CN111455093A

CN111455093A - Universal primer for identifying plant-derived components and application thereof

Info

Publication number: CN111455093A
Application number: CN202010510179.0A
Authority: CN
Inventors: 陈怡文; 崔生辉; 张晓东
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2020-06-08
Filing date: 2020-06-08
Publication date: 2020-07-28

Abstract

The invention discloses a universal primer for identifying plant-derived components and application thereof, wherein the sequence of an upstream primer is shown as SEQ No.1, and the sequence of a downstream primer is shown as SEQ No. 2. the invention adopts an amplicon sequencing analysis method to identify at least 10 plant-derived components such as walnut, peanut, soybean, sesame, hazelnut, almond, coconut, pine nut, pumpkin seed, badam and the like, can solve the problem that the existing PCR and E L ISA detection methods can only carry out one-to-one directional screening on target components, carries out non-directional screening on the plant components of products only through one experiment, reduces the detection working strength, saves the cost of synthetic primers and probes, can be flexibly used for various foods containing allergic components under the condition of saving time and labor material cost, meets the large-batch screening work of samples containing plant components, realizes the development from the detection of doped targets to the screening direction without targets, improves the level of the false detection, and provides an effective technical means for supervision and supervision.

Description

Universal primer for identifying plant-derived components and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a universal primer for identifying plant-derived components and application thereof.

Background

The current domestic standard for detecting plant-derived components is the detection method of 'identification of plant-derived components in plant protein beverage' announced by the State food and drug administration, No. 75 in 2017, the method stipulates primers and probes for screening six plant-derived components of walnut, peanut, soybean, sesame, hazelnut and almond, and the international method of multiple enzyme-linked immunosorbent assay (E L ISA) is used for detecting the plant-derived components.

However, both the two methods are one-to-one directional detection methods, which are difficult to achieve simultaneous detection of multiple targets, cannot meet the real risk point screening, and have high detection time cost and high reagent consumable cost. Meanwhile, many products sold in the market at present, such as foods containing coconut, pine nut, pumpkin seed, badam and other components, are always in the blank field of market supervision because no suitable primers and probes are available. Therefore, a primer and a method which can carry out non-directional screening on a plurality of plant components in a product simultaneously need to be developed, so that various plant-derived components possibly contained in the product can be detected, and the blank of the supervision field is filled.

Disclosure of Invention

In view of the above, the first objective of the present invention is to provide a universal primer for identifying plant-derived components, wherein the sequence of the upstream primer is shown as SEQ No.1, and the sequence of the downstream primer is shown as SEQ No. 2.

The second purpose of the invention is to provide the application of the universal primer in identifying the plant-derived components in the food.

Wherein the plant-derived component comprises one or more of walnut, peanut, soybean, sesame, hazelnut, almond, coconut, pine nut, pumpkin seed and almond.

The third purpose of the invention is to provide a method for identifying plant-derived components in food by applying the universal primer, which comprises the following steps:

(1) extracting total DNA of food to be detected;

(2) and (3) PCR amplification: using total DNA as a template, performing PCR amplification by using the universal primer, and recovering and purifying PCR amplification products;

the PCR amplification conditions were: maintaining at 95 deg.C for 3min, 95 deg.C for 30s, 57 deg.C for 30s, 72 deg.C for 30s, and 12 deg.C; wherein the temperature of 95 ℃ for 30s, the temperature of 57 ℃ for 30s and the temperature of 72 ℃ for 30s are subjected to 40 cycles;

the PCR reaction system comprises 10 × PCR buffer5.0 mu L2 mu L, Primer0.4+0.4 mu L, Taq enzyme 0.3 mu L4 mu L and H₂O37.9μL；

(3) Sequencing the amplicon;

(4) and determining the components.

The fourth purpose of the invention is to provide a kit containing the universal primer.

