Citrus candidatus liberibacter and citrus canker pathogen dual detection kit and application thereof

A technology of citrus canker bacterium and citrus canker sore, applied in the direction of recombinant DNA technology, microbiological determination/inspection, biochemical equipment and methods, etc., can solve the double digital PCR quantitative detection of citrus canker sore and citrus canker bacterium, etc. problems, to achieve the effects of simple absolute quantitative analysis, strong anti-impurity interference ability, good detection accuracy and reproducibility

Active Publication Date: 2017-02-15
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

[0006] According to the relevant research and patent literature search, there is no re

Method used

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  • Citrus candidatus liberibacter and citrus canker pathogen dual detection kit and application thereof
  • Citrus candidatus liberibacter and citrus canker pathogen dual detection kit and application thereof
  • Citrus candidatus liberibacter and citrus canker pathogen dual detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Primer screening and testing.

[0041] Use the primer primer5 software to detect the complementarity between the primers and the annealing temperature of the primers, and select a primer pair with an appropriate annealing temperature and easy identification of the amplified target fragment. The present invention designs two pairs of primers and probes respectively for citrus huanglongbing bacteria and citrus canker sores.

[0042] This example gives the process of screening the best primers, and the alternative primers used for screening are as follows, see Table 1.

[0043] Table 1 Alternative primer sequences for H. citri and X. citri

[0044]

[0045] Alternative primer and probe combinations include: HLB1-F+HLB1-R+HLB1-P, HLB2-F+HLB2-R+HLB2-P, KYB1-F+KYB1-R+KYB1-P, KYB2- F+KYB2-R+KYB2-P, using probe method fluorescent quantitative PCR for specificity analysis, 20 μL reaction system, Premix Ex Taq buffer 10 μL, template 3 μL, primer final concentratio...

Embodiment 2

[0048] Example 2: Primer specificity analysis

[0049] In order to verify the specificity of the double digital PCR quantitative detection method for citrus Huanglongbing bacteria and X. citrus canker sores of the present invention, HLB1-F+HLB1-R+ HLB1-P, and KYB1-F+KYB1- The primer and probe combination of R+KYB1-P is used to detect Huanglongbing and canker disease samples. Use digital quantitative PCR system formula: 2×3D Digital PCR Master Mix 8.0 μL, 10 μM probes and primers HLB1-P, KYB1-P, HLB1-F, HLB1-R, KYB1-F, KYB1-R, 0.4 μL each , 3 μL of citrus disease-like DNA 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 minutes; extension at 58°C for 2 minutes, and denaturation at 98°C for 30 seconds; a total of 40 cycles were performed, and the reaction was stopped at 10°C. The samples of citrus canker, citrus anthracnose, citrus foot rot, citrus decay, citrus split skin, c...

Embodiment 3

[0052] Example 3: Effects of different annealing temperatures on double digital PCR of Huanglongbing citri and X. citri.

[0053] Use HLB1-F+HLB1-R+HLB1-P, and KYB1-F+KYB1-R+KYB1-P primer and probe combinations. Use digital quantitative PCR system formula: 2×3D Digital PCR Master Mix 8.0 μL, 10 μM probes and primers HLB1-P, KYB1-P, HLB1-F, HLB1-R, KYB1-F, KYB1-R, 0.4 μL each , 3 μL of citrus Huanglongbing and citrus canker composite infection-like DNA template, and ddH for the rest 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, and 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.

[0054] Results: At different annealing temperatures, the detection values ​​in the FAM signal channel and the VIC detection channel were not significantly different. According to t...

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Abstract

The invention discloses a citrus candidatus liberibacter and citrus canker pathogen dual detection kit and application thereof. A dual digital PCR quantitative detection agent composition for the citrus candidatus liberibacter and citrus canker pathogen is designed. The kit comprises a citrus disease sample DNA extraction agent, a digital PCR reaction agent, a citrus liberibacter asiaticus and citrus canker positive control sample template and a citrus liberibacter asiaticus and citrus canker negative control sample template. The kit has the advantages of being high in specificity, higher in impurity interference resistance, simple and fast when used for detecting the citrus candidatus liberibacter and citrus canker pathogen, the detection sensitivity is higher than that of an existing real-time fluorescence quantification PCR detection method by about 10 times, and the kit can be used for early-stage qualitative and quantitative detection of the citrus candidatus liberibacter and citrus canker pathogen and epidemiologic investigation.

Description

technical field [0001] The invention belongs to the technical field of plant disease detection, and in particular relates to a dual detection kit for citrus huanglongbing bacteria and citrus canker bacteria and an application thereof. [0002] technical background [0003] Citrus is one of the most important commercial fruits in the world. my country is the main producing country of citrus. The citrus industry plays an important role in the rural society and economy of our country. In recent years, the incidence of citrus huanglongbing and citrus canker in my country's citrus producing areas has been increasing, and the combined infection of the two is increasing, causing economic losses of more than 10 billion yuan to my country's citrus industry every year. At present, there is still a lack of high-efficiency fungicides and disease-resistant varieties for citrus huanglongbing and canker. Once infected with these two diseases, it is necessary to remove the diseased trees in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/159
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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