Method for synchronous detection of sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2)

A technique of synchronous detection and step method, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems affecting the accuracy of detection results, primers cannot distinguish SPFMV and SPVC, short detection cycle, etc., to achieve convenient long-term Time storage, convenient long-distance transportation, and the effect of saving reagent costs

Inactive Publication Date: 2011-11-02
YUNNAN AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

The ELISA method has a short detection cycle and high sensitivity, and can detect large-scale samples at one time. However, because SPFMV, SPVC, SPVG, and SPV2 are very closely related in phylogenetic evolution, they have some serological cross-reactions, which often result in the leakage of some viruses. false positive
Molecular biology technology, especially RT-PCR technology is not only simple to operate, but also has higher specificity and sensitivity than ELISA method. However, ordinary RT-PCR method can only detect one virus at a time. Then it is necessary to carry out a separate detection for each virus, which requires a lot of work and is not efficient
In addition, before the usual RT-PCR detection, it is necessary to use expensive RNA extraction r

Method used

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  • Method for synchronous detection of sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2)
  • Method for synchronous detection of sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2)
  • Method for synchronous detection of sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2)

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0032] Example one:

[0033] 1. Experimental materials

[0034] It was confirmed by a single RT-PCR to be infected with sweet potato G virus (SPVG), sweet potato C virus (SPVC), sweet potato feathery mottle virus (SPFMV), sweet potato virus 2 (SPV2) and multiple infections with SPVG+SPVC, SPVG+SPFMV. , SPVG+SPV2, SPVC+SPFMV, SPVC+SPV2, SPFMV+SPV2, SPVG+SPVC+SPFMV, SPVG+SPVC+SPV2, SPVG+SPFMV+SPV2, SPVC+SPFMV+SPV2 and SPVG+SPVC+SPFMV+SPV2 Sweet potato plants were used as materials, and healthy sweet potato plants were used as negative controls.

[0035] 2. Primer design and synthesis (synthesized by Invitrogen)

[0036] (a) Design and synthesize the universal reverse primer SPFCG2R for detecting SPFMV, SPVC, SPVG and SPV2: 5'-TCGGGACTGAARGAYACGAATTTAA-3', R=A or G, Y=C or T;

[0037] (b) Design and synthesize forward primers SPFF, SPCF, SPGF and SP2F to detect SPFMV, SPVC, SPVG and SPV2 respectively. The primer sequences are as follows:

[0038] SPFF: 5’-GGATTAYGGTGTTGACGACACA-3’,

[0039...

Example Embodiment

[0055] Embodiment two:

[0056] 1. Experimental materials

[0057] The field susceptible sweet potato plants confirmed by a single RT-PCR to be infected with SPVG, SPFMV alone and with SPVC+SPFMV, SPVG+SPFMV+SPV2, SPVC+SPFMV+SPV2 and SPVG+SPVC+SPFMV+SPV2 were used as materials. Healthy sweet potato plants served as negative controls.

[0058] 2. Primer design and synthesis (synthesized by Invitrogen)

[0059] (a) Design and synthesize the universal reverse primer SPFCG2R for detecting SPFMV, SPVC, SPVG and SPV2: 5'-TCGGGACTGAARGAYACGAATTTAA-3', R=A or G, Y=C or T;

[0060] (b) Design and synthesize forward primers SPFF, SPCF, SPGF and SP2F to detect SPFMV, SPVC, SPVG and SPV2 respectively. The primer sequences are as follows:

[0061] SPFF: 5’-GGATTAYGGTGTTGACGACACA-3’,

[0062] SPCF: 5’-GTGAGAAAYCTATGCGCTCTGTT-3’,

[0063] SPGF: 5’-GTATGAAGACTCTCTGACAAATTTTG-3’,

[0064] SP2F: 5’-CGTACATTGAAAAGAGAAACAGGATA-3’,

[0065] Among them, Y=C or T;

[0066] (c) The size of the amplified fragment of...

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Abstract

The invention relates to method for a synchronous detection of sweet potato feathery mottle virus (SPFMV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2), and belongs to the field of plant protection. The method comprises the following steps of respectively designing and synthesizing specific forward primers and a universal reverse primer of SPFMV, SPVC, SPVG and SPV2, extracting total RNA of tissue of a diseased plant through a cetyltrimethyl ammonium bromide (CTAB) method, and carrying out one step quadruplex reverse transcription-polymerase chain reaction (RT-PCR) magnification process to realize a synchronous detection of SPFMV, SPVC, SPVG and SPV2. Primers designed by the method have strong singularities and SPFMV, SPVC, SPVG and SPV2 share areverse primer thus an interaction of primers is reduced. In the invention, a CTAB method is utilized for extracting RNA from tissue of an RNA virus infected plant, thus a quality of RNA is guaranteed and a detection cost is reduced effectively; and the inverse transcription and the multiple PCR are completed in one step thus a detection time is saved. The method has the advantages of rapid detection speed, high efficiency, strong singularity, high sensitivity and low cost and can realize a synchronous detection of four kinds of sweet potato virus thus has wide application prospects.

Description

Technical field: [0001] The invention relates to a method for synchronously detecting SPFMV, SPVC, SPVG and SPV2, which belongs to the field of plant protection. Background technique: [0002] Sweet potato is the seventh largest food crop in the world and the fifth largest food crop in developing countries after rice, wheat, corn and cassava, with an annual output of 127 million tons. China is the world's largest sweet potato producer, with an annual output accounting for 82.7%. Sweet potatoes mainly rely on seedlings and tubers for asexual reproduction, which can easily cause the spread and accumulation of viruses. Virus disease is one of the most restrictive factors for sweet potato production after the sweet potato weevil. The annual loss of sweet potato due to virus infection is as high as 30%-50%. The annual loss of sweet potato virus disease in China is as high as 4 billion yuan. . According to reports, there are more than 20 kinds of viruses infecting sweet potato,...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 李凡陈海如许东林王海宁左瑞娟谭冠林包改丽
Owner YUNNAN AGRICULTURAL UNIVERSITY
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