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Quantitative detection method of tea moth nucleopolyhedrosis virus

A quantitative detection method, nuclear polyhedron technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve the problems of difficult effective monitoring of preparation quality and extensive toxicity index detection, etc. To achieve fine detection of toxicity indicators, to solve accurate qualitative problems, and to shorten the cycle of toxicity evaluation

Active Publication Date: 2017-12-08
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 method of bioassay makes it possible to Irfa During the production and use of NPV virus preparations, there are problems such as extensive detection of toxicity indicators, too long evaluation cycle, and difficulty in effectively monitoring the quality of preparations.

Method used

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  • Quantitative detection method of tea moth nucleopolyhedrosis virus
  • Quantitative detection method of tea moth nucleopolyhedrosis virus
  • Quantitative detection method of tea moth nucleopolyhedrosis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Irfa Obtaining NPV Virus Samples and Extracting Genomic DNA

[0023] Infect the tea moth larvae with the tea moth nuclear polyhedrosis virus, collect the carcasses, and use PBS solution as the buffer solution to homogenize repeatedly. The supernatant is the crude virus extract; the crude virus extract is centrifuged at 35000g for 2 hours, and the precipitate is the purified virus particles; the purified virus particles are washed repeatedly with 1×PBS until milky white; use TaKaRa MiniBEST Viral RNA / The DNA Extraction KitVer.5.0 kit was used for the extraction of the genome of the tea moth nucleopolyhedrosis virus. The steps are as follows: 1) Lysis of the virus ① Take 200 μl of the virus stock solution. ② Add 200 μl of Buffer VGB, 20 μl of Proteinase K and 1.0 μl of CarrierRNA, mix well and incubate in a 56°C water bath for 10 minutes. ③Centrifuge, take the supernatant; add 200μl of absolute ethanol to the supernatant, and mix well by pipetting. 2) Pla...

Embodiment 2

[0024] Embodiment 2: target fragment PCR amplification

[0025] according to Irfa NPV genome sequence (FJ362523) Lef 11 genes, using DNAStar software, designed a pair of specific primers Irfa NPV Lef 11F and primers Irfa NPV Lef 11R, synthesized by Shanghai Sangon Biotechnology Co., Ltd., the primer sequences are: 5'- ACAATGAGAGTGGACCTTACGA-3' (shown in SEQ ID NO: 1) and 5'-ACGCGCTAAAACACGATATGA -3' (shown in SEQ ID NO: 2 shown), amplified Lef 11. PCR reaction uses 25 µL system (template 2 µL, 10×buffer 2.5 µL, dNTP 2 µL, forward and reverse primers 0.5 µL, Tag enzyme 0.25 µL, add sterilized double distilled water to 25 µL), PCR amplification uses two-step method, The procedure is as follows: firstly, pre-denaturation at 94°C for 3min; then 30s at 94°C, 45s at 60°C; 35 cycles, and finally extension at 72°C for 7min. Take 10ul of the PCR product and test it by 1% agarose gel electrophoresis. figure 1 , which is consistent with the expected size. Finally, use th...

Embodiment 3

[0026] Embodiment 3: the preparation of plasmid standard and its concentration determination

[0027]The amplified in embodiment 2 Lef The 11 gene fragments were connected with the vector pGEM-T, transformed into Escherichia coli DH5a, single-clonal strains were picked, and the bacteria were shaken. Use the kit to extract single colony plasmid DNA and carry out PCR identification. The recombinant plasmids identified as positive were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. Compare with the reference sequence to confirm the construction result. After the construction is completed, measure its concentration with a UV spectrophotometer, and dilute 1 µL of positive plasmid 100 times to measure its OD 260 and OD 280 value, calculate the recombinant plasmid concentration and OD 260 / OD 280 Ratio, the recombinant plasmid was diluted to 10 -10 copies / µL, stored in a -20°C freezer.

[0028] For the results of PCR identification of the recombinant plasmid,...

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Abstract

The invention discloses a quantitative detection method of iragoides fasciata nucleopolyhedrovirus and belongs to the technical field of biological agent detection. The method comprises the following steps of designing primer according to a Lef 11 gene of the iragoides fasciata nucleopolyhedrovirus; obtaining an object segment, and performing PCR (polymerase chain reaction) amplification on the object segment to obtain a Lef 11 gene segment; building a recombinant plasmid; building a standard curve used for detecting the iragoides fasciata nucleopolyhedrovirus; extracting a to-be-detected sample genome, performing fluorescent quantitative PCR reaction,calculating copy number of a to-be-detected sample according to the built standard curve, and determining the concentration of the to-be-detected sample. The traditional biological technology is broken through, and the problem on determination of the iragoides fasciata nucleopolyhedrovirus is solved. Compared with the traditional biological detection method, the quantitative detection method has the advantages that detection on virulence indexes is more subtle, the virulence evaluation period is greatly shortened, the quantitative detection method can be widely used for evaluating the quality of a virus preparation, and the production and the application of the iragoides fasciata nucleopolyhedrovirus are regulated.

Description

technical field [0001] The invention belongs to the technical field of biological agent detection, and in particular relates to a quantitative detection method for the tea thorn moth nuclear polyhedrosis virus. Background technique [0002] tea moth nucleopolyhedrosis virus ( Iragoidae fasciata nuclear polyhedrosis virus, Irfa NPV) belongs to the baculoviridae family and belongs to the genus Nucleopolyhedrosisvirus. Irfa NPV is the pathogenic natural enemy of the tea gnat moth, which proliferates rapidly after feeding on the larvae, causing the infection and death of the moth. At present, this virus can be proliferated in large quantities indoors, and can be formulated into products for commercial production. In recent years, Irfa NPV, as a high-efficiency virus insecticide, has been widely used in tea areas of various provinces to effectively control tea moths and produce good social, economic and ecological benefits. Because this virus product is different from gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/70C12Q2561/101C12Q2545/114C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 肖强袁志军付建玉毛腾飞周孝贵
Owner TEA RES INST CHINESE ACAD OF AGRI SCI
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