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LAMP (loop-mediated isothermal amplification) detection primers of banana fusarium wilt bacteria No. 4 microspecies and application thereof

A technology for detecting Fusarium wilt of banana and a primer is applied in the field of molecular biology detection of plant diseases, which can solve the problems of no report on Fusarium wilt of banana, and achieve the effects of guaranteed reliability of results, high specificity and simple operation.

Inactive Publication Date: 2012-09-26
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, LAMP detection is mainly used in the detection of human and animal pathogens, food safety and environmental sanitation. There are very few reports in the detection of plant pathogens, and there are no reports on the detection of Fusarium wilt of bananas at home and abroad.

Method used

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  • LAMP (loop-mediated isothermal amplification) detection primers of banana fusarium wilt bacteria No. 4 microspecies and application thereof
  • LAMP (loop-mediated isothermal amplification) detection primers of banana fusarium wilt bacteria No. 4 microspecies and application thereof
  • LAMP (loop-mediated isothermal amplification) detection primers of banana fusarium wilt bacteria No. 4 microspecies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The present invention will be further described below in conjunction with the embodiments of the accompanying drawings.

[0059] LAMP-specific detection primers for Fusarium wilt race 4:

[0060] F3: 5'-AGGACCTCTTCGAATGGCA-3',

[0061] B3: 5'-GACGCTGCAGCTATGACAA-3',

[0062] FIP: 5'-GGTGGCTCAATAGCCCAGTGAACCGATACCTGTGAAGTCGC-3',

[0063] BIP: 5'-CGACATCATCAGCATCTCCGCTAGCTTTGGCTCTTGTGACAG-3'.

[0064] Banana Fusarium wilt race 4 specific LAMP detection kit, including:

[0065] 1) Primer mixture: 5 pmol each of the outer primers F3 and B3, and 40 pmol each of the inner primers FIP and BIP;

[0066] 2) Reaction mixture: 40mM Tris-HCL, 20mM (NH 4 ) 2 SO 4 , 20 mM KCl, 16 mM MgSO 4 , 0.2% Triton X-100, 1.6M Betaine, 2.8 mM dNTPs;

[0067] 3) 1.0U Bst DNA polymerase.

[0068] The kit also includes a chromogen, which is SYBR green Ⅰ.

[0069] The kit also includes reagents for DNA extraction from diseased banana wilt plant tissue or soil, consisting of 2% CTAB, 10...

Embodiment 2

[0083] Detection of Fusarium wilt race 4 in diseased plant tissues.

[0084] 1) DNA extraction from diseased tissue

[0085] The DNA of Fusarium wilt in tissues was extracted by CTAB method. The specific method is as follows: the diseased plant tissue is ground, 50 mg of the ground plant tissue is taken in a 1.5 ml centrifuge tube, and 900 μl of 2% CTAB (cetyltrimethylammonium bromide) extract is added (the formula of the extract is : 2% CTAB; 100 mmol / L Tris-HCl (tris-hydroxymethylaminomethane hydrochloride), pH 8.0; 20mmol / L EDTA (disodium ethylenediaminetetraacetic acid), pH 8.0; 1.4 mol / L NaCl) and 90 μl 10% SDS (sodium dodecylbenzene sulfonate) and mix well, put in a water bath at 55-60°C for 1.5 h, oscillate and mix once every 10 min, centrifuge (12,000 rpm) for 15 min after 1.5 h in water bath, and take Add the same volume of phenol / chloroform / isoamyl alcohol to the supernatant (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), centrifuge (12,000...

Embodiment 3

[0091] Detection of Fusarium wilt race 4 in diseased soil samples.

[0092] 1) DNA extraction from diseased soil samples

[0093] Take the sieved soil, freeze and dry it for 24-48 hours, add a small amount of quartz sand, pour liquid nitrogen into it and grind it thoroughly, divide the ground soil fine powder into 1.5 ml centrifuge tubes, add 500 μl 0.4% skimmed milk powder solution to each tube , vortex to mix. Centrifuge at 12000 rpm for 15 min. Take the supernatant and add an equal volume of proteinase K buffer to a final concentration of 10 μg / ml proteinase K, and bathe in water at 55°C for 1-3 h. After the water bath, add 1 / 2 volume of 7.5 M NH 4 AC solution, mix up and down. Centrifuge at 12000 rpm for 15 min. Aspirate the supernatant and add 2 times the volume of absolute ethanol to precipitate at -20°C (precipitation time 1.5 h). After the precipitation, centrifuge at 12000 rpm for 15 min. The precipitate was washed with 70% ethanol, poured off, and dried at roo...

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Abstract

The invention belongs to the technical field of biotechnology, and particularly relates to LAMP (loop-mediated isothermal amplification) detection primers of banana fusarium wilt bacteria No. 4 microspecies and an application thereof. The LAMP detection primers of the banana fusarium wilt bacteria No. 4 microspecies in a reaction system are F3, B3, FIP, BIF, F-loop and B-loop, the DNA of the banana fusarium wilt bacteria in an ill plant tissue is extracted, and LAMP isothermal amplification detection is carried out, so that the banana fusarium wilt bacteria can be quickly and specifically detected. The LAMP detection primers of the banana fusarium wilt bacteria No. 4 microspecies disclosed by the invention have the advantages of strong specificity, high sensibility, speediness, high efficiency and easiness in operation, the result of the detection method is reliable, and the primers have high application values in the aspects of on-site rapid detection and early diagnosis of the banana fusarium wilt bacteria.

Description

technical field [0001] The invention belongs to the field of molecular biology detection of plant diseases, and in particular relates to a LAMP detection primer for No. 4 race of Fusarium wilt of banana and application thereof. Background technique [0002] by Fusarium oxysporum Cuban specification ( Fusarium oxysporum f.sp. cubes ) caused by banana fusarium wilt is a devastating soil-borne disease, and it is also one of the import quarantine targets in my country. The disease was first reported in Hawaii, USA in 1904, and caused heavy losses in Panama in 1910. At present, the disease has been widely distributed in banana countries in the world. In mainland my country, the disease was first discovered on Saigon bananas in Guangxi in 1960, and soon the disease occurred in various important focal areas in China. In recent years, banana fusarium wilt has also been found in Fujian, and there is a tendency to continue to spread. The sources of infection are mainly fungal sucke...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 陈庆河李本金翁启勇兰成忠
Owner INST OF PLANT PROTECTION FAAS
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