Method for quantitatively detecting peanut components in sesame butter and sesame paste by utilizing double digital PCR

A quantitative detection, sesame paste technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. achieve the effect of ensuring accuracy

Active Publication Date: 2019-06-28
TECH CENT OF GUANGZHOU CUSTOMS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This behavior seriously hinders fair trade, damages the interests of consumers, and affects food safety.
[0003] "LS / T 3220-2017 Sesame Paste" stipulates that the inspection of sesame paste includes sensory inspection, acid value and fat content and other quality indicators, which cannot effectively identify the authenticity of sesame paste
"SN / T 1961.12-2013 Detection of Allergen Components in Exported Food Part 12: Real-time Fluorescent PCR Method for Detection of Sesame Components", "SN / T 4419.14-2016 LAMP System Detection Method for Common Allergens in Exported Food Part 14: Sesame", " SN / T 1961.2-2007 Detection method for allergens in food Part 2: Real-time fluorescent PCR method for detection of peanut components" and "SN / T 4419.12-2016 Detection method for common allergens in exported food LAMP series Part 12: Peanut" adopted The qualitative detection of sesame and peanuts by real-time fluorescent PCR and isothermal amplification technology can only solve the problem of whether peanut ingredients are contained in sesame paste and sesame paste. "Unintentional pollution" and "intentional addition" for the purpose of adulteration cannot be effectively distinguished, and there are obvious technical limitations, which cannot meet the needs of anti-counterfeit sesame paste and sesame paste

Method used

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  • Method for quantitatively detecting peanut components in sesame butter and sesame paste by utilizing double digital PCR
  • Method for quantitatively detecting peanut components in sesame butter and sesame paste by utilizing double digital PCR
  • Method for quantitatively detecting peanut components in sesame butter and sesame paste by utilizing double digital PCR

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Experimental program
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Effect test

preparation example Construction

[0041] 1. Preparation of samples and extraction of genomic DNA templates: Take 10 g of samples (sesame, peanut, sesame paste, sesame paste filling, etc.), crush and homogenize with a grinder at 1800 rpm for 3 minutes.

[0042] Weigh 30mg of the sample into a 1.5mL centrifuge tube, and extract the sample DNA using the kit method. The kit can be selected: Kurabo QuickGene DNA extraction kit DT-S, Wizard Genomic DNA purification kit (Promega, A1120), PSS nucleic acid automatic extraction instrument, etc. DNA extraction method. Those skilled in the art are familiar with these DNA extraction methods, so as to extract the DNA of the corresponding samples respectively.

[0043] 2. Design and synthesize the primers and probe sequences of the sesame species-specific gene sequences and the primers and probe sequences of the peanut species-specific gene sequences. The nucleotide sequences of the primers and probes are as follows:

[0044]

[0045] 3. Perform double digital PCR reaction...

Embodiment 1

[0093] Example 1: Verification of the Absolute Limit of Quantification of Copy Number

[0094] Samples for testing: In order to verify the absolute limit of quantification of the copy number of this method, the genomic DNA of sesame and peanut were respectively extracted and serially diluted to obtain serial dilutions of 500, 100, 20, 10, 5, 1 copy / microliter of sesame and peanut DNA. Three parallel ddPCR and cdPCR experiments were carried out, and the experimental results are shown in Table 1.

[0095] Table 1 Verification of the absolute limit of quantification (ddPCR and cdPCR) of peanut and sesame DNA copy number

[0096]

[0097] As can be seen from the results shown in Table 1, on the ddPCR platform, when the peanut DNA copy number concentration is 3.77 copies / microliter, the RSD values ​​among the three parallels are all greater than 25%, and when the peanut DNA copy number concentration is greater than or equal to 5.93 copy / microliter, the RSD value between three ...

Embodiment 2

[0098] Example 2: Validation of Quantitative Limit of Copy Number Percentage

[0099] For the sample: In order to verify the quantification limit of copy number percentage of this method, sesame DNA with known copy number concentration was used as the matrix, and peanut DNA with known copy number concentration was mixed, and the copy number percentages of the series were respectively 0.1%, 0.1%, and 0.1%, respectively. 1%, 10%, and 50% mixed samples of sesame and peanut DNA. Three parallel ddPCR and cdPCR experiments were carried out respectively, and the obtained experimental results are shown in figure 1 and figure 2 .

[0100] Depend on figure 1 and figure 2 The results shown show that for the mixed samples of sesame and peanut DNA whose copy number percentages are 0.1%, 1%, 10% and 50%, the detection results on the ddPCR platform are 0.099%, 1.033%, 10.023% and 49.400 respectively %, the RSD value among the three parallels was between 0.58% and 3.11%, and the recove...

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Abstract

The invention provides a method for quantitatively detecting peanut components in sesame butter and sesame paste by utilizing double digital PCR, which comprises the following steps of: adopting a double-channel detection method, utilizing a digital PCR system to simultaneously detect two fluorescent signals, respectively marking probes detected by peanut species specific gene sequences and sesamespecies specific gene sequences as VIC and FAM, calculating copy number concentration of the peanut species specific gene sequences and the sesame species specific gene sequences detected in the samePCR reaction system to obtain DNA copy number percentage of peanuts which account for the sesame and the peanut, and converting to obtain mass percentage of the peanut components which account for the sesame and the peanut components. The method for quantitatively detecting the peanut components in the sesame butter and the sesame paste by utilizing the double digital PCR can accurately and quickly detect the mass percentage content of the peanut components in the sesame butter and the sesame paste, and can provide accurate and reliable technical data for identifying the authenticity of the sesame butter and the sesame paste fillings.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to a method for quantitatively detecting peanut components in sesame paste and sesame paste by double digital PCR. Background technique [0002] According to the regulations in "LS / T 3220-2017 Sesame Paste", sesame paste is a product made from sesame seeds, which is processed by grinding, cleaning and roasting. The only raw material for tahini should be sesame seeds or peeled sesame seeds. Sesame paste, commonly known as Ma Rong, is a stuffing made from sesame, lard and sugar. Because the production process of sesame paste and sesame paste is simple, the market entry threshold is low, and the production enterprises are uneven, some production enterprises adulterate sesame paste and sesame paste in order to reduce costs and obtain higher profits. The most common adulteration method is to blend peanut butter in pure sesame paste and peanut paste in sesame paste...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851
CPCY02A50/30
Inventor 刘津高东微董旭婉李志勇董洁关丽军
Owner TECH CENT OF GUANGZHOU CUSTOMS
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