The invention finds out a proper plant gene sequence based on a DNA bar code technology, designs a universal primer, adopts an amplicon sequencing analysis method to identify at least 10 plant-derived components such as walnut, peanut, soybean, sesame, hazelnut, almond, coconut, pine nut, pumpkin seed, badam and the like, can solve the problem that the conventional PCR and E L ISA detection method can only carry out one-to-one unidirectional screening on target components, carries out non-directional screening on the plant components of a product only through one-time experiment, reduces the detection working strength, saves the cost of a synthetic primer and a probe, can be flexibly used for various foods containing allergic components under the condition of saving time and manpower material cost, meets the screening work of a large batch of samples containing the plant components, realizes the development from the detection with a target to the screening without the target, improves the level of adulteration detection, and provides an effective technical means for supervision.

Drawings

In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.

FIG. 1 shows the results of agarose gel electrophoresis provided in example 1 of the present invention.

FIG. 2 shows the amplification results of rbc L-F327 & rbc L-R638 provided in example 1 of the present invention.

FIG. 3 shows the amplification results of rbc L-F154 & rbc L-R510 provided in example 1 of the present invention.

FIG. 4 shows the amplification results after the double merger of rbc L-F327 & rbc L-R638 provided in example 1 of the present invention.

FIG. 5 shows the amplification results after two-time merger of rbc L-F154 & rbc L-R510 provided in example 1 of the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood, those skilled in the art will now describe the present invention in further detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

The present invention will be described in further detail with reference to specific examples.

Example 1 primer design and screening

1. Design of primers using DNA barcode technology

1.1 primer screening

6 pairs of primers are selected as alternative universal primers (see table 1), the 6 pairs of primers are subjected to PCR amplification by a Roche fluorescence quantitative PCR instrument (model: L ightCycler 480), and the amplification efficiency of the primer pair species is determined according to the Cp value.

TABLE 16 pairs of Primary screening primer sequences

The invention compares the 6 pairs of primers with the base sequences of various plants (walnut, soybean, peanut, hazelnut and almond) on NCBI (NCBI) by using a sequence reader software, and performs base combination according to the principle that R is A/G, Y is C/T, M is A/C, K is G/T, S is C/G, W is A/T, H is A/C/T, B is C/G/T, V is A/C/G, D is A/G/T, and N is A/C/G/T on the basis of the original primers, and the combined primers are shown in Table 2.

TABLE 2 primer sequences after merging

Extracting DNA, namely extracting DNA of walnuts, soybeans, peanuts, hazelnuts and almonds by a CTAB method (see a DNA extraction method), and diluting all the DNA to 1 ng/mu L by virtue of Qubit quantification, wherein the fluorescence quantification PCR reaction comprises the following steps:

fluorescent quantitative PCR reaction conditions:

fluorescent quantitative PCR reaction system:

the results of comparison of the fluorescent quantitative PCR amplification efficiencies are shown in Table 3.

TABLE 3 comparison of fluorescent quantitative PCR amplification efficiency

By comparing the Cp values of 5 species of 6 pairs of primer pairs, 2 pairs of primers rbc L-F154 & rbc L-R510 and rbc L-F327 & rbc L-R638 with good amplification efficiency are preliminarily screened out.

1.2 Secondary screening of Universal primers

Extracting 9 plant components of badam, soybean, hazelnut, peanut, pine nut, coconut, almond, walnut and pumpkin seed by a CTAB method, quantifying by a Qubit, diluting all DNA to 1 ng/mu L. a fluorescent quantitative PCR reaction system is as follows:

the fluorescent quantitative PCR reaction conditions are as follows:

9 plant-derived component rbc L-F327 & rbc L-R63 primer PCR amplification results are shown in Table 3 and figure 2, 9 plant-derived component rbc L-F154 & rbc L-R510 primer PCR amplification results are shown in Table 4 and figure 3. due to the fact that 4 plant-derived components are added in the secondary screening compared with the primary screening experiment, the results show that the amplification efficiency of rbc L-F327 & rbc L-R638 primer on coconut is poor, and the amplification efficiency of rbc L-F154 &5rbc L-R510 on pine nuts is poor.

