Method for rapidly detecting botryosphaeria dothidea based on LAMP (Loop-mediated Isothermal Amplification)

A technology for detection of ringworm bacteria and a detection method, which is applied in the field of LAMP detection primers for apple ringworm bacteria, can solve the problems of cumbersome procedures, low accuracy, and long detection time, and achieve the highest specificity and sensitivity, strong specificity, reliable results

Pending Publication Date: 2018-11-06
SHANDONG YANTAI AGRI SCI & TECH INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to detect and identify apple ringworm in the prior art based mainly on morphological features. The method is time-consuming, cumbersome, empirical, and has low accuracy. It is difficult to monitor and control the occurrence of the disease in time. The transmission 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 relatively long. A method for detecting apple ringworm LAMP technology and a primer composition used are provided. The detection method of the present invention is easy to operate, has strong specificity, high sensitivity, and accurate and reliable results

Method used

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  • Method for rapidly detecting botryosphaeria dothidea based on LAMP (Loop-mediated Isothermal Amplification)
  • Method for rapidly detecting botryosphaeria dothidea based on LAMP (Loop-mediated Isothermal Amplification)
  • Method for rapidly detecting botryosphaeria dothidea based on LAMP (Loop-mediated Isothermal Amplification)

Examples

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Embodiment 1

[0035] Example 1: Design of specific primer composition for detection of ring rot bacterium apple ring mediated isothermal amplification (LAMP) and the specificity verification of primers for ring rot bacterium apple.

[0036] 1. Extraction of genomic DNA of the tested strains

[0037] Genomic DNA of the tested strains (apple ringworm, apple rot fungus, apple anthracnose, apple mold heart fungus) was extracted by CTAB method, the specific method is as follows: Take a small amount of mycelium powder in a 1.5 mL centrifuge tube ( It is better to cover the bottom of the semicircle), add 900 μL 2% CTAB (hexadecyltrimethylammonium bromide) extract (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA, pH 8.0; 1.4 mol / L NaCl) and 90 μL SDS (sodium dodecylbenzenesulfonate) [Note: CTAB, SDS needs to be preheated at 60°C], use a shaker to shake and mix well, and bathe in 60°C water for 20 min (DNA released to buffer), centrifuge at 12000 r / min for 15 min; take 700 μL of supernatant, a...

Embodiment 2

[0050] Embodiment 2: Detection Sensitivity of Apple Ringworm Loop-Mediated Isothermal Amplification (LAMP)

[0051] 1. Preparation of genomic DNA at different concentrations

[0052] Dilute the genomic DNA of apple ringworm with sterile ultrapure water, and prepare a series of concentrations of 10 times the order of magnitude for later use;

[0053] 2. Sensitivity determination and result observation of LAMP detection method

[0054] Using different concentrations of the apple ringworm genomic DNA as a template, the primer composition F3, B3, FIP, BIP, LF, LB was used for LAMP amplification, and the LAMP detection reaction system included 12.5 μL of 2X LAMP Master Mix reaction mixture, 8 U Bst DNA polymerase 0.5 μL, DNA template 1.0 μL, made up to 25 μL with sterilized ultrapure water; LAMP reaction conditions: incubate at 65°C for 60 min, inactivate at 85°C for 10 min; determination of reaction results: visual observation with fluorescent dye or agarose gel electrophoresi...

Embodiment 3

[0057] Embodiment 3: the LAMP detection of apple ringworm bacteria in diseased tissues

[0058] Sample collection: The branches with typical symptoms of apple ring spot and healthy branches were collected from the experimental field of Yantai Academy of Agricultural Sciences and brought back to the laboratory for future use. Branch DNA extraction: DNA was extracted by NaOH rapid cracking method, and the specific process was as follows: Add 10 μL 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 1.5 mL 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.

[0059] Amplification detection and observation: Using the above-mentioned extracted DNA as a template, use the primer composition F3, B3, FIP, BIP, LF, LB for LAMP amplification, the LAMP detection reaction system is 25 μL, inclu...

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Abstract

The invention discloses botryosphaeria dothidea LAMP (Loop-mediated Isothermal Amplification) detection primers and application thereof. A primer composition for detecting botryosphaeria dothidea based on an LAMP technology consists of 6 specific primers, namely F3, B3, FIP, BIP, LF and LB. The invention further provides a botryosphaeria dothidea LAMP detection method. The botryosphaeria dothideaLAMP detection method comprises the following steps: 1), extracting DNA of a sample to be detected; 2), performing loop-mediated isothermal amplification on the DNA of the sample to be detected by using the primer composition provided by the invention; 3), performing result detection, wherein when a chromogenic result is green fluorescence or a trapezoidal electrophoretic band appears, a positiveresult is determined, and when a chromogenic result is orange or orange yellow or no trapezoidal electrophoretic band appears, a negative result is determined. A novel molecular detection method and the novel primer composition are provided for the botryosphaeria dothidea, the botryosphaeria dothidea can be detected quickly, conveniently and efficiently at high specificity and high sensitivity, and a reliable technological and theoretical basis is provided for early diagnosis, detection and rapid import and export inspection of the botryosphaeria dothidea.

Description

technical field [0001] The invention belongs to the technical field of detection, identification and prevention of crop diseases, and in particular relates to a detection primer of apple ringworm LAMP and an application thereof. It can be used for rapid, sensitive and specific molecular detection of apple ring rot, and can be used for early diagnosis of apple ring rot and monitoring and identification of the bacterium. Background technique [0002] apple( Malus sieversii (Led.) Roem. ) fruit is rich in nutrients and rich in zinc. It is known as "wisdom fruit" and "memory fruit". Apples are also rich in nutrients such as protein, polyphenols, and vitamin C. Not only that, apples have a fragrant smell and a sweet taste, which is a high economic value crop loved by producers and consumers. [0003] Apple ring disease ( Botryosphaeria dothidea ) has become an important factor restricting the development of the industry in the high-yield and high-quality cultivation of apples...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 汪少丽王英姿王培松刘保友栾炳辉
Owner SHANDONG YANTAI AGRI SCI & TECH INST
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