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RT-qPCR detection kit and oligonucleotides for detecting grapevine virus E

A grape virus and oligonucleotide technology, which is applied in the field of inspection and quarantine, can solve the problems of grape virus ERT-qPCR detection that has not been reported publicly, and achieve the effect of simple operation

Inactive Publication Date: 2015-08-05
中国检验检疫学会 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, RT-qPCR technology has been widely used in many fields such as virus detection, bacterial detection, phytoplasma detection, genetically modified product detection, nematode detection, etc., but there is no public report on RT-qPCR detection of grape virus E

Method used

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  • RT-qPCR detection kit and oligonucleotides for detecting grapevine virus E
  • RT-qPCR detection kit and oligonucleotides for detecting grapevine virus E
  • RT-qPCR detection kit and oligonucleotides for detecting grapevine virus E

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 detects the design of the special primer of grape virus E and probe

[0039] The gene sequence of grape virus E was obtained from GenBank, and DNAMAN 6.0.40 software was used for comparison and analysis, primers and TaqMan probes were designed, and a pair of primers and a probe were finally determined through screening. The sequence is: Primer GVE-FP: 5'-ACCGCACTGATAGATGATGCAC-3'(SEQ ID No.1); Primer GVE-RP: 5'-TGCCTCGAACACTGTCATGAG-3'(SEQ ID No.2); Probe GVE-P: 5'-CGTGCGGAAGGCAATTGAAAGAGC-3'(SEQ ID No.3). The 5' end of the probe contains a FAM reporter fluorescent dye, and the 3' end contains a quencher fluorescent dye BHQ1. All primers and probes were synthesized by Dalian Bao Biotechnology Company.

Embodiment 2

[0040] Embodiment 2 detects the composition of the RT-qPCR detection kit of grape virus E

[0041] (1) RT-qPCR reaction liquid, it comprises the sense primer shown in sequence listing SEQ ID No.1, the antisense primer shown in sequence listing SEQ ID No.2, the fluorescent probe shown in sequence listing SEQ ID No.3 needle, the 5' end of the probe is labeled with the reporter fluorophore FAM, and the 3' end is labeled with the quencher fluorophore BHQ1;

[0042] (2) Negative control: Infect grape leaves without grape virus E, grind with liquid nitrogen, add DEPC water to fully grind, then centrifuge at 5000rpm for 3min, take supernatant to extract RNA, dissolve in DEPC water;

[0043] (3) Positive control: Infect grape leaves with grape virus E, grind with liquid nitrogen, add DEPC water to fully grind, then centrifuge at 5000rpm for 3min, take the supernatant to extract RNA, and dissolve in DEPC water.

[0044] Further, the above-mentioned RT-qPCR reaction solution also inclu...

Embodiment 3

[0045] Embodiment 3RT-qPCR detects the specificity test of grape virus E

[0046] The specific process includes the following steps:

[0047] 1. Sample source

[0048] Grapevine E virus (GVE), Grapevine fan leaf virus (GFLV), Grapevine leaf roll associated virus GLRaV-1, Grapevine leaf roll associated virus 3 (Grapevine leaf roll associated virus virus 3 GLRaV-3) Arabis mosaic virus ArMV, Tomato ring spot virus ToRSV, Grapevine fleck virus GFV, tested by Beijing Entry-Exit Inspection and Quarantine Bureau Plant Quarantine Experiment Room preservation.

[0049] 2. Extraction of total plant RNA

[0050] For the sample in step 1, extract plant total RNA through RNA extraction kit (purchased from Qiagen Company), and the specific operation is shown in the kit instruction manual;

[0051] 3. Specific detection test

[0052] The total plant RNA obtained in step 1 and step 2 was used as a template, namely: GFLV, GFV, ArMV, ToRSV, GVE, GLRaV-1, GLRaV-3 total RNA, and water was us...

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Abstract

The invention discloses an RT-qPCR detection kit and oligonucleotides for detecting grapevine virus E. Specifically, the invention provides the oligonucleotides for detecting the grapevine virus E; the oligonucleotides are as shown in SEQ ID No.1-SEQ ID No. 3, wherein the SEQ ID No. 1 and the SEQ ID No. 2 are a sense primer and an antisense primer for detecting the grapevine virus E, respectively, and the SEQ ID No. 3 is a fluorescent probe for detecting the grapevine virus E. The invention also provides a detection kit containing the primers and the probe and a detection method using the kit; as a result, the purpose of accurately detecting the GVE in a sample to be tested can be achieved.

Description

technical field [0001] The invention relates to a real-time fluorescent quantitative RT-PCR (RT-qPCR) detection kit and oligonucleotide for detecting grape virus E, belonging to the field of inspection and quarantine. Background technique [0002] Grapevine virus E (GVE) is a new member of the genus Vitivirus, with a length of 725-825nm. The nucleic acid is a single-molecule linear positive-sense ssRNA with a length of about 7.5kb. Rugose wood complex di-sease is a common and serious grape virus disease. The disease causes grape grafting incompatibility, delayed flowering, reduced growth, and even death. So far, five species of Vitivirus related to wrinkled wood complex disease have been reported. Among them, grapevine virus A (Grapevine virus A, GVA) and grapevine virus B (Grapevine virus B, GVB) have been reported in many grape growing regions in the world, while grapevine virus D (Grapevine virus D, GVD), grapevine virus E (Grapevine virus E, GVE) and grapevine virus F...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/6851C12Q1/70C12Q1/701C12Q2531/113C12Q2561/101C12Q2561/113
Inventor 周琦邓丛良边勇赵晓丽张百怡吕玉峰
Owner 中国检验检疫学会
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