TABLE 39 fluorescent quantitative PCR amplification results of primers rbc L-F327 & rbc L-R63 from plant-derived components rbc L-F & rbc L-R63

Pos	Name	Cp	Pos	Name	Cp
						A1	Badam 3	15.21	A2	Pine nuts 3	15.45
B1	Badam 3	14.97	B2	Pine nuts 3	15.44
						C1	Soybean 3	13.92	G2	Almond 3	12.87
D1	Soybean 3	13.99	H2	Almond 3	12.91
						A3	Walnut 3	14.34	C2	Coconut 3	19.44
B3	Walnut 3	14.4	D2	Coconut 3	19.59
						G1	Peanut 3	15.41	E1	Hazelnut 3	14.46
H1	Peanut 3	15.41	F1	Hazelnut 3	14.51
						C3	Pumpkin seeds 3	16.46	E3	Negative control	——
D3	Pumpkin seeds 3	16.36	F3	Negative control	——

TABLE 49 fluorescent quantitative PCR amplification results of primers rbc L-F327 & rbc L-R63 plant-derived component rbc L-F & rbc L-R63

2. Determination of Universal primers

According to the result of 9 plant-derived components, the rbc L-F327 & rbc L-R638 primer degenerates for the second time according to the base sequence of coconut, the rbc L-F154 & rbc L-R510 primer degenerates for the second time according to the base sequence of pine nut, the sequences of the primers after degenerating are shown in Table 5, the result of PCR reaction after degenerating is shown in FIG. 1, and the reaction system and reaction conditions of PCR are the same as those of 1.

TABLE 5 Universal primer sequences after double doubling

The fluorescent quantitative PCR reaction system and the reaction conditions were the same as those in 1.2, the results of PCR amplification after the primers rbc L-F327 & rbc L-R63 were degenerated twice are shown in Table 6 and FIG. 4, and the results of PCR amplification after the primers rbc L-F154 & rbc L-R510 were degenerated twice are shown in Table 7 and FIG. 5.

TABLE 6 rbc L-F327 & rbc L-R638 primer doubling twice and then fluorescent quantitative PCR amplification results

Pos	Name	Cp
			A3	Badam	18.5
A4	Walnut (walnut)	15.8
			B3	Soybean	15.26
B4	Pumpkin seed	16.72
			C3	Hazelnut	15.68
D3	Peanut	17.38
			E3	Pine nut	18.82
E4	Negative control	——
			F3	Coconut	17.65
H3	Almond	15.71

TABLE 7 rbc L-F154 & rbc L-R510 primer doubling twice and then fluorescent quantitative PCR amplification results

Through comparison of amplification efficiency of 2 pairs of primers, the primer rbc L-F154 & rbc L-R510 is better than rbc L-F327 & rbc L-R638, the amplification efficiency of 9 plant-derived ingredients of the primer pair is the closest, and finally, the universal primer is determined as follows:

the sequence of the upstream primer is SEQI No. 1: YGCYGAATCTTCYACYGG

The sequence of the downstream primer is SEQI No. 2: TTWGGTTTRATAGTACATCCYAA

The PCR amplification conditions and reaction system are shown in Table 8 and Table 9, respectively.

TABLE 8 PCR amplification conditions

TABLE 9 PCR reaction System

Example 2 determination of sensitivity of the method for sequencing amplicons

Mixing the three plant-derived components of walnut, peanut and soybean according to different proportions to obtain samples 1-13, wherein the mixing proportions are shown in tables 10 and 11, and the information of the barcode added to the samples is shown in table 12.

TABLE 10 blending of walnut with Soybean sample

TABLE 11 blending of walnut with peanut sample blending information

Table 12 sample addition barcode information

Sample numbering	Upstream barcode	Downstream barcode
			Sample 1	CGATGT	TTAGGC
Sample 2	TGACCA	ACAGTG
			Sample 3	GCCAAT	CAGATC
Sample 4	ACTTGA	GATCAG
			Sample
5	TAGCTT	GGCTAC
			Sample
6	CGATGT	ACAGTG
			Sample
7	GCCAAT	GATCAG
			Sample
8	CGATGT	TTAGGC
			Sample
9	TGACCA	ACAGTG
			Sample
10	GCCAAT	CAGATC
			Sample
11	TAGCTT	GGCTAC
			Sample
12	CGATGT	ACAGTG
			Sample
13	GCCAAT	GATCAG

Samples 1-13 were amplified by PCR, the reaction conditions are shown in Table 8, and the reaction system is shown in Table 9.

