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Double detection kit for citrus anthracnose and citrus foot rot and its application

A citrus anthracnose, double detection technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial determination/inspection, etc. To achieve the effect of simple absolute quantitative analysis, strong resistance to impurity interference, 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

AI Technical Summary

Problems solved by technology

[0005] According to the relevant research and patent literature retrieved, there is no relevant literature report on the dual digital PCR quantitative detection of citrus anthracnose and citrus foot rot

Method used

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  • Double detection kit for citrus anthracnose and citrus foot rot and its application
  • Double detection kit for citrus anthracnose and citrus foot rot and its application
  • Double detection kit for citrus anthracnose and citrus foot rot and its application

Examples

Experimental program
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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 the citrus anthracnose fungus and the citrus foot rot fungus.

[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 P. citrus anthracnose and P. citrus foot rot

[0044]

[0045] Alternative primer and probe combinations include: TJB 1-F+ TJB 1-R+ TJB 1-P, TJB 2-F+ TJB2-R+ TJB 2-P, JFB1-F+ JFB 1-R+ JFB 1-P, JFB 2-F+ JFB 2-R+ JFB2-P, using probe method fluorescent quantitative PCR for specificity analysis, 20 μL reaction system, Premix Ex Taq buffer 1...

Embodiment 2

[0048] Example 2: Primer specificity analysis

[0049] In order to verify the specificity of the quantitative detection method of the present invention to the detection of citrus anthracnose bacteria and citrus foot rot bacteria, use the primers of TJB 1-F+ TJB 1-R+ TJB 1-P and JFB1-F+ JFB 1-R+ JFB 1-P respectively Combined with the probe to detect citrus anthracnose and citrus foot rot disease samples. Use digital quantitative PCR system formula: 2×3D Digital PCRMaster Mix 8.0μL, 10μM probes and primers TJB 1-F, TJB 1-R, TJB 1-P, JFB1-F, JFB 1-R, JFB1-P each 0.2 μL, 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 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 citrus canker disease, citrus anthracnose, citrus foot rot, citrus decay, ci...

Embodiment 3

[0052] Example 3: Effects of different annealing temperatures on double digital PCR reactions of citrus anthracnose and citrus foot rot.

[0053] Primer and probe combinations of TJB 1-F+ TJB 1-R+ TJB 1-P, and JFB1-F+ JFB 1-R+ JFB 1-P were used. Use digital quantitative PCR system formula: 2×3D Digital PCR Master Mix 8.0μL, 10μM probes and primers TJB 1-F, TJB 1-R, TJB 1-P, JFB1-F, JFB 1-R, JFB 1- 0.2 μL for each of P, 3 μL of DNA templates for composite infection of citrus anthracnose and citrus foot rot, and 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, 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 or VIC detection channel had little differenc...

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Abstract

The invention discloses a dual detection kit for citrus anthracnose bacteria and citrus foot rot bacteria and an application thereof. The invention designs a double digital PCR quantitative detection reagent composition for citrus anthracnose bacteria and citrus foot rot bacteria. The kit contains: citrus disease-like DNA extraction reagents, digital PCR reaction reagents, citrus anthracnose and citrus foot rot positive control sample templates and negative control sample templates. The detection of citrus anthracnose bacteria and citrus foot rot by using the kit of the present invention has the advantages of strong specificity, better anti-impurity interference, simplicity and speed, detection sensitivity comparable to real-time fluorescence quantitative PCR detection method, etc., and can be used for citrus anthracnose bacteria and citrus foot rot Early qualitative and quantitative detection of pathogens and epidemiological investigations.

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 anthracnose and citrus foot rot and an application thereof. technical background [0002] Citrus anthracnose and citrus foot rot are two important diseases on citrus, and their pathogens are all mycelial pathogens. Colletotrichum gloeosporioides Anthracnose is a fungus of the genus Anthracnose, which can not only cause disease in various parts of the above-ground parts of citrus, cause citrus trees to weaken and reduce production and income, but also cause a large number of fruits to rot during storage. Phytophthora capsici Phytophthora oomycetes can cause symptoms such as citrus stem base rot, glue flow, leaf spot, stem dieback and root rot. Recent surveys have found that the incidence of these two pathogenic bacteria in citrus producing areas in my country has been increasing (B. P. Cheng et al. 2014, PlantDisease;...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12Q1/06C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/16C12Q2537/143C12Q2563/107
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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