Primer pair and method for detecting or assisting in detecting southern tomato virus based on one-step RT-PCR technology

An auxiliary detection and primer pair technology, applied in the field of plant viruses, can solve problems such as loss, fruit shrinkage, and plant chlorosis

Inactive Publication Date: 2021-04-09
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tomato symptoms directly caused by STV are not yet clear, but it can be highly compounded with tomato chlorosis virus, tomato mosaic virus, tomato yellow leaf curl virus, tomato spotted wilt virus, etc., causing plant chlorosis, yellowing, decline, Symptoms such as fruit shrinkage, causing serious losses

Method used

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  • Primer pair and method for detecting or assisting in detecting southern tomato virus based on one-step RT-PCR technology
  • Primer pair and method for detecting or assisting in detecting southern tomato virus based on one-step RT-PCR technology
  • Primer pair and method for detecting or assisting in detecting southern tomato virus based on one-step RT-PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, design and synthesis of primers

[0058] After a large number of sequence analysis, sequence design, artificial screening optimization, and effect verification, the specific primer pair for identifying southern tomato virus was obtained. The specific primer pair consists of STV-812-F and STV-812-R. The primer sequences are as follows:

[0059] STV-812-F: 5'-GGACACTAGGGCTATTCCATTCTC-3' (SEQ ID No.1);

[0060] STV-812-R: 5'-CATAGCCCCTCAACAGTTTAGCC-3' (SEQ ID No.2);

[0061] Among them, STV-812-F is a forward primer, and STV-812-R is a reverse primer.

[0062] The theoretical amplification product size of the specific primer pair is 812bp, and its nucleotide sequence is shown in SEQ ID No.3 in the sequence listing.

Embodiment 2

[0063] Embodiment 2, establishment of identification method

[0064] One, the method for identifying whether the virus to be tested is southern tomato virus

[0065] 1. Extract the total RNA of the virus to be tested.

[0066] 2. Using the total RNA extracted in step 1 as a template, use the primer pair designed in Example 1 to amplify by one-step RT-PCR.

[0067] The reaction system is as follows (25 μl): Primer STV-812-F 0.5 μl (10 μmol / L), Primer STV-812-R 0.5 μl (10 μmol / L), PrimeScript 1Step Enzyme Mix 1 μl, 2x 1step Buffer (Dye Plus) 12.5 μl , template 100ng (that is, the total RNA extracted in step 1), and make up the remainder with RNase-free water to 25ul. Among them, PrimeScript1StepEnzyme Mix and 2x 1step Buffer (Dye Plus) are from Takara's PrimeScript TM One StepRT-PCR Kit Ver.2 (Dye Plus), Cat. No. RR057A.

[0068] The reaction conditions are as follows:

[0069] 1. React at 50°C for 30 minutes

[0070] 2. React at 94°C for 2 minutes

[0071] 3. React at 9...

Embodiment 3

[0087] Embodiment 3, specificity experiment

[0088] The viruses to be tested are as follows: southern tomato virus (STV), cucumber mosaic virus (CMV), potato virus Y (PVY), pepper mild mottle virus (PMMoV) ), tomato chlorosis virus (ToCV), tomato mosaic virus (ToMV), tomato spotted wilt virus (tomato spotted wilt virus, TSWV).

[0089] The detection is carried out according to "II. Method for identifying whether the plant material to be tested is infected with southern tomato virus" in Example 2.

[0090] see results figure 1 . figure 1Among them, lane 1 corresponds to the positive control sample containing southern tomato virus; lane 2 corresponds to the tomato sample containing CMV (tomato leaves artificially inoculated with CMV); lane 3 corresponds to the tomato sample containing potato virus Y (artificially inoculated Tomato leaves of potato Y virus); Swimming lane 4 corresponds to pepper samples containing pepper light mottle virus (pepper leaves artificially inoculat...

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Abstract

The invention discloses a primer pair, an RT-PCR reagent and an RT-PCR kit for detecting or assisting in detecting southern tomato virus based on a one-step RT-PCR technology, and application and a method of the primer pair, the RT-PCR reagent and the RT-PCR kit. The primer pair is composed of a primer STV-812-F and a primer STV-812-R; the STV-812-F is (a1) or (a2) shown as follows: (a1) a single-stranded DNA molecule shown in SEQ ID No. 1 in a sequence table, and (a2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the SEQ ID No.1 and has the same function as the SEQ ID No. 1; and the STV-812-R is (a3) or (a4) as follows: (a3) a single-stranded DNA molecule shown in SEQ ID No.2 in the sequence table, and (a4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the SEQ ID No.2 and has the same function as the SEQ ID No. 2. The primer pair can specifically amplify the southern tomato virus, so that whether tomato seeds or plants carry the virus diseases or not can be identified.

Description

technical field [0001] The invention relates to the field of plant virus technology, relates to biological detection technology, in particular to primer pairs, RT-PCR reagents, RT-PCR kits and applications and methods for detecting or assisting detection of southern tomato virus based on one-step RT-PCR technology. Background technique [0002] Southern tomato virus (STV) belongs to the genus Amalgavirus of the Amalgaviridae family, and its genome is a double-stranded RNA. It is a new viral disease on tomato in recent years. In 2005, it was first found on tomatoes in southwest Mexico and northeast Mississippi. my country first discovered the virus in tomatoes in Xinjiang in 2011, and then in Shouguang, Shandong, and tomato samples in Beijing also detected the virus. Tomato symptoms directly caused by STV are not yet clear, but it can be highly compounded with tomato chlorosis virus, tomato mosaic virus, tomato yellow leaf curl virus, tomato spotted wilt virus, etc., causing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2521/107
Inventor 邱艳红徐秀兰张海军田文赵桂新
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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