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Citrus tristeza virus and citrus exocortis viroid dual detection kit and application thereof

A technology for dual detection of citrus decay virus, applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problem of double digital PCR quantitative detection of citrus decay virus and citrus split skin virus, etc. , to achieve the effects of simple absolute quantitative analysis, good detection accuracy and reproducibility, and strong anti-impurity interference ability

Active Publication Date: 2016-12-21
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Relevant research and patent literature search did not find relevant literature reports on dual digital PCR quantitative detection of citrus decay virus and citrus split skin virus

Method used

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  • Citrus tristeza virus and citrus exocortis viroid dual detection kit and application thereof
  • Citrus tristeza virus and citrus exocortis viroid dual detection kit and application thereof
  • Citrus tristeza virus and citrus exocortis viroid dual detection kit and application thereof

Examples

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Embodiment 1

[0035] Example 1: Preliminary screening and detection of primers.

[0036] Primer primer5 software was used to detect the complementarity between primers and primer annealing temperature, and select primer pairs with close annealing temperature and easy to distinguish the amplified target fragments. The present invention designs two pairs of primers and probes for citrus decay virus and citrus split peel virus respectively. This example presents the process of screening the best primers, and the candidate primers for screening are as follows, see Table 1.

[0037] Table 1. Alternative primer sequences for citrus decay virus and citrus split virus

[0038]

[0039] Primer and probe combinations for alternatives include: STB1-F+ STB1-R+ STB1-P, STB2-F+STB2-R+STB2-P, LPB1-F+LPB1-R+LPB1-P, LPB2-F+ LPB2-R+LPB2-P, using the probe method fluorescence quantitative PCR to compare the detection effect of primer and probe combination respectively, 20μL reaction system, 10μL Premix E...

Embodiment 2

[0042] Example 2: Primer Specificity Analysis

[0043] In order to verify the specificity of the double digital PCR quantitative detection method for citrus decay virus and citrus crack peel virus of the present invention for the detection of citrus decay virus and citrus crack peel virus, STB1-F+STB1-R+STB1-P, and LPB1-F were used respectively. The combination of primers and probes of +LPB1-R+LPB1-P was used to detect citrus decay virus and citrus split peel virus disease samples. Use digital quantitative PCR system recipe: 2×3D Digital PCR Master Mix 8.0μL, 10μM probe and primers STB1-F, STB1-R, STB1-P and LPB1-F, LPB1-R, LPB1-P each 0.2μL, Citrus disease-like cDNA template 3μL, the rest use ddH 2 O make up to 16 μL. The digital PCR amplification reaction program was as follows: pre-denaturation at 96 °C for 10 min; denaturation at 98 °C for 30 s; extension at 60 °C for 2 min, a total of 40 cycles were performed, and the reaction was stopped at the final 10 °C. The citrus...

Embodiment 3

[0046] Example 3: Effects of different annealing temperatures on the duplex digital PCR reaction.

[0047] Primer and probe combinations of STB1-F+ STB1-R+ STB1-P and LPB1-F+LPB1-R+LPB1-P were used. Use digital quantitative PCR system formula: 2×3D Digital PCR Master Mix 8.0μL, 10μM probe and primers STB1-F, STB1-R, STB1-P, LPB1-F, LPB1-R, LPB1-P each 0.2μL, Citrus decay virus and citrus split peel virus co-infected disease-like cDNA template 3 μL, the rest were treated with ddH 2 O make up to 16 μL. The PCR amplification reaction program was: pre-denaturation at 96 °C for 10 min; denaturation at 98 °C for 30 s; then annealing and extension at a temperature gradient (56, 58, 60, 62) for 2 min for a total of 40 cycles; the reaction was stopped at the final 10 °C.

[0048] RESULTS: Under different annealing temperatures, the detected values ​​in the FAM signal channel and the VIC detection channel were not significantly different. The annealing extension temperature of 60°C wa...

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Abstract

The invention discloses a citrus tristeza virus and citrus exocortis viroid dual detection kit and application thereof, and designs a dual digital PCR quantitative detection reagent composition for citrus tristeza viruses and citrus exocortis viroids. The kit contains a virus sample RNA extraction reagent, a cDNA reverse transcription synthesis reagent, a digital PCR reagent, a citrus decline and citrus exocortis positive control sample template and a citrus decline and citrus exocortis negative control sample template. The kit has the advantages of high specificity, higher impurity interference resistance, convenience, high speed, detection sensitivity equivalent to that of an existing real-time fluorescent quantitative PCR detection method and the like when being used for detecting the citrus tristeza viruses and the citrus exocortis viroids, and can be used for early qualitative and quantitative detection of the citrus tristeza viruses and the citrus exocortis viroids, epidemiological survey and the like.

Description

technical field [0001] The invention belongs to the technical field of plant disease detection, and in particular relates to a dual digital PCR quantitative detection kit for citrus decay virus and citrus split skin virus and an application thereof. technical background [0002] Citrus decay disease and citrus cracking disease are two important viral diseases on citrus. Citrus decay virus is a positive-sense single-stranded RNA virus belonging to the genus Cyclovirus. The virus is mainly transmitted by aphids and diseased scions. It has destroyed more than 100 million citrus plants and caused great harm to the citrus industry. Citrus split skin virus belongs to the genus Potato tuber viroid, a low molecular weight covalently closed circular RNA virus without a protein coat and a regular geometric structure; the virus can be grafted or pruned using tools Mechanical propagation causes longitudinal bark cracking at the rootstock, resulting in weakened tree vigor, dwarfing of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2563/107C12Q2537/143C12Q2545/113
Inventor 程保平彭埃天赵弘巍宋晓兵凌金锋陈霞
Owner PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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