Sequencing the amplified product by amplicon, and adding soybean amplicon according to a certain proportion as shown in Table 13, and adding peanut amplicon according to a certain proportion as shown in Table 14. The results show that the number of reads in the amplicon sequencing result shows a decreasing trend as the concentration of the template DNA is reduced.

TABLE 13 sequencing results with scaled additions of soybean amplicons

TABLE 14 sequencing results with proportional addition of peanut amplicons

Example 3 testing of primer suitability Using authentic samples

The universal primer and the identification method are adopted to test 10 batches of beverage containing plant components at the same time.

The method comprises the following steps:

(1) and (3) extracting total DNA of the food to be detected, namely adding 1m L isopropanol into 1m L plant beverage, uniformly mixing, standing at room temperature for 5min, centrifuging at 12500rpm for 20min at a high speed, removing supernatant, carrying out sample nucleic acid precipitation, and repeating the operation for three times.

Adding 600u L CTAB Buffer solution and 10ul protease K into a sample, shaking and mixing uniformly, shaking and incubating at 56 ℃ for 30min, adding 500ul phenol, namely a mixed solution of 25:24:1 of chloroform and isoamylol, shaking strongly, centrifuging at 12000g for 15min, sucking supernatant into a new centrifuge tube, adding isopropanol (below 0 ℃) with the same volume, centrifuging at 12000g for 20min after shaking uniformly, discarding supernatant, adding 500ul Buffer AP1 and 4ul RNase A, incubating at 65 ℃ at 950rpm for 10min, adding 130ul Buffer P3, mixing uniformly, placing on ice for 5min, centrifuging at 20000g for 5min, transferring lysate into Ashrededder protein, centrifuging at 20000g for 2min, transferring the centrifuged filtrate into a new EP tube with 1.5ml, adding 1.5-fold AW1, mixing uniformly, adding Dn into Min 650 min, incubating min, transferring the centrifuged filtrate into 6500 min, adding 6500g DNA into a new EP tube, centrifuging the DNA, adding 6500g DNA into a filtration solution with 500ul DNA centrifugation instrument, adding 6500, centrifuging the filtration solution with AW protein A, adding 6500, centrifuging the filtration solution with 500ul DNA, adding 6500, centrifuging the filtration solution, adding the filtration solution with 500ul DNA, diluting the filtration solution with 500ul DNA, centrifuging the filtration solution, adding the filtration concentration of 500ul DNA, adding the filtration solution, adding the filtration.

(2) And (3) PCR amplification: using total DNA as a template, performing PCR amplification by using the universal primer, and recovering and purifying PCR amplification products; the PCR amplification conditions were in agreement with tables 8 and 9.

(3) Sequencing: the sequencing instrument used for sequencing was Illumina Novaseq 6000, and the sequencing strategy was PE250, i.e. paired-end sequencing.

The results are shown in Table 15, and Table 16 shows that various plant-derived components contained in the beverage can be measured by using the universal primers and method of the present invention.

TABLE 1510 batch vegetable protein beverage amplicon sequencing results

TABLE 1610 batch vegetable beverage sample information

In conclusion, the invention applies the DNA barcode technology and the target region amplicon sequencing technology, selects the proper plant universal primer to carry out second-generation sequencing on the PCR product with the specific length, and analyzes the plant-derived information according to the difference in the sequence. According to the research, the detection range of the plant-derived components is enlarged, and meanwhile, the plant components of the product are subjected to non-directional screening only through one-time experiment, so that the detection working strength is reduced, the cost of synthesizing primers and probes is saved, the screening work of large-batch samples containing the plant components can be met, and the defect of market supervision is filled. With the development of the scientific and technological level, the second-generation sequencing technology tends to be perfect, and the prior art needs to be upgraded urgently, so that the development from the detection with targets to the screening without targets is realized, the limitation of the PCR technology can be broken through by the technology, and the level of adulteration detection is improved. According to the currently completed adulteration model and the test result of the sample containing the real plant components, the method is confirmed to be suitable for the detection of the real sample.

