Molecular detection method and application for zearalenone toxin

A technology for zearalenone and toxins, applied in the directions of application, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of expensive equipment, time-consuming and complicated operation

Inactive Publication Date: 2012-05-16
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the detection method of zearalenone is mainly the chemical analysis method of phase chromatography-mass spectrometry (LC / MS), but this method involves the extraction and purification of the toxin, and the operation is time-consuming and complicated, and requires expensive equipment

Method used

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  • Molecular detection method and application for zearalenone toxin
  • Molecular detection method and application for zearalenone toxin
  • Molecular detection method and application for zearalenone toxin

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Embodiment 1

[0035] Example 1 Preparation Example

[0036] 1. Collection of relevant strains for detection

[0037] Graminea strains are shown in Table 1 after tissue isolation and single spore purification culture of wheat, barley and maize scab ear samples collected from different regions in China, and identified by morphological standard procedures.

[0038] Table 1 The Fusarium graminearum bacterial strain that the present invention adopts

[0039]

[0040] Note: This table is the result determined by LC / MS chemical analysis.

[0041] 2. Establishment of basic detection methods

[0042] 2.1. Preparation of Fusarium graminearum spore suspension

[0043] 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 7.5...

Embodiment 2 application Embodiment 1

[0058] Detection and identification of ZEN toxin produced by Fusarium graminearum 5035

[0059] After the Fusarium graminearum strain 5035 (see Table 1) was activated and cultured on a PDA plate for 5 days, it was placed on a PDA (recipe: 200 g of potatoes, 20 g of glucose, 15 g of agar, added distilled water to 1 L, and sterilized by high pressure steam at 121° C. for 20 min. After activating and culturing on the plate for 5 days, pick the edge mycelium and inoculate it 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. 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...

Embodiment 3 application Embodiment 2

[0065] Detection and Identification of ZEN Toxin Produced by Fusarium graminearum Strain 12002

[0066] The chemical detection of ZEN toxin produced by Fusarium graminearum strain 12002 (see Table 1), the total DNA extraction of strain mycelium, the reaction system and amplification method of specific PCR amplification using primer pairs PKS4-P1 / 2 and PKS13-P1 / 2 The growth conditions are the same as the toxin detection and analysis steps of strain 5035 in "Example 1". Chemical analysis showed that the strain 12002 produced ZEN toxin, and the primer pair PKS4-P1 / 2 was used to amplify a 271bp DNA fragment. The detection results were completely consistent, and the inventive effect was as follows: figure 2 Swimming lane 2 is shown. Utilize primer pair PKS13-P1 / 2 to amplify the DNA fragment of 302bp, the detection result is consistent, and its invention effect is as follows image 3 Swimming lane 2 is shown.

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Abstract

The invention belongs to the plant examination quarantine technical field, concretely relates to a molecular detection method and an application for fusarium toxin ZEN. The invention is characterized in that by aiming at zearalenone toxin (ZEN), two pairs of PCR primers of PKS4-P1 and PKS4-P2 as well as PKS13-1 and PKS13-P12 are designed through PKS4 and PKS 13 gene sequence information, 271bp and 302bp specific fragments are obtained by separating the cereal fusarium genome capable of generating ZEN toxin, by using PCR reaction, the agarose gel electrophoresis is employed for detecting the product, and the cereal fusarium capable of generating ZEN toxin detection can be carried out according to the information of the product fragments. The molecular detection method and application for zearalenone toxin can be used for detecting and analyzing the ZEN toxin in cereal, 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 method for inspection and quarantine of fusarium toxin zearalenone, which is related to molecular detection technology. Background technique [0002] Fusarium head blight or scab (Fusarium head blight or scab) caused by Fusarium graminearum is an important disease that harms wheat, barley, corn and other crops worldwide. Toxic secondary metabolites, namely Fusarium toxins, degrade food or feed quality. Fusarium toxins have high stability and are not easy to degrade, so they can enter the food chain along with food and feed processing, threatening the health of humans and animals. Zearalenone (ZEN) is one of the most important fusarium toxins produced by Fusarium graminearum, and is widely found in moldy corn, sorghum, wheat and other cereal crops and milk products. Zearalenone is an estrogen-like polyketide chemical, which has strong reprod...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/11C12Q1/68C12Q1/04
Inventor 廖玉才王建华李和平张静柏黄涛瞿波
Owner HUAZHONG AGRI UNIV
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