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: 2014-04-02
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 testing

[0037] Fusarium graminearum strains obtained from wheat, barley and corn head blight samples collected from different regions of China after tissue isolation, monospore purification and identification through morphological standard procedures are shown in Table 1.

[0038] Table 1 Fusarium graminearum strains used in the present invention

[0039]

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

[0041] 2. Establishment of basic testing methods

[0042] 2.1. Preparation of Fusarium graminearum spore suspension

[0043] The purified and identified Fusarium graminearum strains (Table 1) were activated on PDA medium (recipe: 200g potato, 20g glucose, 15g agar, distilled water to make the volume to 1L, and use after autoclaving at 121℃ for 20min) After culturing for 5 days, pick the marginal hyphae and inoculate them in CMC liquid medium (formulation: carboxymethyl cellulos...

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, the PDA (recipe: 200g potato, 20g glucose, 15g agar, distilled water was added to make the volume to 1L, 121°C autoclave for 20min) Use) After 5 days of activation and cultivation on the plate, pick the marginal hyphae and inoculate them in CMC liquid medium (formulation: carboxymethyl cellulose 7.5g, NH 4 NO 3 0.5g, KH 2 PO 4 0.5g, MgSO 4 ·7H 2 O 0.25g, yeast extract 0.5g, add distilled water to make the volume to 1L, use after autoclaving at 121℃ for 20min, pH 7.0) after shaking culture (25℃, 150rpm) to produce conidia, use a hemocytometer to count. Adjust the spore concentration to 5×10 4 A / ml is used for inoculation. In the flowering period of the susceptible wheat variety "Annong 8455", the wheat ears were sprayed evenly with a micro sprayer, and the spray concentration p...

Embodiment 3 application Embodiment 2

[0065] Detection and identification of ZEN toxin produced by Fusarium graminearum strain 12002

[0066] Fusarium graminearum strain 12002 (see Table 1) chemical detection of ZEN toxin production, extraction of total DNA from the strain's hyphae, reaction system and amplification for specific PCR amplification of PKS4-P1 / 2 and PKS13-P1 / 2 using primers The increasing 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 a 271bp DNA fragment was amplified by primer pair PKS4-P1 / 2, and the detection results were completely consistent. The effect of the invention is as follows figure 2 The lane 2 is shown. The primer pair PKS13-P1 / 2 was used to amplify a 302 bp DNA fragment, and the detection results were consistent. The effect of the invention is as follows image 3 The 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 specifically relates to an inspection and quarantine method for the fusarium toxin zearalenone, which is related to molecular detection technology. Background technique [0002] Fusarium head blight or scab caused by Fusarium graminearum is an important disease that harms wheat, barley, corn and other crops worldwide. It not only severely affects crop yields, but also Fusarium can produce a lot of harmful effects on humans and animals. The toxic secondary metabolite, fusarium toxin, reduces the quality of food or feed. Due to the high stability of the Fusarium toxin and the resistance to degradation, it can enter the food chain along with the food and feed processing process, thereby threatening the health of humans and animals. Zearalenone (ZEN) is one of the most important fusarium toxins produced by Fusarium graminearum. It is widely present in moldy corn, sorghum, whea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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