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Qualitative detection method of tea geometrid nuclear polyhedrosis virus

A nuclear polyhedron and qualitative detection technology, which is applied to antiviral immunoglobulins, measuring devices, instruments, etc., can solve the problems of difficult to effectively monitor the quality of preparations and extensive detection of toxicity indicators, and achieve fine detection of toxicity indicators, The effect of shortening the cycle of toxicity evaluation and standardizing production and application

Active Publication Date: 2013-07-10
TEA RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This traditional bioassay method has caused problems in the production and use of EoNPV virus preparations, such as extensive detection of toxicity indicators, long evaluation cycles, and difficulty in effectively monitoring the quality of preparations.

Method used

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  • Qualitative detection method of tea geometrid nuclear polyhedrosis virus
  • Qualitative detection method of tea geometrid nuclear polyhedrosis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the preparation of tea geometrid nuclear polyhedrosis virus polyhedrin monoclonal antibody

[0038] 1. Isolation and purification of EoNPV virus particles

[0039] Infect the school-age larvae with the tea geometrid nuclear polyhedrosis virus EoNPV (Zhejiang Hangzhou strain), collect and grind the infected dead larvae; filter the larger particles with 5 layers of cheesecloth, and use an appropriate amount of newly prepared lysate 0.1mol / L Na 2 CO 3, 0.05mol / L NaCl (pH=10.3) to crack the polyhedron for 1-2h; neutralize the lye with 50% HCl, centrifuge at 3000g for 5min to remove the unlysed polyhedron; ultracentrifuge at 160000g for 1.5h to precipitate EoNPV Virus particles; finally, the purified virus particles were dissolved with PBS and stored in a refrigerator at 4°C until use.

[0040] 2. Immunization of mice

[0041] The isolated and purified tea geometrid nuclear polyhedrosis virion was used as an antigen and mixed fully with an equal amount of F...

Embodiment 2

[0057] Embodiment 2, ELISA detection

[0058] Take EoNPV biological agents and chemical pesticides, use 0.9% physiological saline to dilute the samples at 1:10, 1:100, 1:1000, 1:10000, mix them with a pipette, and set 3 for the diluted samples Repeat, and set up a negative control, with 1 × PBS as a negative control. Add 100 μL / well of the diluted samples in the above configuration to the wells of the microtiter plate. Coat overnight in a refrigerator at 4°C or for 2-3 hours at 37°C.

[0059] A. Use 1×PBST to wash the microtiter plate continuously for 3 times, each time for 5 minutes;

[0060] B. Prepare 2%-4% skimmed milk powder, add 150 μL 2%-5% skimmed milk powder to the wells, and block at 37°C for 1 hour.

[0061] C. Use 1×PBST to wash the microtiter plate continuously for 3 times, each time for 5 minutes;

[0062] D. Dissolve the prepared EoNPV monoclonal antibody in 2%-5% skimmed milk powder, dilute it at 1:1000, add 100 μL to each well, and incubate at 37°C for 1-2...

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Abstract

The invention relates to a qualitative detection method of a tea geometrid nuclear polyhedrosis virus, which belongs to the technical field of the detection of biological preparations and is characterized by comprising the following steps of: firstly, preparing a polyhedrin monoclonal antibody of the tea geometrid nuclear polyhedrosis virus, then coating a sample to be detected and detecting by using an indirect Elisa method. The invention breaks through a traditional biological assay technology by means of an immunological means and solves the problem of accurate qualification of the tea geometrid nuclear polyhedrosis virus. Compared with a traditional biological assay method, the virulence index detection of the method is finer, the virulence evaluation period is greatly shortened, the method can be widely used for estimating the quality of a virus preparation and specification of the production and the application of a tea geometrid virus.

Description

technical field [0001] The invention belongs to the technical field of biological agent detection, and in particular relates to a qualitative detection method of virus preparations. Background technique [0002] Ectropis obliqua nucleopolyhedrovirus (EoNPV) belongs to the family Baculoviridae and belongs to the genus Nucleopolyhedrovirus. The virus was first discovered in my country in 1977. EoNPV is the pathogenic natural enemy of the tea geometrid. It proliferates rapidly in the worm after being fed by the larvae, causing the infection and death of the worm. At present, this virus can be proliferated in large quantities indoors, and then formulated into products for commercial production. In recent years, EoNPV has been widely promoted and used as a highly effective virus insecticide in tea areas of various provinces to carry out effective biological control of tea geometrids. Produced good social, economic and ecological benefits. Because this virus product is different...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569C07K16/08
Inventor 肖强付建玉杜军利唐美君
Owner TEA RES INST CHINESE ACAD OF AGRI SCI
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