and the midgut of the silkworm infected with the densovirus BmDNV is taken in a mortar, and 1 mL of sterilized wa...

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Abstract

The invention discloses a rapid PCR ((Polymerase Chain Reaction) testing method of a silkworm densovirus BmDNV, which comprises the steps of: rapidly extracting a template DNA of the silkworm densovirus BmDNV by adopting a boiling precipitation method, and designing a specifc primer to test the silkworm densovirus BmDNV through the PCR method. The rapid PCR testing method of the silkworm densovirus BmDNV can be used for effectively extracting the template DNA of the silkworm densovirus BmDNV and testing the silkworm densovirus BmDNV rapidly and accurately, is applicable to small-amount sample operation, and is simple to operate; the DNA sample prepared by using the method can meet the requirement of the PCR method on molecular diagnosis of the silkworm densovirus BmDNV; and the rapid PCR testing method of the silkworm densovirus BmDNV lays the foundation and provides technical reservation for early diagnosis and prediction check of BmDNV by applying a PCR technology.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis and identification, in particular to a method for rapid diagnosis of silkworm densovirus and genome cloning, sequencing and analysis. Background technique [0002] Bombyx mori densovirus (BmDNV) is a common viral disease virus in silkworm cocoon production in my country, which seriously threatens the continuous and stable production of silk industry. [0003] In the early 1970s, serological methods were used abroad for early diagnosis of silkworm virus diseases. This method can be diagnosed at the early stage of silkworm infection (24h ~ 48h after infection with the virus). In the late 1970s, my country formally started the research on serological early diagnosis of silkworm virus disease. Wang Yuxing et al. (Wang Yuxing, Cao Yisun, Qian Yuanjun, etc. Enzyme Convective Immunoelectrophoresis for Early Diagnosis of Bombyx mori Concentrated Nuclear Disease. Sericulture Science, 1983, 9(...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 刘吉平王永宾
Owner SOUTH CHINA AGRI UNIV
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