A high-throughput detection method for carbendazim-resistant strains of Fusarium graminearum based on liquid-phase chip technology and its application

A technology of Fusarium graminearum and drug resistance, which is applied to the detection primer and probe combination of Fusarium graminearum to carbendazim-resistant strains and the field of detection, and achieves high sensitivity, high throughput, and reduced error. Effect

Inactive Publication Date: 2017-06-09
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
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Problems solved by technology

However, there is no report on the detection of pathogen resistance

Method used

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  • A high-throughput detection method for carbendazim-resistant strains of Fusarium graminearum based on liquid-phase chip technology and its application
  • A high-throughput detection method for carbendazim-resistant strains of Fusarium graminearum based on liquid-phase chip technology and its application
  • A high-throughput detection method for carbendazim-resistant strains of Fusarium graminearum based on liquid-phase chip technology and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Implementation example 1: Specificity experiment for detection of drug-resistant Fusarium graminearum by liquid-phase chip method

[0062] In order to verify the specificity of the LAMP method, 35 strains of Fusarium graminearum identified by the colony diameter method were used as test objects. Among them, 15 strains were carbendazim-sensitive strains and 20 were resistant strains. Among the resistant strains, the numbers of the three drug-resistant mutants were 16 in F167Y, 2 in E198K, and 2 in F200Y (Table 1).

[0063] Table 1 list of tested strains

[0064] Strain number Types of source P8H8 F167Y Jiangsu P8A9 F167Y Jiangsu P8B9 F167Y Jiangsu P8C9 F167Y Jiangsu P8D9 F167Y Jiangsu P8E9 F200Y Jiangsu P10H5 F167Y Jiangsu P10A6 F167Y Jiangsu P10B6 E198Q Jiangsu P10C6 F167Y Jiangsu P10D8 F167Y zhejiang P10E8 F167Y zhejiang P10F8 E198Q Jiangs...

Embodiment 2

[0101] Implementation example 2: Sensitivity experiment of liquid chip method for detection of drug-resistant Fusarium graminearum

[0102] In order to verify the sensitivity of the LAMP method, extract sensitive strains P10G9, resistant strains P10F8, P10G8, and P11F3 (Table 1), and use a spectrophotometer to measure the DNA concentration (100ng / μL), and perform 10-fold serial dilution with ultrapure water, Set 5 template DNA concentration gradients of 0.001, 0.01, 0.1, 1, 10ng / reaction.

[0103] The result is as figure 1 , the minimum detection limit of 167m, 167w, 200w, 198m, 198w is 0.1ng / reaction, and the detection limit of probe 200m is 1ng / reaction.

Embodiment 3

[0104] Implementation example 3: Identification of resistance of Gibberella wheat to carbendazim in Nantong City, Jiangsu Province in 2014

[0105] Isolation of strains:

[0106] According to the method of Zhang Hao (2008) et al., single spores were isolated from ears of wheat head blight collected in Nantong, Jiangsu, and a total of 20 Fusarium graminearum strains were obtained.

[0107] Template DNA extraction:

[0108] Pick a small amount of hyphae on the surface of the sample with a sterilized toothpick or tip, put it into a PCR tube, add 50 μL Buffer A solution (100 mM NaOH, 2% 20), incubate at 95°C for 10 minutes. Add 50 μL of BufferB solution (100 mM Tris-HCl, 2 mM EDTA), shake and mix, centrifuge at 12000 rpm for 15 s, and take 1 μL of the supernatant as the template for amplification.

[0109] Amplification of the target fragment:

[0110] The PCR system using primers tub2F and tub2R to amplify the target sequence is as follows: 20 μl system contains 0.5 μM of ea...

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Abstract

The invention discloses a combination of primers and probes for detection of carbendazim-resistant strains of Fusarium graminearum and a detection method thereof. The primer-probe combination consists of forward primer tub2F, reverse primer tub2R, three drug-resistant mutants and wild-type probes 167m, 198m, 200m, 167w, 198w, and 200w. The detection method of the present invention can simultaneously detect three different types of drug-resistant mutations, and has high throughput, strong specificity and scalability, high signal-to-noise ratio, high sensitivity, high degree of automation, strong practicability, and can meet the requirements of anti-carbendazim and The rapid detection of Fusarium valley provides a basis for scientific and rational drug use.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a combination of primers and probes for detection of Fusarium graminearum strains resistant to carbendazim and a detection method thereof. Background technique [0002] Head blight of wheat, caused by Fusarium graminearum, is a devastating disease with frequent outbreaks in Asia, Europe, Canada and the northern United States. The pathogen can rapidly spread on the ears of wheat just weeks before harvest, hindering seed formation and shrinking the kernels, causing serious economic losses. In addition, the pathogen Fusarium metabolizes and produces trichothecenes to contaminate food and feed, which can also cause severe diseases and decreased immunity in humans and animals (Pestka and Smolinski 2005), prevent eukaryotic protein synthesis (Ueno et al., 1973). Therefore, cause the major hidden danger on the food safety. [0003] Due to the lack of effective disease-resista...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04C12R1/77
Inventor 张昊冯洁徐进许景升
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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