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Method for establishing DNA (deoxyribonucleic acid) fingerprint spectra of peanut aspergillus flavus in different producing areas with ISSR (inter-simple sequence repeat) molecular marker technology

A technology of DNA fingerprinting and Aspergillus flavus, which is applied in the field of food safety, and achieves the effect of stable method, easy mastering and good primer specificity

Inactive Publication Date: 2015-12-16
张初署 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to meet the needs of the origin traceability management of peanut aflatoxin pollution, fill the gap in the DNA traceability technology for peanut aflatoxin contamination from different origins in my country, and provide a method for constructing DNA fingerprints for the different origins of peanuts in my country. And the DNA fingerprint of the origin attribute of peanuts obtained by this method, so as to provide theoretical and reference basis and technical support for the traceability of the origin of peanut Aspergillus flavus pollution

Method used

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  • Method for establishing DNA (deoxyribonucleic acid) fingerprint spectra of peanut aspergillus flavus in different producing areas with ISSR (inter-simple sequence repeat) molecular marker technology
  • Method for establishing DNA (deoxyribonucleic acid) fingerprint spectra of peanut aspergillus flavus in different producing areas with ISSR (inter-simple sequence repeat) molecular marker technology
  • Method for establishing DNA (deoxyribonucleic acid) fingerprint spectra of peanut aspergillus flavus in different producing areas with ISSR (inter-simple sequence repeat) molecular marker technology

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1—the inventive method is stable, reproducible, easy to master

[0037] According to the method of the present invention, a strain of Aspergillus flavus is carried out three parallels of the ISSR mark of R1 primer, and its collection of figures is shown in figure 1 . Where M is a DNA marker. figure 1 It can be seen that the method of the present invention is stable, has good reproducibility, clear background and high band brightness.

Embodiment 2

[0038] Embodiment 2—The present invention can trace the source of Aspergillus flavus pollution in the main peanut production area of ​​China to a single production area or a smaller area

[0039] According to the method of the present invention, 24 strains of Aspergillus flavus isolated from different regions are subjected to ISSR markers of R1, R2, and R3 primers, and the results are clustered and analyzed, and the map figure 2 . From figure 2 It can be seen that the bacterial strains in each region can be separated from the bacterial strains in other regions by this method. Therefore, the present invention can trace the source of Aspergillus flavus pollution in the main peanut producing regions of China to a single producing region or a smaller area.

Embodiment 3

[0040] Example 3—Peanut Aspergillus flavus sample: 7 strains of Aspergillus flavus isolated from peanuts produced in Xiangyang Village, Baoshan Town, Jiaonan City, Qingdao City, Shandong Province

[0041] (1) Isolation of 7 strains of Aspergillus flavus: isolate, purify and identify Aspergillus flavus from peanuts produced in Xiangyang Village, Baoshan Town, Jiaonan City, Qingdao City, Shandong Province.

[0042] Take 10 g of peanuts from different origins, add 90 mL of 0.1% peptone sterile water (w / v), shake at room temperature for 30 minutes, and make a 10-1 bacterial suspension; take 0.1 mL of the bacterial liquid, and spread it on the DG18 medium, Incubate in the dark at 30°C for 5 days. Pick Aspergillus flavus with yellow spores and perform secondary streak isolation on DG18 medium until a single colony is obtained. Pick a single colony of Aspergillus flavus on the MEA slant test tube medium, culture it at 30°C for 3 days, and then store it at 4°C. The identification of...

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Abstract

The invention discloses a method for establishing DNA (deoxyribonucleic acid) fingerprint spectra of peanut aspergillus flavus in different producing areas with an ISSR (inter-simple sequence repeat) molecular marker technology. The method comprises steps as follows: aspergillus flavus DNA extraction, ISSR-PCR (inter-simple sequence repeat-polymerase chain reaction) and electrophoretogram analysis of reaction products, wherein primers adopted in the ISSR-PCR include an R1 primer, an R2 primer and an R3 primer, the R1 primer is AGA GAG AGA GAG AGA GG, the R2 primer is CAC ACA CAC ACA CAC AA, and the R3 primer is AGA GTT GGT AGC TCT TGA TC. The method is stable, has good reproducibility and is easy to master; the selected primers have good specificity, aspergillus flavus contamination of main peanut producing areas in China can be traced back to a single producing area or a district with a smaller range, and the method can be taken as an origin traceability theoretical method and technical support for the aspergillus flavus contamination of the main peanut producing areas in China.

Description

technical field [0001] The invention relates to a method for establishing DNA fingerprints of Aspergillus flavus in peanuts from different origins by using ISSR molecular marker technology, which belongs to the field of food safety. Background technique [0002] Aflatoxin (Aflatoxin, AFT) is a class of secondary metabolites mainly produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. In my country, aflatoxin is mainly produced by Aspergillus flavus. Aflatoxin has the "three-cause" effects of carcinogenicity, teratogenicity, and cell mutation. Only a dose of 0.294 mg / kg can cause acute poisoning death in sensitive animals, and is one of the main factors inducing malignant tumors such as primary hepatocellular carcinoma. , can lead to liver necrosis and canceration in the shortest 24 weeks. [0003] Peanuts are extremely susceptible to infection by Aspergillus flavus. Once the infected Aspergillus flavus encounters a suitable environment, it will rapidly g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 张初署孙杰毕洁于丽娜许婷婷张玉凤彭娅萍
Owner 张初署
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