Method for potato leafroll virus (PLRV) detection, and special antibody adopted by method

A leaf-roll virus and potato technology, applied in the biological field, can solve the problems affecting potato quality and yield, compound infection, etc., and achieve the effects of great application value, good specificity and high accuracy.

Inactive Publication Date: 2019-01-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

PLRV can not only infect alone, but also often in combina...

Method used

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  • Method for potato leafroll virus (PLRV) detection, and special antibody adopted by method
  • Method for potato leafroll virus (PLRV) detection, and special antibody adopted by method
  • Method for potato leafroll virus (PLRV) detection, and special antibody adopted by method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053]Embodiment 1, the preparation of polyclonal antibody

[0054] 1. Construction of recombinant plasmid pDB.His.MBP-PLRV-MP

[0055] 1. Using the RNA of PLRV as a template, the 5'- CATATG TCAATGGTGGTGTACAA-3' (the underline is the recognition site of restriction endonuclease Nde I) and 5'- CTCGAG TCCGCGCTTGATAAG-3' (the underline is the recognition site of the restriction endonuclease Xho I) was used as a primer for PCR amplification, and a PCR amplification product of about 480bp was obtained.

[0056] 2. Ligate the PCR amplification product obtained in step 1 with the cloning vector pMD19-T to obtain the recombinant plasmid pT-PLRV-MP.

[0057] 3. Take the recombinant plasmid pT-PLRV-MP, digest it with restriction endonucleases Nde I and Xho I, and recover a fragment of about 480 bp.

[0058] 4. Take the vector pDB.His.MBP, digest it with restriction endonucleases Nde I and Xho I, and recover the vector skeleton of about 6.5 kb.

[0059] 5. Ligate the restriction fr...

Embodiment 2

[0085] Embodiment 2, polyclonal antibody detects PLRV

[0086] SDS-PAGE and Western blot were performed on the total protein of the leaves of the tobacco system infected with PLRV, and the polyclonal antibody prepared in step 3 of Example 1 was used as the primary antibody to detect PLRV. Specific steps are as follows:

[0087] 1. Extract the total protein of the leaves of the N. benthamiana system leaves infected by PLRV.

[0088] 2. After step 1 is completed, the total leaf protein is subjected to SDS-PAGE, and then the protein is transferred to a nitrocellulose membrane by electrotransfer (200mA, 85min).

[0089] 3. After completing step 2, place the nitrocellulose membrane in TBST buffer containing 5% (w / v) skimmed milk powder, and block at 37° C. for 1 hour.

[0090] 4. After completing step 3, add the dilution of the polyclonal antibody prepared in step 3 in Example 1, incubate at 37°C for 1 hour, and then wash the membrane with TBST buffer 3 times, 10 minutes each tim...

Embodiment 3

[0094] Embodiment 3, titer determination of polyclonal antibody

[0095] 1. Extract the total protein from leaves of N. benthamiana leaves infected with PLRV or healthy N. benthamiana leaves (as a negative control).

[0096] 2. Take the polyclonal antibody prepared in Step 3 of Example 1, and add TBST buffer for dilution to obtain a dilution. The dilution times of the diluent are 4000, 8000, 10000, 16000, 20000, 32000, 40000, 64000, 80000 and 128000, respectively.

[0097] 3. Perform SDS-PAGE and Western blot on the total protein extracted in step 1, and use the dilution obtained in step 2 as the primary antibody to detect PLRV.

[0098] For some Western blot results, see figure 2 In A (from left to right, the dilution ratios of the dilutions are 4000, 8000, 10000, 16000, 20000, 32000, 40000, 64000, 80000 and 128000).

[0099] 4. Extract the total protein from the inoculated leaves of N. benthamiana or healthy N. benthamiana leaves (as a negative control) infected by PLRV....

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Abstract

The invention discloses a method for potato leafroll virus (PLRV) detection, and a special antibody adopted by the method. The method and the antibody provided by the invention have the advantages that a PLRV-MP protein, of which an amino acid sequence is as shown by Sequence 4 or Sequence 6 in a sequence table, is firstly prepared, and the PLRV-MP protein is then used as an immunogen for boosterimmunization of animals to obtain a polyclonal antibody; PLRV detection through western blot, in which the polyclonal antibody is used as a primary antibody, achieves high accuracy, high sensitivity and high specificity; and whether a sample to be tested contains PLRV or not can be detected, so that significant application value is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting potato leafroll virus and a special antibody thereof. Background technique [0002] Potato leafroll virus (PLRV) is a representative species of the genus Polerovirus and belongs to the family Luteoviridae. Potato leafroll caused by PLRV occurs widely in potato growing areas all over the world, and it is a worldwide viral disease that leads to severe reduction in potato yield. PLRV can not only infect alone, but also often in combination, which seriously affects the quality and yield of potatoes. Potato yield loss in northern my country is generally 20%-50%, and can reach 70%-80% in severe cases; it is widely distributed in southern regions such as Guizhou, Sichuan, Yulin, Guangxi and other places. [0003] PLRV spreads in a persistent non-proliferative form by the vector aphid, which can infect the phloem and some non-phloem tissues of Solanaceae ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6854
Inventor 张宗英韩成贵王颖杨芳李大伟于嘉林王辉
Owner CHINA AGRI UNIV
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