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RPA detection primer for sweet potato leaf roll virus, detection method and application

A technology for detection of sweet potato leafroll virus and primers, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., which can solve the problems of expensive instruments and long detection cycle for detection, and achieve easy operation , reliable results and broad application prospects

Pending Publication Date: 2020-03-27
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The technical problem to be solved in the present invention is to provide a kind of RPA detection primer of sweet potato leafroll virus for the long detection cycle required by the traditional detection method of sweet potato leafroll virus in the prior art, detection needs expensive instruments and the like and detection methods and applications. The primer has the advantages of rapidity, sensitivity, and specificity, and can be used to develop a rapid detection kit suitable for on-site detection in field sweet potato production, so as to ensure the healthy production of sweet potato

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  • RPA detection primer for sweet potato leaf roll virus, detection method and application
  • RPA detection primer for sweet potato leaf roll virus, detection method and application
  • RPA detection primer for sweet potato leaf roll virus, detection method and application

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Effect test

Embodiment 1

[0031] The RPA detection method of a kind of sweet potato leafroll virus that the present invention relates to, according to the relatively conservative AV1 gene sequence of SPLCV published by GenBank as the target gene, primers are designed by using primer 5 primer design software, and a pair of specific primers are designed. Using Recombinase Polymerase (Recombinase Polymerase), rapid RPA amplification detection of the target gene under constant temperature conditions.

[0032] Wherein, the primer pair is specifically:

[0033] Upstream primer F: 5'-AGGCTGAACTTCGAGACAGCTATCGTGCCCTAC-3', as shown in SEQ ID No:1;

[0034] Downstream primer R: 5'-AAGACCTGCATTCTATCCCTCAGATCCATTCGGAT-3', as shown in SEQ ID No:2.

[0035] Specific steps are as follows:

[0036] Step 100: According to the relatively conserved AV1 gene sequence of SPLCV published by GenBank as the target gene, primers are designed by using primer 5.0 primer design software, and a pair of specific primers are used....

Embodiment 2

[0043] Specific verification of RPA:

[0044] Extract the total DNA of positive samples infected with sweet potato leafroll virus (SPLCV), the total DNA of positive samples of tomato yellow leafroll virus (TYLCD), the positive samples of sweet potato feather mottle virus (SPFMV), the positive samples of sweet potato G virus (SPVG), The cDNA of the positive sample of sweet potato chlorotic dwarf virus (SPCSV) was specifically detected by the RPA reaction system.

[0045] The results showed that 5 μL of the product was analyzed by 2% agarose gel electrophoresis, and the specific results were as follows: figure 2 As shown, only SPLCV detection results have specific bands, and other samples do not produce specific bands, indicating that the established RPA detection method has good specificity for the detection of SPLCV.

Embodiment 3

[0047] In order to investigate the sensitivity of the RPA detection method of sweet potato leafroll virus of the present invention, the applicant adopts 10-fold concentration serial dilution method to dilute the extracted sweet potato leafroll virus DNA into a concentration of 10 ng μL respectively for the DNA sample in step 200 of embodiment 1 -1 , 1ng μL -1 , 100pg μL -1 , 10pg μL -1 , 1pg μL -1 , 100fg μL -1 , 10fg μL -1 ; 1fg μL -1 ; A total of 8 different concentration gradients. Perform the RPA reaction according to step 400 in the first embodiment.

[0048] After the RPA reaction, 5 μl of the amplified product was detected by 2% agarose gel electrophoresis. If the RPA characteristic band appeared, it was judged as positive, and if no amplified band appeared, it was judged as negative. Test results such as image 3 Shown: Sensitive detection of sweet potato leafroll virus RPA, the characteristic band of RPA appeared in agarose gel electrophoresis, and the detecti...

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Abstract

The invention discloses an RPA detection primer for a sweet potato leaf roll virus, a detection method and an application. The RPA detection primer is specially used for specific molecular detection of the sweet potato leaf roll virus. The detection primer is as follows: an upstream primer F is as shown in SEQ ID No: 1; and a downstream primer R is as shown in SEQ ID No: 2. The detection method comprises the following steps: extracting plant DNA infected with the sweet potato leaf roll virus, carrying out RPA isothermal amplification for 20 min, and after completion of amplification, carryingout agarose gel electrophoresis so as to generate a specific band. The RPA isothermal amplification technology established for the sweet potato leaf curl virus provided by the invention has the advantages of rapid and highly-efficient amplification, good specificity, easy and convenient operation, no need of special instruments and the like, and provides a technical basis for early diagnosis of the sweet potato leaf curl virus.

Description

technical field [0001] The invention belongs to the technical field of detection, identification and prevention of crop diseases, and in particular relates to an RPA detection primer, detection method and application of sweet potato leafroll virus. Background technique [0002] Sweet potato is an asexually propagated crop that is propagated by roots or seedlings. It is often infected by viruses. After the virus infects sweet potatoes, it will be passed on from generation to generation, thus affecting the normal growth and development of sweet potatoes, and the germplasm and species degeneration. And sweet potato leaf curl virus (Sweet potato leaf curl virus, SPLCV) is one of main viruses that can infect sweet potato transmitted by whitefly. Seriously, the production of sweet potato was cut off, causing huge economic losses. [0003] Sweet potato leafroll virus is a member of the Begomovirus genus of the family Geminiviridae, and SPLCV is a typical DNA virus transmitted by w...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844
Inventor 李华伟邱思鑫邱永祥张鸿李国良罗文彬刘中华许泳清汤浩纪荣昌林赵淼许国春
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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