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LAMP primer for rapidly detecting apple brown spot bacteria, and detection method and kit thereof

A technology of apple brown spot fungus and detection method, which is applied in the field of LAMP primers and its detection for rapid detection of apple brown spot fungus, can solve the problems of low accuracy, strong experience, cumbersome procedures, etc., and achieve specificity improvement and high sensitivity , the effect of simple operation

Inactive Publication Date: 2020-05-05
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the shortcomings of the prior art detection methods for brown spot pathogen of apples, such as time-consuming, cumbersome procedures, strong experience, and low accuracy, the present invention provides LAMP primers for rapid detection of brown spot pathogen of apples, a detection method and a kit thereof, The detection method has fast detection speed, simple operation, strong specificity, high sensitivity, and accurate and reliable results.

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  • LAMP primer for rapidly detecting apple brown spot bacteria, and detection method and kit thereof
  • LAMP primer for rapidly detecting apple brown spot bacteria, and detection method and kit thereof
  • LAMP primer for rapidly detecting apple brown spot bacteria, and detection method and kit thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1 Primer Design and Synthesis

[0049] According to the specificity of the ITS (Internal Transcribed Spacer) sequence (the sequence is shown in SEQ ID No.5 in the sequence listing) of apple brown spot fungus (Marssonina coronaria), the LAMP primer online design software PrimerExplorer (http: / / primerexplorer.jp / lampv5e / index .html) Design a set of LAMP primers specific to P. spp. and carry out sequence synthesis. The LAMP primers include outer primers F3 and B3, inner primers FIP and BIP, and the primer sequences are respectively:

[0050] F3: 5'-GTGAATTTTCTCCAAAAAAAATGGT-3'; (SEQ ID No.1)

[0051] B3: 5'-ATTGCGCGTGTTTGTTTG-3'; (SEQ ID No.2)

[0052] FIP: 5'-AGGTGATACAAAGAGGCTATGTCAT-TGTATGAAAAAACCTTTTTGCG-3'; (SEQ ID No. 3)

[0053] BIP: 5'-AATGTATGAGTGGATGAGTGTCAG-CAGATGACAAAAGCACAAGA-3'; (SEQ ID No. 4);

[0054] The primers synthesized above were diluted with sterilized double-distilled water to 10 μmol / L respectively, and the primers were stored in a -20°C...

Embodiment 2

[0055] The LAMP detection of embodiment 2 apple brown spot bacteria

[0056] 1. Extraction of Genomic DNA of Apple Brown Spot Bacteria to be Tested

[0057] Take a small amount of sample to be tested in a centrifuge tube containing 400 μL of DNA lysate (0.15M Tris-HCl, 0.04M EDTA-Na2, 0.2M NaCl, 3mM SDS, PH=8.0), add a steel ball, shake at 65Hz for 1min, and centrifuge at 12000rpm for 2min , take 200 μL of the supernatant, add 400 μL of absolute ethanol, let stand at -20°C for 30 minutes, centrifuge at 12000 rpm for 2 minutes, discard the supernatant, dry it in the air, and add 30 μL of sterile double-distilled water to dissolve the DNA.

[0058] 2. Establishment of LAMP reaction detection system:

[0059] Using the genomic DNA of the sample to be tested extracted above as a template, use primers F3, B3, FIP, and BIP for LAMP amplification. The LAMP detection reaction system takes 26 μL as an example. The specific reaction system includes:

[0060] 10 μM F3: 0.5 μL

[0061]...

Embodiment 3

[0076] The specificity test of embodiment 3LAMP primer

[0077] Establish the LAMP reaction detection system according to the method for embodiment 2, simultaneously to apple brown spot fungus (Marssonina coronaria), apple rot fungus (Valsa mali), apple ring spot fungus (Botryosphaeria dothidea), apple anthracnose fungus (Colletotrichumgloeosporioides), apple spotted leaf fall bacterium ( Alternaria mali), Sclerotium rolfsii, and Venturia inaequalis were tested by LAMP, and they were numbered a1-a7 respectively. Simultaneous experiments were carried out using sterile double distilled water as a negative control template. .

[0078] Test result: if figure 1 As shown, only the reaction product in the reaction tube of sample a1 (Apple brown spot bacteria) is sky blue, and the electrophoresis result is a trapezoidal band, and the detection shows a positive result; the detection results of the other 6 samples and the negative control are all negative. The result shows that the 3 ...

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Abstract

The invention discloses a loop-mediated isothermal amplification (LAMP) primer for rapidly detecting apple brown spot bacteria, and a detection method and kit thereof. The LAMP primer consists of a pair of outer primers F3 and B3 and a pair of inner primers FIP and BIP. The forward outer primer F3 is shown as SEQ ID No.1, the reverse outer primer B3 is shown as SEQ ID No.2, the forward inner primer FIP is shown as SEQ ID No.3, and the reverse inner primer BIP is shown as SEQ ID No.4. Through adoption of the two pairs of specific LAMP primers for carrying out LAMP amplification on a sample to be detected, rapid detection of apple brown spot bacteria can be realized. The technology has the advantages of simple operation, high sensitivity and specificity and high detection speed, does not need expensive instruments and equipment, and is suitable for early diagnosis of apple brown spot in fields and detection and identification of pathogenic bacteria.

Description

technical field [0001] The invention relates to the technical field of methods for detecting apple brown spot bacteria, in particular to LAMP primers for rapid detection of apple brown spot bacteria, a detection method and a kit. Background technique [0002] As the saying goes "an apple a day keeps the doctor away". Apple is sweet and sour, delicious and nutritious, and it is an indispensable nutritious fruit in people's daily life. At present, my country has become the world's largest apple producer, with apple planting area and output accounting for more than 50% of the world. Apple brown spot, also known as green-edge brown spot, is caused by Marssonina coronaria, which occurs in every apple planting area in my country, and is the main disease that causes early leaf defoliation of fruit trees. In the years when the apple brown spot disease is serious, the disease can cause early leaf fall of fruit trees, tree vigor is weak, fruits cannot be matured normally, and fruit ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6844
Inventor 任维超刘娜李保华练森王彩霞
Owner QINGDAO AGRI UNIV
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