While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that the described embodiments may be modified in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are illustrative in nature and should not be construed as limiting the scope of the invention.

Sequence listing

<110> institute for testing and examining Chinese food and drug

<120> universal primer for identifying plant-derived components and application thereof

<160>22

<170>SIPOSequenceListing 1.0

<210>1

<211>18

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>1

ygcygaatct tcyacygg 18

<210>2

<211>23

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>2

ttwggtttra tagtacatcc yaa 23

<210>3

<211>18

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>3

atgcgatact tggtgtga 18

<210>4

<211>18

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>4

tagccccgcctgacctga 18

<210>5

<211>20

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>5

gcagaatccc gcgaatcatc 20

<210>8

<211>18

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>8

cggatatctt ggctcttg 18

<210>6

<211>19

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>6

ccgcttagtg atatgctta 19

<210>7

<211>31

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>7

ggtrcsgagt caaatgctag aaaattcatt t 31

<210>9

<211>25

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>9

cgtacagtac ttttgtgttt acgag 25

<210>10

<211>25

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>10

cgtacagtac ttttgtgttt acgag 25

<210>11

<211>18

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>11

ataagaaacg gtccctcc 18

<210>12

<211>18

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>12

tgccgaatct tctactgg 18

<210>13

<211>24

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>13

ttcggtttaa tagtacagcc caat 24

<210>14

<211>18

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>14

atgygatamy tggtgtga 18

<210>15

<211>17

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>15

tarycccgcc tgacctg 17

<210>16

<211>20

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>16

gcaraatccc gygaaycatc 20

<210>17

<211>18

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>17

cggatatctc ggctctyg 18

<210>18

<211>19

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>18

csgcttattg atatgctta 19

<210>19

<211>24

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>19

gagtcaaatg ytrsaaaatt catt 24

<210>20

<211>25

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>20

cgyrcagtac ttttgtgytt acgag 25

<210>21

<211>24

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>21

tgtttacttc cattgtvggt aatg 24

<210>22

<211>18

<212>DNA

<213> Artificial Sequence (artificial Sequence)

<400>22

ataygaaacg gtctctcc 18

Claims

1. The universal primer for identifying the plant-derived components is characterized in that the sequence of an upstream primer is shown as SEQ No.1, and the sequence of a downstream primer is shown as SEQ No. 2.

2. The universal primer for identifying plant-derived components according to claim 1, wherein the plant-derived components comprise one or more of walnuts, peanuts, soybeans, sesames, hazelnuts, almonds, coconuts, pine nuts, pumpkin seeds and almonds.

3. Use of the universal primer for identifying plant-derived components as claimed in claim 1 or 2 for identifying plant-derived components in food.

4. Use according to claim 3, characterized in that the authentication method comprises the following steps:

(1) extracting total DNA of food to be detected;

(3) sequencing the amplicon;

(4) and determining the components.

5. The use of claim 4, wherein the PCR amplification conditions of step (2) are: maintaining at 95 deg.C for 3min, 95 deg.C for 30s, 57 deg.C for 30s, 72 deg.C for 30s, and 12 deg.C; wherein the temperature of 95 ℃ for 30s, the temperature of 57 ℃ for 30s and the temperature of 72 ℃ for 30s are subjected to 40 cycles.

6. The use of claim 4, wherein the PCR reaction system of step (2) is 10 × PCR buffer5.0 μ L2 μ L, Primer0.4+0.4 μ L, Taq enzyme 0.3 μ L4 μ L, H₂O37.9μL。

7. A kit comprising the universal primer for identifying plant-derived components according to claim 1 or 2.