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Primer group for detecting plantain lily plant virus and application thereof

A plant virus and primer set technology, applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem of detection of hosta plant virus X that has not been seen yet

Inactive Publication Date: 2013-06-05
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Due to the advantages of short reaction time, no need for special instruments, high detection sensitivity and good specificity, the LAMP method overcomes the shortcomings of the currently used DAS-ELISA and RT-PCR methods. Report on Hosta virus X

Method used

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  • Primer group for detecting plantain lily plant virus and application thereof
  • Primer group for detecting plantain lily plant virus and application thereof
  • Primer group for detecting plantain lily plant virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Preparation and application of RT-LAMP kit for detecting hosta plant virus X

[0041] 1. Preparation of RT-LAMP primer set for detection of Hosta virus X

[0042] The loop-mediated isothermal amplification primer set for detecting Hosta plant virus X in this embodiment consists of primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6. These six primers were prepared according to the following method: According to the nucleotide sequence of Hosta virus X reported by NCBI (GenBank: NC_011544.1), the specific region of the virus nucleic acid was found through software DNAMAN analysis as a template reference sequence , using the online LAMP primer design software primer Explorer V4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html) to design and synthesize primers, and the sequences are shown in Table 1.

[0043] Table 1 LAMP primers used to detect Hosta virus X

[0044] Primer name

Sequence (5′→3′)

Position on NC_011544.1

FIP

...

Embodiment 2

[0070] Embodiment 2, the specificity analysis that is used to detect the RT-LAMP kit of hosta plant virus X

[0071]In order to verify the specificity of the RT-LAMP kit for the detection of Hosta virus X prepared in Example 1, this example selects the same genus as Hosta virus X and the potato virus X (PVX) which is different from the genus but can Arabidopsis mosaic virus (ArMV) infecting common host plants was used as a control virus.

[0072] 1. Extraction of total RNA by nano-magnetic bead method

[0073] (1) Obtaining the sample preparation solution: referring to the method in Step 3 of Example 1, from Hosta spp. Three sample preparations were prepared from leaves of tobacco (Nicotiana benthamiana) and leaves of quinoa (Chenopodium quinoa) infected with Arabidopsis mosaic virus (ArMV).

[0074] (2) Obtaining total RNA: referring to the relevant method in Step 3 of Example 1, obtain 3 total RNAs from the 3 sample preparation solutions obtained in Step (1), respectively,...

Embodiment 3

[0078] Example 3. Sensitivity analysis of the RT-LAMP kit for detecting Hosta plant virus X

[0079] In this example, the sensitivity of the RT-LAMP kit for detecting Hosta virus X prepared in Example 1 was analyzed. details as follows:

[0080] 1. Extraction of total RNA by nano-magnetic bead method

[0081] (1) Obtaining the sample preparation solution: referring to the method in step 3 of Example 1, the sample preparation solution was prepared from the hosta (Hosta spp.) leaf sample infected with Hosta plant virus X (HVX).

[0082] (2) Obtaining total RNA: refer to the relevant method in step 3 of Example 1, and obtain total RNA from the sample preparation solution obtained in step (1).

[0083] 2. RT-LAMP amplification

[0084] Dilute the total RNA obtained in step 1 (concentration: 121ng / μl) by 10 times, respectively with 10 -1 -10 -6 A total of six dilutions of the total RNA sample solution were used as templates, and the reaction system (25 μL) of (a1) was prepared...

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Abstract

The invention discloses a primer group for detecting plantain lily plant viruses and an application of the primer group. The primer group disclosed by the invention comprises a primer 1, a primer 2, a primer 3, a primer 4, a primer 5 and a primer 6, or comprises the primer 1, the primer 2, the primer 3 and the primer 4, wherein the nucleotide sequences of the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6 are respectively a sequence 1, a sequence 2, a sequence 3, a sequence 4, a sequence 5 and a sequence 6. Tests show that the effective amplification can be accomplished within 15min by using the primer group; the result can be judged by naked eyes through turbidity precipitation reaction or fluorochrome color reaction, no expensive or large-sized instruments are needed, a plantain lily plant virus X in a sample can be rapidly detected on site, the requirements on rapid quarantine inspection at a port are met, and powerful guarantees for effectively controlling the incoming and outflow of the plantain lily plant virus X and for prompting the development of Chinese flower industry are provided.

Description

technical field [0001] The invention belongs to the technical field of biological quarantine and identification, and relates to a primer set for detecting hosta plant virus X and an application thereof. Background technique [0002] Hosta virus X (HVX) is a virus belonging to the family Alphaflexiviridae (Alphaflexiviridae), and it is a virus that specifically infects plants of the genus Hosta (Hosta Tratt). H. plantaginea Aschers.) and purple calyx (H. ventricosa Stearn) and other hosta plants such as mosaic, mottled and leaf dehydration symptoms. HVX can be transmitted through seeds, seedlings and gardening tools. Countries that have seen HVX reports so far include the Netherlands, France, South Korea, and the United States. After HVX was introduced into the United States, it has caused serious harm to American hosta plants, resulting in huge product losses and seriously affecting the economic benefits of hosta plants. The United States has listed HVX as a regulated non...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 张永江辛言言魏梅生马洁朱水芳李桂芬李曼
Owner CHINESE ACAD OF INSPECTION & QUARANTINE