CN114350831A - Primer probe and method for detecting cocoa-derived components in cocoa powder - Google Patents
Primer probe and method for detecting cocoa-derived components in cocoa powder Download PDFInfo
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Abstract
The present invention relates to a real-time fluorescent PCR detection method for qualitatively detecting cocoa-derived components in cocoa powder, which comprises using specific oligonucleotide primers and fluorescently labeled probes for cocoa components in cocoa powder. The invention also relates to application of specific oligonucleotide primers and fluorescent probes for cocoa components in cocoa powder in qualitative detection of cocoa-derived components. The real-time fluorescent PCR detection method can accurately and sensitively detect the cocoa-derived components in the cocoa powder.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an oligonucleotide primer and a fluorescence labeling probe for detecting cocoa-derived components, a real-time fluorescence PCR detection method for detecting cocoa-derived components, and application of a specific oligonucleotide primer and a specific probe for detecting cocoa-derived components in a sample in detection of cocoa-derived components.
Background
Cocoa powder is a cocoa product made directly from cocoa beans. The cocoa powder has unique flavor and strong fragrance, is rich in various nutrient elements and active ingredients such as carbohydrate, cocoa butter, polyphenol, protein and the like, and is an important raw material for manufacturing chocolate. With the rapid development of cocoa processing industry, the phenomenon of adulteration of counterfeit or inferior cocoa powder exists in domestic and foreign markets, which not only influences the health benefits of consumers, but also damages the order of ordered markets.
In order to realize the authenticity identification of the cocoa powder, a document establishes a quick detection method for mixing the carob bean powder in the cocoa powder based on a near infrared spectrum technology, and a research establishes a visual detection method for mixing the peanut powder in the cocoa powder by adopting a near infrared hyperspectral technology. Although the pretreatment by the spectroscopy is simple and the detection speed is high, the requirement on the representativeness of the sample is high, and a statistical model needs to be constructed. The modern biotechnology analyzes the sources and characteristics of raw materials and products from the gene level by the characteristics of convenience, rapidness, accuracy and the like, has strong specificity and high sensitivity, and is widely applied to food identification research such as food type identification, origin tracing and the like. At present, the conventional PCR detection method for mixing exogenous plant sources such as soybean meal, sesame meal, peanut shells, chestnut shells and the like into cocoa powder is researched and established. However, the conventional PCR technique requires agarose gel electrophoresis, EB staining and other steps, and the operation is time-consuming and labor-consuming. In recent years, real-time fluorescent PCR is a gold-labeled method for identifying animal and plant source components in food, has strong specificity and high sensitivity, and forms various national standards, industrial standards and the like.
At present, no method for quickly, specifically and sensitively detecting cocoa-derived components in cocoa powder is available at home and abroad.
Therefore, there is a need in the art for a rapid, specific, and sensitive method for detecting cocoa-derived components in cocoa powder.
Disclosure of Invention
It is an object of the present invention to provide specific oligonucleotide primers and fluorescently labeled probes for accurate qualitative detection of cocoa-derived components.
Another objective of the present invention is to provide a real-time fluorescence PCR detection method for accurately and qualitatively determining cocoa-derived components.
Still another object of the present invention is to provide the use of specific oligonucleotide primers and probes for cocoa-derived components in the accurate qualitative detection of cocoa-derived components in food.
