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LAMP (Loop-mediated Isothermal Amplification) primers for rapidly assaying tea tree anthracnose pathogens and assay method

An anthracnose fungus, tea tree technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of long detection time, long method time-consuming, cumbersome procedures, etc., to achieve rapid detection and applicability Good, the effect of shortening the operation time

Active Publication Date: 2018-03-06
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The object of the present invention is to provide a kind of LAMP primer and detection method for rapid detection of tea tree anthracnose bacteria, for the detection and identification of tea tree anthracnose bacteria in the prior art is mainly based on morphological characteristics, the method is time-consuming, cumbersome, empirical and accurate It is difficult to timely monitor the occurrence of diseases and control the spread and prevalence of pathogenic bacteria, and the existing PCR molecular detection needs to rely on expensive instruments such as amplification instruments, and the detection time is long. The tea tree anthracnose bacteria The latest molecular detection method, LAMP detection of tea tree anthracnose bacteria, short detection cycle, high accuracy, high sensitivity, the detection results can be observed with naked eyes

Method used

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  • LAMP (Loop-mediated Isothermal Amplification) primers for rapidly assaying tea tree anthracnose pathogens and assay method
  • LAMP (Loop-mediated Isothermal Amplification) primers for rapidly assaying tea tree anthracnose pathogens and assay method
  • LAMP (Loop-mediated Isothermal Amplification) primers for rapidly assaying tea tree anthracnose pathogens and assay method

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Design of specific primers for the detection of tea tree anthracnose bacteria loop-mediated isothermal amplification (LAMP) and verification of primer specificity

[0032] 1. Extraction of genomic DNA of the tested strains

[0033] The genomic DNA of the tested strain (Table 1) was extracted by the CTAB method. The specific method was as follows: Take a small amount of mycelium powder in a 1.5 mL centrifuge tube (it is better that the mycelium powder just covers the bottom of the semicircle), add 900 µL of 2% CTAB ( cetyltrimethylammonium bromide) extract (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA, pH 8.0; 1.4 mol / LNaCl) and 90 µL SDS (ten Sodium dialkylbenzene sulfonate) [Note: CTAB, SDS needs to be preheated at 60°C], use a shaker to shake and mix, 60°C water bath for 1h (DNA is released into the buffer), 12000 r min -1 Centrifuge for 15 min; take 700 µL of the supernatant, add an equal volume of phenol, chloroform, isoamyl alcohol (25:24:1), sh...

Embodiment 2

[0045] Example 2: Determination of detection sensitivity of tea plant anthracnose bacteria loop-mediated isothermal amplification (LAMP)

[0046] 1. Preparation of genomic DNA at different concentrations

[0047] Dilute the genomic DNA of tea plant anthracnose bacteria with sterile ultrapure water, and prepare a series concentration of 10 times order of magnitude for subsequent use;

[0048] 2. Sensitivity determination and result observation of LAMP detection method

[0049] Using different concentrations of tea tree anthracnose bacterial genomic DNA as a template, the outer primers F3 / B3 and inner primers FIP / BIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including 5 μM outer primers F3 and 1.0 μL B3, 40 μM internal primers FIP and BIP each 1.0 μL, LAMP reaction mixture [40 mM Tris-HCl, 20 mM (NH 4 ) 2 SO 4 , 20 mM KCl, 16 mM MgSO 4 , 1.6 mol / L Betaine (Betaine), 2.0 mM dNTPs, 0.2% Trion X-100] 12.5 μL, 8 U Bst Polymerase 1.0 μL, D...

Embodiment 3

[0052] Example 3: LAMP detection of tea tree anthracnose bacteria in diseased tissues

[0053] Sample collection: Collect tea tree leaves with typical symptoms of anthracnose and healthy leaves from Fu'an, Wuyishan, and Shaxian County in Fujian and bring them back to the laboratory for later use;

[0054] Plant tissue DNA extraction: DNA was extracted by NaOH rapid cleavage method, the specific process is as follows: add 10µL 0.5 mol / L NaOH to each mg of plant tissue, fully grind the tissue into a paste in a mortar and transfer it to a 1.5mL centrifuge tube centrifuge at 12,000 rpm for 6 min, take 5 µL of the supernatant and add 495 µL 0.1 mol / L Tris-HCl (pH=8.0) to mix well, and take 1.0 µL as a PCR template for amplification.

[0055] LAMP amplification detection and observation: Using the above-mentioned extracted DNA as a template, use the outer primer F3 / B3 and the inner primer FIP / BIP to perform LAMP amplification. The LAMP detection reaction system is 25 μL, including 5...

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Abstract

The invention provides LAMP (Loop-mediated Isothermal Amplification) primers for rapidly assaying tea tree anthracnose pathogens and an assay method. Aimed at the beta-tubulin(TUB2) gene sequence of tea tree anthracnose pathogens, the invention designs and screens out a set of specific assay primer group, and the primer group consists of four specific primers F3, B3, FIP and BIP. The primers screened out by the invention have high sensitivity and strong specificity, the established assay method ha the advantages of high accuracy, strong specificity, easiness in operation, short assay time, simple instruments and equipment and the like, and is applicable to the high-sensitivity rapid assay, identification and early disease diagnosis of tea tree anthracnose pathogens in diseased tissues andpathogen-carrying plants, and for the early monitoring of tea tree anthracnose in agricultural production, the invention is of great significance for the determination of optimal prevention and control time.

Description

technical field [0001] The invention belongs to the field of detection, identification and prevention of crop diseases, and in particular relates to a detection primer and detection method of tea anthracnose pathogen LAMP and its application in early diagnosis of tea anthracnose, monitoring and identification of the pathogen. Background technique [0002] As one of the three major non-alcoholic beverages, tea contains a variety of chemical components that are beneficial to the human body, which can effectively reduce the chance of human disease, and is deeply loved by consumers all over the world. my country is a big tea producing country and also a big tea exporting country. The production of tea is very important in the economic development of our country. At present, China has 20 provinces (municipalities, autonomous regions) producing tea, with an economic output value of 2.6 billion U.S. dollars. Tea trees ( Camellia sinensis ) has become one of the important economic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 兰成忠游泳阮宏椿吴玮姚锦爱
Owner INST OF PLANT PROTECTION FAAS
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