Primer combination for detecting succulent plant stem fusarium moniliforme based on LAMP (Loop-mediated Isothermal Amplification) and application thereof

A succulent and primer combination technology, which is applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of long detection time, cumbersome procedures, low accuracy, etc., and achieve reliable results and sensitivity. High and strong specificity

Inactive Publication Date: 2018-12-11
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to detect and identify the succulent stem rot pathogen mainly based on morphological characteristics in the prior art. The method takes a long time, the procedure is cumbersome, the experience is strong, and the accuracy is low. It is difficult to monitor and identify the disease in time. To control the spread and prevalence of pathogenic bacteria, while PCR molecular detection needs to rely on expensive instruments such as amplification instruments, and the detection time is relatively long. A primer combination based on loop-mediated isothermal amplification technology (LAMP) for the detection of succulent stem rot pathogens is provided. And its application, the LAMP detection of succulent stem rot bacteria has the advantages of short detection cycle, high accuracy, high sensitivity, and naked eye observation of the detection results, etc.

Method used

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  • Primer combination for detecting succulent plant stem fusarium moniliforme based on LAMP (Loop-mediated Isothermal Amplification) and application thereof
  • Primer combination for detecting succulent plant stem fusarium moniliforme based on LAMP (Loop-mediated Isothermal Amplification) and application thereof
  • Primer combination for detecting succulent plant stem fusarium moniliforme based on LAMP (Loop-mediated Isothermal Amplification) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Design of specific primer combinations for detection of succulent stem rot by loop-mediated isothermal amplification (LAMP) and verification of primer specificity

[0034] 1. Extraction of genomic DNA of the tested strains

[0035] When used to detect pure cultures of pathogenic bacteria, the CTAB method was used to extract genomic DNA. 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 2 % CTAB (cetyltrimethylammonium bromide) extract (which contains 2% CTAB; 100 mmol·L -1 Tris-HCl, pH 8.0; 20 mmol L -1 EDTA, pH 8.0; 1.4 mol L -1 NaCl) and 90 µL SDS (sodium dodecylbenzenesulfonate) [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 ...

Embodiment 2

[0046] Example 2: Detection Sensitivity of Loop-Mediated Isothermal Amplification (LAMP) for Stem Rot of Succulents

[0047] 1. Preparation of Genomic DNA at Different Concentrations

[0048] The genomic DNA of succulent stem rot fungus was diluted with sterile ultrapure water (DDW), and prepared into a series of 10-fold concentrations for future use.

[0049] Sensitivity determination of detection method and result observation

[0050] Genomic DNA of succulent stem rot fungus at different concentrations was used as a template, and the outer primer F3 / B3 and inner primer FIP / BIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 8.0 mM MgSO 4 , 50 mM KCl, 0.8 mM Betaine, 1.75 mmol L -1 dNTPs, 8 U BstDNA polymerase, 1.6 μmol L -1 FIP and BIP, 0.2 μmol L -1 For F3 and B3, 1 μL template DNA (10 ng·μL -1 , 1 ng·μL -1 , 100 pg·μL -1 , 10 pg·μL -1 , 1 pg·μL -1 , 100 fg·μL -1 , 10 fg·μL -1 ,...

Embodiment 3

[0053] Example 3: LAMP detection of succulent stem rot pathogen in diseased tissue

[0054] Sample collection: Collect the roots and stems of succulent plants with typical symptoms of stem rot disease (2-6 cm from the ground surface) and healthy plant roots and stems from Jiuhu Town, Longhai, Zhangzhou, Fujian and other places, and bring them back to the laboratory for future use.

[0055] Extraction of DNA from plant tissue: DNA was extracted by NaOH rapid cleavage method, the specific process was as follows: Add 10 µL of 0.5 mol L to each mg of plant tissue -1 NaOH, thoroughly grind the tissue into a paste in a mortar, transfer 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 of 0.1 mol·L -1 Tris-HCl (pH=8.0) was mixed evenly, and 1.0 µL was used as a PCR template for amplification.

[0056] Amplification detection and observation: Using the above-mentioned extracted DNA as a template, the outer primer F3 / B3 and ...

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Abstract

The invention provides a primer combination for detecting succulent plant stem fusarium moniliforme based on LAMP (Loop-mediated Isothermal Amplification) and application thereof. The LAMP primer combination for detecting the succulent plant stem fusarium moniliforme consists of 1 pair of outside primers F3/B3 and a pair of inside primer FIP/BIP, wherein the F3/B3 and FIP/BIP primer sequences areshown as SEQ ID NO. 1 to 4. By adopting the detection system disclosed by the invention, the succulent plant stem fusarium moniliforme can be detected rapidly, conveniently and efficiently at high specificity and high sensitively through incubation at the constant temperature of 65 DEG C for 1 hour without complex and expensive instruments, and in-situ detection of the succulent plant stem fusarium moniliforme can be met well. The detection system can be widely applied to early diagnosis of field diseases of establishment units as well as monitoring and identification of viruses, and a reliable technological and theoretical basis is provided for prevention and control of the succulent plant stem fusarium moniliforme.

Description

technical field [0001] The invention belongs to the technical field of disease detection, identification and prevention of horticultural crops, and in particular relates to a primer combination and application thereof for loop-mediated isothermal amplification (Loop-mediated Isothermal Amplification, LAMP) detection of succulent stem rot pathogens, which can be used for succulent stem rot The rapid, sensitive and specific molecular detection of rot pathogen can be used for early diagnosis and monitoring identification of succulent stem rot. Background technique [0002] Succulentsrefers to higher plants with hypertrophied plant vegetative organs, usually with three vegetative organs of roots, stems, and leaves and three reproductive organs of flowers, fruits, and seeds. In gardening, it is also called succulent plant or succulent flower, but the name succulent plant is the most commonly used. Succulents are popular all over the world as potted plants, and most of the common...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/107
Inventor 姚锦爱黄鹏陈汉鑫余德亿
Owner INST OF PLANT PROTECTION FAAS
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