Aiming at the above purpose, the invention provides the following technical scheme:
the inventor designs an oligonucleotide primer pair and a probe capable of specifically identifying cocoa-derived components according to a cocoa microsatellite DNA sequence, and can efficiently and specifically amplify a short section of cocoa-specific gene fragment from sample DNA. According to one embodiment of the invention, the invention provides specific oligonucleotide primer pairs and fluorescently labeled probes for detecting cocoa-derived components by a real-time fluorescent PCR method, wherein the primer pairs and the probes are designed according to the characteristic that the sequences of microsatellite DNA have difference in different species. The primer pair consists of an upstream primer and a downstream primer, wherein the upstream primer is a primer consisting of Cocoa-F: TGGACATGCACTGATGAGAGA (SEQ ID No.1), the downstream primer being Cocoa-R: GCTTTCAGTCCTTTGCTT (SEQ ID No. 2); the probe is Cocoa-P: GAGAGGGTGGGAACCTTAGC (SEQ ID No.3), a fluorescence quenching group TAMRA is connected to the 3 'end of the probe, and a fluorescence reporter group FAM is connected to the 5' end. In one embodiment, the invention provides a cocoa-specific detection composition comprising a specific oligonucleotide primer pair and a probe. In a preferred embodiment, the invention provides a composition for qualitatively detecting cocoa-derived components by a real-time fluorescent PCR method, wherein the composition comprises a cocoa-specific oligonucleotide primer pair and a probe, wherein the cocoa-specific primer pair consists of an upstream primer and a downstream primer, the base sequence of the upstream primer is SEQ ID No.1, and the base sequence of the downstream primer is SEQ ID No. 2; the base sequence of the probe is SEQ ID No.3, the 3 'end of the probe is connected with a fluorescence quenching group TAMRA, and the 5' end of the probe is connected with a fluorescence reporting group FAM.
According to another embodiment of the invention, the invention provides a real-time fluorescence PCR qualitative detection method of cocoa-derived components, which comprises using specific oligonucleotide primer pairs and probes for cocoa-derived components, wherein the primer pairs and probes are designed according to the characteristic that the sequences of microsatellite DNA have difference in different species. In one embodiment, in the method for real-time fluorescence PCR qualitative detection of cocoa-derived component of the present invention, the used cocoa-specific oligonucleotide primer pair consists of an upstream primer and a downstream primer, wherein the base sequence of the upstream primer is SEQ ID No.1, and the base sequence of the downstream primer is SEQ ID No. 2; the base sequence of the probe is SEQ ID No.3, the 3 'end of the probe is connected with a fluorescence quenching group TAMRA, and the 5' end of the probe is connected with a fluorescence reporting group FAM. In one embodiment, the PCR amplification conditions are 95 ℃ for 10 min; at 95 ℃ for 15s and 60 ℃ for 1min, for 40 cycles.
According to yet another embodiment of the present invention, the present invention provides the use of specific oligonucleotide primers and probes for the detection of cocoa-derived components in food products by real-time fluorescent PCR method. In a preferred embodiment, the invention provides specific oligonucleotide primer pairs SEQ ID No.1 and SEQ ID No.2 and a specific probe SEQ ID No.3 of cocoa-derived ingredients in cocoa powder, wherein a fluorescence quenching group TAMRA is connected to the 3 'end of a cocoa microsatellite DNA probe, and a fluorescence reporter group FAM is connected to the 5' end of the cocoa microsatellite DNA probe.
According to the invention, the cocoa DNA sequence is compared and analyzed based on the detection of the cocoa DNA and according to the characteristic that the micro-template DNA sequence has difference in different species. Primers were designed based on these sequences and real-time fluorescent PCR was used to qualitatively detect cocoa-derived components in samples.
Real-time fluorescent quantitative PCR is that on the basis of a conventional PCR method, a probe or a fluorescent dye which is fluorescently labeled is added, a fluorescent signal emitted by the probe or the dye is enhanced along with the accumulation of a PCR product, and a fluorescent monitoring system can receive the fluorescent signal, namely, one fluorescent molecule is formed every time one DNA chain is generated, so that the complete synchronization of the accumulation of the fluorescent signal and the formation of the PCR product is realized. Therefore, the whole PCR reaction process can be monitored in real time, and the initial copy number of the sample to be detected can be finally detected, so that the cocoa-derived components contained in the cocoa powder to be detected can be detected.
The real-time fluorescent PCR detection method adopts complete closed-tube detection, does not need PCR post-treatment, and avoids cross contamination and false positive. The method skillfully utilizes the high-efficiency DNA amplification of the PCR technology, the specificity of nucleic acid hybridization and the rapidness and the sensitivity of the fluorescence detection technology, and has the advantages of simple operation, time and labor saving, reliable result, accuracy and sensitivity and the like. The PCR detection method can be used for qualitative detection, and has the characteristics of simplicity, rapidness, specificity and sensitivity, and is suitable for detection of cocoa-derived components in cocoa powder in markets at home and abroad and the like.
