Molecular identification method of trichothecene type-B toxins of fusarium

A technology of trichothecenes and deoxynivalenol, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, microbial measurement/inspection, etc., and can solve the problems of high cost, cumbersome identification process, The actual application of toxin detection is far from the problem

Inactive Publication Date: 2012-05-09
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chandler et al. established a molecular identification method for the specific identification of 3-AcDON toxin based on the Tri7 gene (Chandler et al.Development of PCR assays to Tri7 and Tri13trichothecene biosynthetic genes, and characterization of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis.Physiol.and Mol.Plant Pathol., 2003, 62:355-367); Jennings et al. applied 7 pairs of primers to analyze the situation of 153 Fusarium flavum producing DON and its acetylation type, NIV toxin (Jennings et al.Determination of deoxynivalenol and nivalenol chemotypes of Fusariumculmorum isolates from England and Wales by PCR assay. Plant Pathol., 2004, 53: 182-190), the identification process is cumbersome and expensive
Due to the above limitations, these methods are far from the practical application of toxin detection in food and food safety.

Method used

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  • Molecular identification method of trichothecene type-B toxins of fusarium
  • Molecular identification method of trichothecene type-B toxins of fusarium
  • Molecular identification method of trichothecene type-B toxins of fusarium

Examples

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preparation example Construction

[0040] 2.1. Preparation of Fusarium graminearum spore suspension

[0041] Activate the purified and identified Fusarium graminearum strains (Table 1) on PDA medium (recipe: potato 200g, glucose 20g, agar 15g, add distilled water to 1L, use after autoclaving at 121°C for 20min) After cultivating for 5 days, pick the edge hyphae and inoculate them in CMC liquid medium (formula: carboxymethyl cellulose 7.5g, NH 4 NO 3 0.5g, KH 2 PO 4 0.5g, MgSO 4 ·7H 2 O 0.25g, 0.5g of yeast extract, add distilled water to make the volume to 1L, and use it after high-pressure steam sterilization at 121°C for 20min, pH 7.0). Adjust the spore concentration to 5×10 4 per milliliter for inoculation.

[0042] 2.2. Flowering inoculation and toxin chemical analysis

[0043] During the flowering stage of the susceptible wheat variety "Annong 8455", the wheat ears were evenly sprayed with a micro-sprayer, and the spray concentration per ear was 5×10 4 Each milliliter of Fusarium graminearum spore...

Embodiment 1

[0059] Detection and Identification of Toxin Produced by Fusarium graminearum B51066

[0060] Put the purified and identified Fusarium graminearum strain B51066 (see Table 1) on PDA medium (recipe: potato 200g, glucose 20g, agar 15g, add distilled water to 1L, use after autoclaving at 121°C for 20min) After activating the culture for 5 days, pick the edge mycelium and inoculate it in the CMC liquid medium (formula: carboxymethyl cellulose 7.5g, NH 4 NO 3 0.5g, KH 2 PO 4 0.5g, MgSO 4 ·7H 2 O 0.25g, 0.5g of yeast extract, add distilled water to make the volume to 1L, and use it after high-pressure steam sterilization at 121°C for 20min, pH 7.0). Adjust the spore concentration to 5×10 4 per milliliter for inoculation. At the flowering stage of the susceptible wheat variety "Annong 8455", the ears of wheat were evenly sprayed with a micro-sprayer, with about 1ml of spore liquid per ear, and 30 ears of wheat were inoculated for each strain. Then put on a plastic bag to mois...

Embodiment 2

[0065] Detection and Identification of Toxin Produced by Fusarium graminearum 5035

[0066] The detection and identification of the toxin produced by Fusarium graminearum 5035 (Table 1), the total DNA extraction of the mycelium of the strain 5035, the specific PCR reaction system and the amplification conditions were all the same as the toxin detection and analysis steps of the strain B51066 in "Example 1". Chemical analysis showed that the strain produced 15-AcDON toxin, multiple PCR amplification obtained a specific fragment of 279bp toxin, the results were completely consistent, and the inventive effect was as follows: image 3 Lane 7 of the .

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Abstract

The invention belongs to the technical field of plant inspection and quarantine, and specifically relates to a molecular detection method of type-B toxins of fusarium and application thereof. The molecular detection method of the invention is characterized by comprising the steps of designing a pair of multiplex PCR primers through Tril 3 gene information aiming at the trichothecene type-B toxins, separating the fusarium graminearum for generating 3-AcDON, 15-AcDON and NIV toxins to obtain the specific segments of 334bp, 279bp and 497bp, using the PCR reaction, detecting the product by the agarose gel electrophoresis, and directly detecting the type of the toxins generated by the fusarium graminearum according to the size of the segment of the product. The invention is applicable for the detection of type-B toxins of fusarium in the grain, feed and food safety.

Description

technical field [0001] The invention belongs to the technical field of plant inspection and quarantine, and in particular relates to a molecular identification method of fusarium trichothecene family B toxins. Background technique [0002] Fusarium head blight (Fusarium head blight or scab) caused by Fusarium is an important disease that harms wheat, barley, corn and other crops worldwide. The active trichothecenes can reduce the quality of food or feed. Fusarium toxin has high stability and is not easy to degrade. It can enter the food chain along with food and feed processing, and then threaten the health of humans and animals. These toxins can inhibit the protein synthesis of eukaryotic cells and destroy the immune system of humans and animals. After poisoning, they are often accompanied by symptoms such as vomiting, diarrhea, and dizziness. Some toxins may even cause cancer. Trichothecenes are the most important Fusarium toxins, mainly including A and B. According to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/11C12Q1/68C12Q1/04
Inventor 廖玉才李和平王建华张静柏黄涛
Owner HUAZHONG AGRI UNIV
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