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Nucleic acid test strip for detecting cucumber green mottle mosaic viruses and application of nucleic acid test strip

A green mottled flower and leaf virus technology, applied in the direction of microbial-based methods, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of unfavorable grass-roots unit promotion and use, low sensitivity, and long time-consuming, etc., to achieve high sensitivity High efficiency, high detection efficiency, simple and convenient operation

Active Publication Date: 2016-12-14
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 viruses and application of nucleic acid test strip
  • Nucleic acid test strip for detecting cucumber green mottle mosaic viruses and application of nucleic acid test strip
  • Nucleic acid test strip for detecting cucumber green mottle mosaic viruses and application of nucleic acid test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Design and synthesis of primers

[0076] After a large number of sequence analysis, sequence design, manual screening optimization, and effect verification, a set of specific primers for the 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 (sequence 1): 5'-CCTCAACGGTCCTGTGTTG-3';

[0078] CGMMV-MBF (sequence 2): 5'-Biotin-GGCCTATCTTCGTTTCGCT-3';

[0079] CGMMV-DF (sequence 3): 5'-CTTAGCTCCACGGATACGC-3';

[0080] CGMMV-CPR (sequence 4): 5'-AACCTCAATGACCCTATTAGCGGCCTATCTTCGTTTCGCT-3';

[0081] CGMMV-BR (sequence 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 the detection probe 1. CGMMV-DF is the detection probe 2. CGMMV-CPR is a c...

Embodiment 2

[0084] Example 2, the establishment of the method

[0085] 1. 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, the primer set designed in Example 1 was used to carry out reverse transcription cross primer isothermal amplification.

[0088] Reaction system (20 μl): primer CGMMV-CPR 1.5 μmol / L, biotin-labeled primer CGMMV-MBF 0.9 μmol / L at 5’ end, CGMMV-DF 0.9 μmol / L with 6-FAM label at 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, template 1 μl, and the balance is sterile water.

[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 to the instructions). If both t...

Embodiment 3

[0095] Example 3. Specificity experiment

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

[0097] Cucumber green mottle mosaic virus (CGMMV): Reference: Chen Xiaoyu, Zhang Yongjiang, Li Guifen, etc. Research on the treatment effect of cucumber green mottle mosaic virus [J]. Plant Quarantine, 2010, 24(4 ): 16-19..

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

[0099] Pepper mild mottle virus (PMMoV-S): Reference: 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 Tobacco mosaic virus [J]. Hubei Agricultural Science, 2015, 54(11): 2624-2627..

[0101] Tomato mosaic ...

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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, and specifically designs a nucleic acid test strip for detecting cucumber green mottle mosaic virus and uses the test strip to identify the detection of cucumber green mottle mosaic virus or the detection of cucumber green mottle mosaic virus containing cucumber green mottle virus A method of plant material for mosaic virus. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV) belongs to the genus Tobamovirus 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 my country, sweet potato crops such as Liaoning and Beijing suffered heavy losses. Therefore, it is of great significance to strengthen the detection of the virus and...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
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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