Drawings
FIG. 1 shows the results of specific detection of cocoa components by real-time fluorescent PCR using specific oligonucleotide primer pairs SEQ ID No.1 and SEQ ID No.2 and SEQ ID No.3, wherein the amplification curve of cocoa samples is above the baseline and 11 plant samples of potato, corn, rice, millet, mung bean, buckwheat, soybean, hazelnut, sesame, walnut, etc. and blank control (sterile water) are below the baseline.
FIG. 2 is a graph showing the sensitivity of real-time fluorescent PCR specific detection of cocoa components, in which a 10-fold gradient of cocoa DNA solution was diluted sequentially to 50 ng/. mu.L, 5 ng/. mu.L, 0.5 ng/. mu.L, 0.05 ng/. mu.L, 0.005 ng/. mu.L, 0.0005 ng/. mu.L and a blank (sterile water).
FIG. 3 shows the results of the detection of commercial cocoa samples using the real-time fluorescent PCR method established in the present invention. Above the baseline are positive control (cacao DNA), commercial cacao powder 3 parts (1#, 2#, 3#) and blank control (sterile water).
Detailed Description
The present invention will be further described by way of examples, but the present invention is not limited to only the following examples.
Example 1
This example is a specificity and sensitivity evaluation of the primer pairs and probes of cocoa by the following assay.
By detecting the microsatellite DNA sequence, the specificity and detection sensitivity of the cocoa primer probe combination can be determined. The reaction system is as follows: fast Start Universal PCR Master Mix 12.5. mu.L; probe (10. mu.M) 0.5. mu.L; 0.5. mu.L of each of the upstream and downstream primers (10. mu.M); 5 mu L of template DNA; sterile water was added to a total volume of 25. mu.L. The reaction program is 95 ℃ for 10 min; 15s at 95 ℃; 60 ℃ for 1min, 40 cycles.
The sequences of the primers and probes used for detecting cacao are as follows:
the primer sequences are SEQ ID No.1 and SEQ ID No.2, the probe sequence is SEQ ID No.3, the 3 'end is connected with a fluorescence quenching group TAMRA, and the 5' end is connected with a fluorescence reporter group FAM.
The detection main instruments used:
micropipettes (10. mu.L, 100. mu.L, 1000. mu.L), fluorescent quantitative PCR instruments, high-speed bench centrifuges, and the like.
Detecting main reagents:
chloroform, isopropanol, CTAB lysate (20g/L CTAB, 1.4mol/L NaCl, 0.1mol/L Tris, 0.02mol/L Na)2EDTA), CTAB precipitation solution (5g/L CTAB, 0.04mol/L NaCl), 1.2mol/L NaCl, Fast Start Universal Probe Master Mix (Rox); the primers and probes were synthesized by Biotechnology engineering (Shanghai) Inc.
The detection comprises the following main steps:
1 DNA extraction
Detecting a sample: (1) 12 samples such as cocoa, potato, corn, rice, millet, mung bean, buckwheat, soybean, hazelnut, sesame, walnut and the like are used for specificity analysis; (2) diluting the extracted cocoa DNA solution with sterile water to concentrations of 50 ng/. mu.L, 5 ng/. mu.L, 0.5 ng/. mu.L, 0.05 ng/. mu.L, 0.005 ng/. mu.L and 0.0005 ng/. mu.L respectively for analyzing the detection sensitivity of the primer probe combination; (3) 3 portions of cocoa powder samples sold in the market are detected by the established real-time fluorescent PCR method, and the feasibility of the method is verified.
Weighing 0.1g of sample powder into a clean 2.0mL centrifuge tube, adding 1.5mL CTAB lysate, inverting at 65 ℃ for 1h, and mixing for several times; 8000rpm for 15min, taking 1mL of supernatant into 1 clean 2.0mL centrifuge tube, adding 700 μ L of chloroform, violently mixing for 30s, 14500rpm for 10min, respectively taking 650 μ L of supernatant into a clean 2.0mL centrifuge tube, adding 1300 μ L of CTAB precipitation, violently mixing for 30s, and standing at room temperature for 1 h; 14500rpm for 10min, discarding the supernatant, adding 350 μ L1.2M NaCl, shaking vigorously for 30s, adding 350 μ L chloroform, mixing vigorously for 30s, 14500rpm for 10 min; respectively taking 320 mu L of supernatant, adding 0.8 times volume of isopropanol, mixing uniformly, then removing the supernatant at minus 20 ℃ for 1h, 14500rpm for 20min, adding 500 mu L of 70% ethanol, mixing uniformly, then 14500rpm for 20min, removing the supernatant, airing to air dry, adding 100 mu L of sterile water for dissolving, and storing at 4 ℃ for later use.
