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Method for detecting potential pollution risk of aflatoxin in peanuts and products thereof by using real-time fluorescent quantitative PCR (polymerase chain reaction) technology

A real-time fluorescence quantitative and aflatoxin technology, which is applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems of inaccurate judgment standards and inability to make quantitative judgments, and achieve high sensitivity and safe and reliable detection results , good stability

Inactive Publication Date: 2015-11-04
张初署 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention patent (201210584285.9) discloses a method for analyzing the production trend of aflatoxin B1 in peanut meal by using multiplex PCR technology. The production trend of aflatoxin B1 is determined by the richness of the bands. Judging the production trend of aflatoxin B1 has the disadvantage that the judgment standard is not accurate and cannot be quantitatively judged

Method used

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  • Method for detecting potential pollution risk of aflatoxin in peanuts and products thereof by using real-time fluorescent quantitative PCR (polymerase chain reaction) technology
  • Method for detecting potential pollution risk of aflatoxin in peanuts and products thereof by using real-time fluorescent quantitative PCR (polymerase chain reaction) technology
  • Method for detecting potential pollution risk of aflatoxin in peanuts and products thereof by using real-time fluorescent quantitative PCR (polymerase chain reaction) technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0026] Example 1——The real-time fluorescent quantitative PCR technology is a method for detecting the risk of aflatoxin contamination in peanuts and its products with good specificity.

[0027] According to the AFLR gene sequence published by Genebank, the Primer Premier5.0 software was used to design specific primers, and its sequence was: upstream primer 5'-AACCTGATGACGACTGATAT-3', downstream primer 5'-AGCACCTTGAGAACGATAA-3', as can be seen from Figure 1 , using the designed primers to do Real-time PCR on Aspergillus flavus, there is no miscellaneous peak, and the product has good parallelism.

[0028] The specificity of Aspergillus flavus toxin-producing bacteria and Aspergillus flavus non-toxin-producing bacteria (without AFLR gene) was verified, and the results showed that the primers only amplified the strains containing the AFLR gene, but did not amplify the strains without the AFLR gene. There was amplification, which indicated that the primer had strong specificity an...

Embodiment 3

[0031] Example 3 - The method of real-time fluorescent quantitative PCR technology to detect the risk of aflatoxin contamination in peanuts and its products has good stability

[0032] Dilute the DNA of Aspergillus flavus toxin-producing bacteria to a concentration of 3×10-13 mg / ml, and repeat 6 times. The results are shown in Table 1. The coefficient of variation is 2.67%, indicating good repeatability.

[0033] Table 1 The repeatability test results of real-time quantitative PCR

[0034]

Embodiment 4

[0035] Example 4—Establishing a real-time fluorescent quantitative PCR relationship model between the risk of aflatoxin contamination in peanuts and the afIR gene of Aspergillus flavus

[0036] Correlation analysis was carried out between the aflatoxin content in peanut samples and the Ct value of Real-time PCR, and the correlation coefficient was -0.718, a significant negative correlation; the mathematical relationship equation was: y=-0.0852x+27.257.

[0037] Table 2 The content of aflatoxin in peanut and the Ct of fluorescent quantitative PCR

[0038]

[0039] Note: C is the aflatoxin content, and Ct is the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold.

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Abstract

The invention discloses a method for detecting potential pollution risk of aflatoxin in peanuts and products thereof by using real-time fluorescent quantitative PCR (polymerase chain reaction) technology. The method utilizes a quartering process to collect the samples, utilizes a benzyl chloride process to extract the DNA (deoxyribonucleic acid), and designs the specific primer by using the Aspergillus flavus toxin production key gene AFLR as the target gene to perform the fluorescent quantitative PCR, judges the aflatoxin pollution risk of the sample according to the fluorescent quantitative Ct value, and establishes a relation model between the aflatoxin pollution risk and sample fluorescent quantitative Ct value. The method can quickly and accurately judge the potential pollution risk of aflatoxin in the peanuts and products thereof, has the advantages of high specificity, high stability and high sensitivity, is simple and quick for operation, and is suitable for mass detection.

Description

technical field [0001] The invention relates to a method for detecting the potential contamination risk of aflatoxin in peanuts and products by using real-time fluorescent quantitative PCR technology, belonging to the field of food safety and detection. Background technique [0002] The planting area of ​​peanuts in China is 5 million hectares, with an annual output of 14 million tons, accounting for more than 40% of the world's total peanut production, ranking first in the world, and is one of the important economic crops in my country. At present, the level of aflatoxin pollution in peanuts and their products in my country has reached 23-500 μg / kg, which greatly exceeds the limit value of the national standard, seriously affecting the edible safety and international trade of peanut products, and aflatoxin pollution has become a The biggest limiting factor restricting the development of peanut industry and food safety. Aflatoxin pollution in peanuts is serious, mainly becau...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q2561/113C12Q2561/101C12Q2545/114C12Q2531/113
Inventor 张初署于丽娜孙杰毕洁
Owner 张初署
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