Dual detection kit for citrus decay virus and citrus split skin virus and its application

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

Active Publication Date: 2019-05-21
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Dual detection kit for citrus decay virus and citrus split skin virus and its application
  • Dual detection kit for citrus decay virus and citrus split skin virus and its application
  • Dual detection kit for citrus decay virus and citrus split skin virus and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Primer screening and testing.

[0036] Use the primer primer5 software to detect the complementarity between the primers and the annealing temperature of the primers, and select the primer pair that has a close annealing temperature and the amplified target fragment is easy to distinguish. The present invention designs two pairs of primers and probes respectively for citrus decay virus and citrus cracking skin virus. This example gives the process of screening the best primers, and the alternative primers used for screening are as follows, see Table 1.

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

[0038]

[0039] Alternative primer and probe combinations 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, use probe method fluorescent quantitative PCR to conduct comparative analysis of the detection effect of primer and probe combination, 20 μL reaction s...

Embodiment 2

[0042] Example 2: Primer specificity analysis

[0043] In order to verify the specificity of the citrus decay virus and citrus cracking skin virus double digital PCR quantitative detection method of the present invention to the detection of citrus decaying virus and citrus cracking skin virus, use STB1-F+STB1-R+STB1-P, and LPB1-F respectively The primer and probe combination of +LPB1-R+LPB1-P is used to detect citrus decay virus and citrus split skin virus disease samples. Use digital quantitative PCR system formula: 2×3D Digital PCR Master Mix 8.0 μL, 10 μM probes and primers STB1-F, STB1-R, STB1-P and 0.2 μL each of LPB1-F, LPB1-R, LPB1-P, 3 μL of citrus disease-like cDNA template, and ddH for the rest 2 O to 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 10°C. The samples of...

Embodiment 3

[0046] Example 3: Effects of different annealing temperatures on double digital PCR reactions.

[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 8.0 μL, 10 μM probes and primers STB1-F, STB1-R, STB1-P, LPB1-F, LPB1-R, LPB1-P 0.2 μL each, Citrus decay virus and citrus cracking skin virus composite infection disease-like cDNA template 3 μL, the rest with ddH 2 O to make up to 16 μL. The PCR amplification reaction program was as follows: pre-denaturation at 96°C for 10 min; denaturation at 98°C for 30 s; then annealing and extension for 2 min under a temperature gradient (56, 58, 60, 62) respectively, for a total of 40 cycles; finally, the reaction was stopped at 10°C.

[0048] Results: At different annealing temperatures, the detection values ​​in the FAM signal channel and the VIC detection channel were not significantly different. According to the experim...

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Abstract

The invention discloses a dual detection kit for citrus decay virus and citrus split skin virus and its application. The invention designs a double digital PCR quantitative detection reagent composition for citrus decay virus and citrus cracking skin virus. The kit includes: disease-like RNA extraction reagents, cDNA reverse transcription synthesis reagents, digital PCR reaction reagents, positive control sample templates and negative control sample templates for citrus decay disease and citrus cracking disease. The application of the kit of the present invention to detect citrus decay virus and citrus cracking skin virus has the advantages of strong specificity, better resistance to impurity interference, simplicity and speed, detection sensitivity comparable to existing real-time fluorescent quantitative PCR detection methods, etc., and can be used for citrus decay virus and citrus split skin virus. Early qualitative and quantitative detection and epidemiological investigation of citrus split skin virus.

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 Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
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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