Nucleic acid test strip for detecting cucumber green mottle mosaic virus and its application

A green mottled flower and leaf virus technology, which is applied in the directions of microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc. Simple and convenient, high sensitivity, high detection efficiency

Active Publication Date: 2019-12-20
CHINESE ACAD OF INSPECTION & QUARANTINE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The existing detection methods for cucumber green mottle mosaic virus are mainly based on the observation of pathogenic symptoms and RT-PCR, etc., which are time-consuming, have low sensitivity or require expensive operating instruments, which are not conducive to the promotion and use in grassroots units

Method used

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  • Nucleic acid test strip for detecting cucumber green mottle mosaic virus and its application
  • Nucleic acid test strip for detecting cucumber green mottle mosaic virus and its application
  • Nucleic acid test strip for detecting cucumber green mottle mosaic virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, design and synthesis of primers

[0076] After a large number of sequence analysis, sequence design, artificial screening optimization, and effect verification, a set of specific primers for identification of cucumber green mottle mosaic virus was finally obtained. The specific primer set consists of primer CGMMV-BF, primer CGMMV-MBF, primer CGMMV-DF, primer CGMMV-CPR and primer CGMMV-BR.

[0077] CGMMV-BF (SEQ ID NO: 1): 5'-CCTCAACGGTCCTGTGTTG-3';

[0078] CGMMV-MBF (SEQ ID NO: 2): 5'-Biotin-GGCCTATCTTCGTTTCGCT-3';

[0079] CGMMV-DF (SEQ ID NO: 3): 5'-CTTAGCTCCACGGATACGC-3';

[0080] CGMMV-CPR (SEQ ID NO: 4): 5'-AACCTCAATGACCCTATTAGCGGCCTATCTTCGTTTCGCT-3';

[0081] CGMMV-BR (SEQ ID NO: 5): 5'-GTGGGATTGCTAGGATCTA-3'.

[0082] The 5' end of CGMMV-MBF was labeled with biotin, and the 5' end of CGMMV-DF was labeled with 6-FAM.

[0083] CGMMV-BF is a forward displacement primer. CGMMV-MBF is detection probe 1. CGMMV-DF is detection probe 2. CGMMV-CPR i...

Embodiment 2

[0084] Embodiment 2, establishment of method

[0085] One, the method for identifying whether the virus to be tested is cucumber green mottle mosaic virus

[0086] 1. Extract the total RNA of the virus to be tested.

[0087] 2. Using the total RNA extracted in step 1 as a template, use the primer set designed in Example 1 to perform reverse transcription and cross-primer constant temperature amplification.

[0088] Reaction system (20 μl): 1.5 μmol / L of primer CGMMV-CPR, 0.9 μmol / L of primer CGMMV-MBF with biotin label at the 5' end, 0.9 μmol / L of primer CGMMV-DF with 6-FAM label at the 5' end, Primer CGMMV-BF 0.3μmol / L, primer CGMMV-BR 0.3μmol / L, dNTP 0.4mmol, 10×Thermopol buffer 2μl, AMV reverse transcriptase 10 units, Bst DNA polymerase 8 units, MgSO 4 2 mmol, 1 μl template, and sterile water as the rest.

[0089] Reaction conditions: 59°C, 90min.

[0090] 3. Take the product of step 2, and use a disposable nucleic acid detection device for detection (operate according...

Embodiment 3

[0095] Embodiment 3, specificity experiment

[0096] The viruses to be tested are as follows:

[0097] Cucumber green mottle mosaic virus (CGMMV): References: Chen Xiaoyu, Zhang Yongjiang, Li Guifen, etc. Research on the effectiveness of cucumber green mottle mosaic virus outbreaks[J]. Phytosanitary, 2010, 24(4 ):16-19..

[0098] Cucumber mosaic virus (CMV): References: Li Guifen, Zhu Shuifang, Zhou Qun et al. Identification of Cucumber mosaic virus isolates infecting Echinacea purpurea [J]. Plant Protection, 2007, 33(1) :54-56..

[0099] Pepper mild mottle virus (Pepper mild mottle virus, PMMoV-S): References: Zhang Yongjiang, Li Guifen, Ma Jie, etc. Detection of pepper mild mottle virus in pepper seeds [J]. Anhui Agricultural Sciences, 2007, 35(14) :4228-4229..

[0100]Tobacco mosaic virus (TMV): References: Zhang Yongjiang, Ma Rongqun, Xin Yanyan, etc. Establishment of a barcode identification method for the genus Tobacco mosaic virus [J]. Hubei Agricultural Sciences, 2...

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Abstract

The invention discloses a nucleic acid test strip for detecting cucumber green mottle mosaic viruses and an application of the nucleic acid test strip. A specific primer group comprises a primer CGMMV-BF, a primer CGMMV-MBF, a primer CGMMV-DF, a primer CGMMV-CPR and a primer CGMMV-BR which are sequentially shown as a sequence 1 to 5 in a sequence table. The specific primer group is used for identifying the cucumber green mottle mosaic viruses or detecting whether a plant sample to be detected contains the cucumber green mottle mosaic viruses or not. The nucleic acid test strip has the advantages of good specificity, high sensitivity, simplicity and convenience in operation, high detection efficiency and the like, and can meet the basic requirements of port and field detection.

Description

technical field [0001] The invention belongs to the technical field of plant quarantine, relates to biological detection technology, specifically designs a nucleic acid test strip for detecting cucumber green mottle mosaic virus and uses the test strip to identify cucumber green mottle mosaic virus detection or containing cucumber green mottle mosaic virus Mosaic virus plant material method. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus genus of the family Tymoviridae and is an important quarantine pest in my country. It is mainly transmitted through soil and seeds. Significant threat to cucurbit crops such as cucumbers, watermelons and melons. Around 2005, with the import of melon seeds into our country, the sweet melon crops in Liaoning and Beijing suffered heavy losses. Therefore, it is of great significance to strengthen the detection of the virus and do a good job in epidemic prevention and control to reduce the los...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/94
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2521/107C12Q2565/625
Inventor 张永江霍亚云赵文军李为民朱罗罗胡林尤其敏
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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