2 primers and probes for real-time fluorescent PCR detection
The primer sequences are SEQ ID No.1 and SEQ ID No. 2;
the probe sequence is SEQ ID No.3, the 3 'end is connected with a fluorescence quenching group TAMRA, and the 5' end is connected with a fluorescence reporting group FAM.
3, real-time fluorescent PCR reaction system:
Fast Start Universal PCR Master Mix 12.5μL
probe (10. mu.M) 0.5. mu.L
0.5. mu.L of upstream primer (10. mu.M)
Downstream primer (10. mu.M) 0.5. mu.L
Template DNA 5. mu.L
Adding sterile water to make the total volume to be 25 μ L
Note: setting corresponding blank control in each PCR detection (using ultrapure water for preparing a reaction system to replace a DNA template to detect whether a reagent is polluted);
4 real-time fluorescent PCR reaction parameters:
95℃ 10min
95℃ 15s
60℃ 1min
40 cycles.
Note: the reagents and reaction parameters of PCR should be adjusted properly for different instruments.
As shown in fig. 1, when the assay of the cocoa microsatellite DNA sequence was specifically performed by real-time fluorescent PCR, the samples were: no amplification curve appears in 11 samples such as potato, corn, rice, millet, mung bean, buckwheat, soybean, sesame, walnut and the like and blank control (sterile water), and the primer probe designed in the experiment is specific to the cocoa sample.
To determine the detection limit of the combination of cocoa-specific primer probes, the extracted cocoa DNA solutions were diluted with sterile water to concentrations of 50 ng/. mu.L, 5 ng/. mu.L, 0.5 ng/. mu.L, 0.05 ng/. mu.L, 0.005 ng/. mu.L, and 0.0005 ng/. mu.L, respectively, and real-time fluorescence PCR amplification was performed under the above conditions, and the results are shown in FIG. 2. The specific amplification curves were observed at cocoa DNA concentrations of 50 ng/. mu.L, 5 ng/. mu.L, 0.5 ng/. mu.L, 0.05 ng/. mu.L, and 0.005 ng/. mu.L, while no specific amplification curve appeared at concentrations below 0.005 ng/. mu.L. The result shows that the detection limit of the real-time fluorescence PCR detection method established by the patent for detecting the cocoa component is 0.005 ng/. mu.L.
3 portions of cocoa powder samples (1# -3#) sold in the market are detected by the established real-time fluorescence PCR method, and the feasibility of the method is verified (figure 3). The results indicate that the method can be used to detect cocoa-derived components in cocoa powder.
While particular embodiments of the present invention have been described, those skilled in the art will recognize that many changes and modifications may be made thereto without departing from the scope or spirit of the invention. Accordingly, it is intended to cover all such changes and modifications that fall within the scope of the appended claims and equivalents thereof.
Claims (4)
1. Specific oligonucleotide primer pairs and fluorescently labeled probe compositions for detecting Cocoa-derived components by a real-time fluorescent PCR method, wherein the primer pairs are Cocoa-F: TGGACATGCACTGATGAGAGA, the downstream primer is Cocoa-R: GCTTTCAGTCCTTTGCTT, respectively; the probe is Cocoa-P: GAGAGGGTGGGAACCTTAGC are provided.
2. The composition of claim 1, wherein the probe has a fluorescence quencher TAMRA attached to its 3 'end and a fluorescence reporter FAM attached to its 5' end.
3. A real-time fluorescent PCR method for qualitative detection of cocoa-derived components, said method comprising using the primer probe composition of claims 1-2.
4. Use of the primer probe composition of claims 1-2 for detecting cocoa-derived components in cocoa powder.
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