Banana wilt bacterium molecule detecting genes and detecting method thereof

A technology for the detection of Fusarium wilt of banana and molecular detection, which can be used in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., and can solve the problems of long cycle, poor specificity and low sensitivity.

Inactive Publication Date: 2008-06-25
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a banana fusarium wilt biological detection method in the prior art for the required cycle long, poor specificity, low sensitivity Specific molecular detection gene of Fusarium wilt and rapid detection and diagnosis method of Fusarium wilt of banana with reliable results, easy operation and high sensitivity

Method used

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  • Banana wilt bacterium molecule detecting genes and detecting method thereof
  • Banana wilt bacterium molecule detecting genes and detecting method thereof
  • Banana wilt bacterium molecule detecting genes and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] PCR Amplification of Specific Primers FOC-F / FOC-R of Fusarium wilt of Banana.

[0061] A detection kit for detecting genes of Fusarium wilt of banana, including the following components:

[0062] Its specific primer and its sequence are:

[0063] FOC-F: 5’-ATA TGA ATG ACT CGT GGC ACG-3’

[0064] FOC-R: 5’-GCT GGG AAT GCG ACG GTA T-3’

[0065] Its kit PCR reaction system 25μl, including 1 times PCR reaction buffer, 1.5mM MgCl 2 , 0.2mMdNTPs, 1.5U Taq DNA polymerase, primer FOC-F / FOC-R each 50pmol and 10ng template DNA, d.d.H 2 O to make up 25 μl. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 64°C for 30 sec, extension at 72°C for 30 sec, and a total of 30 cycles; extension at 72°C for 10 min.

[0066] Specificity of detection: In addition to the 364bp product that can be specifically amplified from the banana Fusarium wilt strains from Hainan, Guangdong and Fujian provinces where Fusari...

Embodiment 2

[0072] Detection of Fusarium wilt in diseased plant tissues.

[0073] Take the diseased tissue of banana wilt and the banana tissue of artificial inoculation to extract DNA by CTAB method, and take 1 μl of DNA to carry out PCR amplification according to the method implemented by the above kit. The PCR reaction system of the kit is 25 μl, including 1 times of PCR reaction buffer solution, 1.5mM MgCl 2 , 0.2mM dNTPs, 1.5U Taq DNA polymerase, primer FOC-F / FOC-R each 50pmol and 10ng template DNA, d.d.H 2 O to make up 25 μl. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 64°C for 30 sec, extension at 72°C for 30 sec, and a total of 30 cycles; extension at 72°C for 10 min. The amplified product was detected by electrophoresis, and the results were shown in image 3 , a clear specific band with a molecular weight of 364bp was seen, so it was judged that both the pathogenic tissue of banana wilt and the...

Embodiment 3

[0075] Detection of Fusarium wilt in diseased soil samples.

[0076] 1) DNA extraction from diseased soil samples:

[0077] 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 1.5ml centrifuge tubes, add 500μl 0.4% skimmed milk powder solution to each tube, Vortex to mix. Centrifuge at 12000rpm for 15min. 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 1-3h. After the water bath, add 1 / 2 volume of 7.5M NH 4 AC solution, mix up and down. Centrifuge at 12000rpm for 15min. Aspirate the supernatant and add 2 times the volume of absolute ethanol to precipitate at -20°C (precipitation time 1.5h). After the precipitation, centrifuge at 12000rpm for 15min. The precipitate was washed with 70% ethanol, poured off, and dried at room temperature. Th...

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Abstract

The invention discloses a fusarium oxysporum f.sp.cubense molecular detecting gene and detecting method thereof, which is especially applicable to high sensitive quick molecular detection of fusarium oxysporum f.sp.cubense. The detecting gene is a specific 404bp sequence of the fusarium oxysporum f.sp.cubense, and homologous sequences are not found in a Genbank database. A pair of primers FOC-F / FOC-R is designed to specifically amplify 364bp specific amplified products on pure DNA of the fusarium oxysporum f.sp.cubense, germ-carrying tissues and soils. The invented detecting gene and primers can be used for detecting the fusarium oxysporum f.sp.cubense in banana germ carrying tissues and soils quickly, sensitively and specifically in productive practice; meanwhile, can be used for the early diagnosis for field fusarium oxysporum f.sp.cubense and the monitoring and identification of germs.

Description

Technical field: [0001] The invention relates to a molecular detection gene of Fusarium wilt of banana and its detection method, which is specially used for high-sensitivity and rapid molecular detection of Fusarium wilt of banana, and can be used for early diagnosis of Fusarium wilt of banana in the field and monitoring and identification of pathogens, and belongs to the control and identification of crop diseases and plant diseases. The field of quarantine technology. Background technique: [0002] Fusarium oxysporum f.sp.cubense is caused by Fusarium oxysporum f.sp.cubense, and it is a three-category quarantine disease in my country. The disease was first reported in Hawaii, USA in 1904, and it caused heavy losses in Panama in 1910. At present, the disease is widely distributed in banana countries in the world (Stover, R.H. 1962. Fusarial Wilt (PanamaDisease) of Bananas and Other Musa Species. CMI, Kew, Surrey, UK; Koening et al. Fusariumoxysporum f.sp. Cubense consists ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12N15/31
Inventor 陈庆河翁启勇赵健李本金兰成忠邱荣洲吕新
Owner INST OF PLANT PROTECTION FAAS
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