Universal primer for plant-derived component identification and application of universal primer

A plant-derived, universal primer technology, applied in the field of bioengineering, can solve the problems of high detection time cost and reagent consumables cost, difficulty in simultaneous detection of multiple targets, and inability to meet risk point screening, saving time and labor. Material cost, reduced inspection work intensity, cost saving effect

Inactive Publication Date: 2020-07-28
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, these two methods are one-to-one directional detection methods, which are difficult to detect multiple targets at the same time, cannot meet the real risk point screening, and the cost of detection time and reagent consumables is h

Method used

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  • Universal primer for plant-derived component identification and application of universal primer
  • Universal primer for plant-derived component identification and application of universal primer
  • Universal primer for plant-derived component identification and application of universal primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Primer Design and Screening

[0025] 1. Using DNA barcoding technology to design primers

[0026] 1.1 Primer screening

[0027] Select 6 pairs of primers as alternative universal primers (see Table 1), use the 6 pairs of primers to perform PCR amplification with a Roche fluorescent quantitative PCR instrument (model: LightCycler 480), and determine the amplification of the primers to species according to the Cp value efficiency.

[0028] Table 1 6 pairs of primary screening primer sequences

[0029]

[0030] According to the DNA barcode technology, the present invention compares the 6 pairs of primers with the base sequences of various plants (walnut, soybean, peanut, hazelnut and almond) on NCBI with sequencher software, and based on the original primers, according to R=A / G, Y=C / T, M=A / C, K=G / T, S=C / G, W=A / T, H=A / C / T, B=C / G / T, V =A / C / G, D=A / G / T, N=A / C / G / T principle to carry out base merger, and the primers after merger are shown in Table 2.

[0031] ...

Embodiment 3

[0087] Embodiment 3 tests the suitability of primer with real sample

[0088] The universal primer and identification method of the present invention are used to simultaneously test 10 batches of beverages containing plant components.

[0089] Methods as below:

[0090] (1) Extract the total DNA of the food to be tested: take 1 mL of plant beverage and add 1 mL of isopropanol, mix well, place at room temperature for 5 min, centrifuge at 12500 rpm for 20 min at high speed, and discard the supernatant. The sample nucleic acid precipitation was carried out, and the operation was repeated three times in total.

[0091] Add 600uL CTAB buffer and 10ul proteinase K to the sample, shake and mix, and incubate at 56°C for 30min with shaking. Add 500ul of a mixture of phenol:chloroform:isoamyl alcohol=25:24:1, shake vigorously, and centrifuge at 12000g for 15min. Aspirate the supernatant into a new centrifuge tube, add an equal volume of isopropanol (below 0°C), shake evenly, centrifu...

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Abstract

The present invention discloses a universal primer for plant-derived component identification and an application of the universal primer. An upstream primer sequence of the universal primer is shown as SEQ No.1 and a downstream primer sequence of the universal primer is shown as SEQ No.2. An amplicon sequencing analysis method is used to identify at least 10 plant-derived components of walnuts, peanuts, soybeans, sesame seeds, hazelnuts, apricot kernels, coconuts, pine nuts, pumpkin seeds, Badam, etc. and can solve a problem that current PCR and ELISA detection methods can only conduct one-to-one targeted screening of the targeted components, only one experiment is needed to conduct non-targeted screening of the plant components of the product, intensity of detection work is reduced, costof synthezing primers and probes is saved, and in the case of saving time, labor and material costs, the universal primer can be flexibly used in a variety of food containing allergenic ingredients tomeet screening work of large quantities of plant component-containing samples, realizes development from target detection to target-free screening, improves a level of adulteration detection and provides an effective technical means for supervision.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a general primer for identification of plant-derived components and its application. Background technique [0002] Foods containing plant ingredients have a high market share at present. At present, the domestic standard for testing plant-derived ingredients is the detection method of "Identification of Plant-derived Components in Plant Protein Beverages" announced by the State Food and Drug Administration in 2017 No. 75. This method is suitable for screening walnuts, peanuts, soybeans, and sesame The primers and probes used for the six plant-derived ingredients of , hazelnut and almond are specified. In the world, enzyme-linked immunosorbent assay (ELISA) is often used to detect plant-derived components. [0003] However, these two methods are one-to-one directional detection methods, which are difficult to detect multiple targets at the same time, cannot meet the real ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6869C12N15/11
CPCC12Q1/6895C12Q1/6869C12Q2531/113C12Q2563/185
Inventor 陈怡文崔生辉张晓东
Owner NAT INST FOR FOOD & DRUG CONTROL
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