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Primer pair for amplifying watermelon latent virus and application thereof

A latent virus and primer pair technology, applied in the field of RT-PCR reagents, RT-PCR kits, and biological detection, can solve the problems of lack of virus specificity and sensitivity

Active Publication Date: 2021-06-22
BEIJING AGRI TECH PROMOTION STATION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of specific and sensitive primers and detection methods for the virus

Method used

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  • Primer pair for amplifying watermelon latent virus and application thereof
  • Primer pair for amplifying watermelon latent virus and application thereof
  • Primer pair for amplifying watermelon latent virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Design and synthesis of primers

[0060] Based on the genome of watermelon latent virus with GenBank accession number KY081285, after a large number of sequence analysis, sequence design, manual screening optimization, and effect verification, a specific primer pair for identifying watermelon latent virus was obtained for the conserved region RdRP encoded by dsRNA1. The specific primer pair consisted of CILCV-588-F and CILCV-588-R. The primer sequences are as follows:

[0061] CiLCV-588-F: 5'-TCCAGACGTTGGCTACACAC-3' (SEQ ID NO.1);

[0062] CiLCV-588-R: 5'-ATTGCGAACCTCTCAGGTGG-3' (SEQ ID NO.2);

[0063] Among them, CiLCV-588-F is the forward primer, and CiLCV-588-R is the reverse primer.

[0064] The theoretical amplification product size of the specific primer pair is 588bp, its nucleotide sequence is shown in SEQ ID NO.3 in the sequence listing, and the corresponding RNA sequence is shown in SEQ ID NO.4 in the sequence listing.

[0065] 5'-TCCAGACGTTGGCT...

Embodiment 2

[0067] Embodiment 2: establishment of identification method

[0068] One, the method for identifying whether the virus to be tested is watermelon latent virus

[0069] 1. Use the TRIZOL method or other kits that can be used to extract viral RNA to extract the total RNA of the sample to be tested according to the instructions.

[0070] 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.

[0071] The reaction system was as follows (25 μl): Primer CiLCV-588-F 0.5 μl (10 μmol / L), Primer CiLCV-588-R 0.5 μl (10 μmol / L), PrimeScript 1Step Enzyme Mix 1 μl, 2x 1stepBuffer (Dye Plus) 12.5 μl, Template 1 μL (that is, the total RNA extracted in step 1, the concentration is 100ng-200ng / μL), and the balance is made up to 25μl with RNase-free water. Among them, PrimeScript 1StepEnzyme Mix and 2x 1step Buffer (Dye Plus) are from Takara's PrimeScript TM One Step RT-PCR Kit Ver.2 (Dye Plus), Cat. No. RR057A.

[0...

Embodiment 3

[0091] Embodiment 3: specificity experiment

[0092] The viruses to be tested are as follows:

[0093] Cucumber green mottle mosaic virus, cucumber mosaic virus, melon endogenous RNA virus, melon chlorotic virus, squash mosaic virus, tomato spotted wilt virus, watermelon mosaic virus, zucchini yellow mosaic virus.

[0094] The test samples of plants that have been confirmed to be infected with the above-mentioned viruses are tested according to "2. Method for identifying whether the plant material to be tested is infected with watermelon latent virus" in Example 2.

[0095] see results figure 1 . figure 1 Among them, lane 1 corresponds to the positive control watermelon sample containing watermelon latent virus; lane 2 corresponds to the detection of the amplification product of the watermelon sample containing cucumber green mottle mosaic virus; lane 3 corresponds to the detection of the amplification product of the watermelon sample containing cucumber mosaic virus. Ampli...

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Abstract

The invention discloses a primer pair for amplifying watermelon latent virus genome segments. The primer pair comprises a first primer and a second primer, the nucleic acid sequence of the first primer is as shown in SEQ ID NO. 1; and the nucleotide sequence of the second primer is as shown in SEQ ID NO. 2. The invention further discloses a reagent and a kit comprising the primer pair and application of the reagent and the kit. The invention also discloses a method for detecting whether a to-be-detected plant material is infected with the watermelon latent virus or not and a method for detecting whether the to-be-detected virus is the watermelon latent virus or not by using the primer pair. The primer pair is high in sensitivity and good in specificity when used for detecting the watermelon latent viruses.

Description

technical field [0001] The invention belongs to the technical field of plant viruses, relates to biological detection technology, in particular to primer pairs, RT-PCR reagents, RT-PCR kits and applications and methods for detecting watermelon latent viruses based on one-step RT-PCR technology. Background technique [0002] Citrullus lanatus cryptic virus (CiLCV) is an important disease virus newly discovered on watermelon in recent years, and it is a member of the genus Deltapartitivirus in the family Partitiviridae. Consists of two dsRNA fragments. Among them, dsRNA-1 has a full length of 1603nt and contains an ORF encoding RdRp; dsRNA-2 has a full length of 1466nt and contains an ORF encoding CP. The virus was first discovered in Israel in 2013, first reported in Henan, my country in 2017, and detected on watermelons in Beijing in 2019. The virus can be transmitted from seeds to offspring seedlings with high efficiency, and the seeds of most watermelon varieties can tra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2521/107Y02A50/30
Inventor 赵立群邱艳红徐秀兰田文张海军张晓飞刘慧温常龙
Owner BEIJING AGRI TECH PROMOTION STATION
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