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Lamp primer composition and application thereof for detecting cucumber wilt

A technology of cucumber fusarium wilt bacteria and primer composition, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of cumbersome procedures, low accuracy, and long detection time, and achieve amplification Good effect, strong specificity and high sensitivity

Active Publication Date: 2019-10-01
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to detect and identify cucumber wilt pathogen mainly based on morphological characteristics in the prior art. The method is time-consuming, complicated procedures, strong experience, low accuracy, and it is difficult to monitor and control the pathogenic bacteria in time for the occurrence of the disease. The spread and prevalence of the problem, and the existing PCR molecular detection needs to rely on expensive instruments such as amplification instruments, and the detection time is long. A new molecular detection method for cucumber fusarium wilt is provided, and LAMP detection is performed on cucumber fusarium wilt. Short cycle, high accuracy, high sensitivity, visual inspection results

Method used

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  • Lamp primer composition and application thereof for detecting cucumber wilt
  • Lamp primer composition and application thereof for detecting cucumber wilt
  • Lamp primer composition and application thereof for detecting cucumber wilt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Design of specific primer composition and primer specificity verification for the detection of Cucumber Fusarium wilt by loop-mediated isothermal amplification (LAMP)

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

[0035] The genomic DNA of the tested strains (Table 1) was extracted by the CTAB method. The specific method is as follows: Take a small amount of mycelium powder in a 1.5mL 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),...

Embodiment 2

[0049] Example 2: Detection Sensitivity of Cucumber Fusarium Wilt Loop-Mediated Isothermal Amplification (LAMP)

[0050] 1. Preparation of genomic DNA at different concentrations

[0051] Dilute the genomic DNA of Fusarium wilt of cucumber with sterile ultrapure water, and prepare a series concentration of 10 times of magnitude for future use;

[0052] 2. Sensitivity determination and result observation of LAMP detection method

[0053] Using different concentrations of Fusarium wilt genomic DNA as a template, the outer primer F3 / B3 and inner primer FIP / BIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including 5 μM outer primers F3 and B3, 1.0 μL each, 40 μM 1.0 μL of each inner primer FIP and BIP, 12.5 μL of LAMP reaction mixture, 8 U Bst 1.0 μL of polymerase, 1.0 μL of DNA templates with different concentrations, made up to 25 μL with sterilized ultrapure water; LAMP reaction conditions: incubate at 63 ℃ for 60 min; determination of react...

Embodiment 3

[0056] Example 3: LAMP detection of Fusarium wilt pathogen in diseased tissues

[0057] Sample collection: collect typical roots and stems (4-6cm from the ground surface) and healthy roots and stems of cucumber wilt disease symptoms from Fuzhou, Xiapu, and Fuan in Fujian and bring them back to the laboratory for later use;

[0058] 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 of 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 outer primer F3 / B3 and inner primer FIP / BIP for LAMP amplification, the LAMP detection reaction system is 25 μL, i...

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Abstract

The invention discloses a LAMP primer composition for detecting fusarium wilt of cucumbers (Fusarium oxysporum f. sp. cucumerinum) and application of the LAMP primer composition. The LAMP primer composition for detecting fusarium wilt of cucumbers consists of a pair of outer side primers F3 / B3 and a pair of inner side primers FIP / BIP. The invention further provides a LAMP detecting method for fusarium wilt of cucumbers by using the primers. The method comprises the following specific steps: (1) extracting a to-be-detected sample genome DNA; (2) establishing a LAMP reaction system; and (3) detecting results. The LAMP reaction system is implemented at the isothermal condition of 63 DEG C, and the fusarium wilt of cucumbers can be rapidly, conveniently and efficiently detected at high specificity and high sensitivity by incubation for 1 hour. A complex instrument is not required, field detection on the fusarium wilt of cucumbers can be met well, the LAMP primer composition for detecting the fusarium wilt of cucumbers can be used for early diagnosis of field diseases and monitoring and identifying of germs, and a reliable technology and theoretical basis are provided for prevention and treatment of the fusarium wilt of cucumbers.

Description

technical field [0001] The invention belongs to the technical field of crop disease detection, identification and prevention, in particular to a LAMP primer composition for detecting cucumber wilt pathogen and its application, which can be used for rapid, sensitive and specific molecular detection of cucumber wilt pathogen, and can be used for cucumber at the same time Early diagnosis of Fusarium wilt and monitoring and identification of pathogens. Background technique [0002] by Cucumber wilt fungus ( Fusarium oxysporum f. sp. cucumerinum Cucumber wilt caused by Owen), also known as wilting disease, vine cutting disease, dead seedling disease, is a systemic disease that is transmitted by soil, invades from roots or rhizomes, and parasitizes in vascular bundles. It is a relatively common disease in cucumber production. It is one of the diseases that are difficult to control. Due to the characteristics of rapid onset, severe disease, and rapid spread, the disease often cau...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
Inventor 兰成忠吴玮姚锦爱阮宏椿
Owner INST OF PLANT PROTECTION FAAS
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