Primer probe composition for detecting mint component, application and detection method

Abstract

The invention provides a primer probe composition for detecting mint components, application and a detection method, relates to the technical field of molecular biological detection, and designs a primer probe group capable of specifically detecting the mint components on the basis of taking rpl16 genes and psbA genes of mint as targets. By using the primer probe group provided by the invention, the mint component can be specifically detected, and the detection sensitivity of the primer probe group to the mint component can reach 0.01 fg / mu L. The detection method provided by the invention has relatively strong specificity and repeatability, and can provide an effective technical means for detection of the mint component in the perfume component detection process.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biological detection, and in particular to a primer-probe composition for detecting mint components, and its application and detection method. Background Art

[0002] Mint and its extracts occupy a vital position in the flavoring industry, serving as key ingredients in the production of cooling agents and flavoring agents for foods, beverages, cosmetics, and pharmaceuticals. Different products in the flavoring industry require varying proportions and purity levels of mint ingredients, making precise component testing of mint raw materials and their products crucial.

[0003] Real-time PCR (Quantitative Real-time PCR) is a method that adds fluorescent dyes or fluorescent probes to conventional PCR technology, using the accumulated fluorescent signal to monitor the entire PCR process in real time and quantitatively analyze templates of unknown concentrations using a standard curve. As an internationally recognized standard for nucleic acid quantification, real-time PCR is the preferred method for rapidly measuring gene expression levels. It not only represents a significant leap from qualitative to quantitative PCR, but also offers greater sensitivity, specificity, reproducibility, and automation compared to conventional PCR.

[0004] It is particularly important to develop a method based on real-time fluorescence quantitative PCR technology to specifically detect mint components.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a primer-probe combination for detecting mint components, so as to at least solve one of the technical problems existing in the prior art.

[0007] A second object of the present invention is to provide a reagent for detecting mint components.

[0008] A third object of the present invention is to provide a kit for detecting mint components.

[0009] A fourth object of the present invention is to provide the use of the above-mentioned primer-probe combination, reagent or kit in detecting mint components.

[0010] A fifth object of the present invention is to provide a method for detecting mint components.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0012] The present invention provides a primer-probe combination for detecting mint components, comprising at least one of the following primer pairs:

[0013] Primer pair for rpl16 detection: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NOs. 1 and 2, respectively, and the nucleotide sequence of the probe P1 is shown in SEQ ID NO. 3;

[0014] Primer pair for psbA detection: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NOs. 7 and 8, respectively, and the nucleotide sequence of the probe P2 is shown in SEQ ID NO. 9.

[0015] The present invention targets the rpl16 and psbA genes of mint and designs a primer and probe set capable of specifically detecting mint components. Using the primer and probe set of the present invention, mint components can be specifically detected with a sensitivity of 0.01 fg / μL. The detection method of the present invention exhibits strong specificity and reproducibility, providing an effective technical means for detecting mint components in flavoring assays.

[0016] Furthermore, each probe is independently modified with a luminescent group and a quenching group.

[0017] This embodiment does not limit the specific fluorescent reporter group and fluorescent quencher group contained in the probe, and any group that can be connected to the probe and play a corresponding characterization role can be used.

[0018] Furthermore, the 5' end of the fluorescent probe P1 is labeled with a fluorescent group FAM, and the 3' end is labeled with a quencher group MGB; the 5' end of the fluorescent probe P2 is labeled with a fluorescent group FAM, and the 3' end is labeled with a quencher group MGB.

[0019] Furthermore, the working concentrations of the rpl16 detection primer pair and the psbA detection primer pair are independently 8 to 12 μM, for example, but not limited to 8, 9, 10, 11 or 12 μM, or a range between any two of the above points, preferably 10 μM.

[0020] By limiting the working concentrations of primers and probes, we can avoid degradation of primers or probes due to too low working concentrations, and avoid the generation of primer dimers due to too high concentrations. At the same time, within the preferred working concentration range, we can ensure maximum utilization of primers or probes and avoid waste.

[0021] The present invention also provides a reagent for detecting mint components, wherein the reagent comprises the primer-probe combination.

[0022] The present invention also provides a kit for detecting mint components, wherein the kit comprises the above-mentioned primer-probe combination or reagent.

[0023] Furthermore, the kit further comprises at least one of a PCR reaction premix, a positive control reference substance, a negative control reference substance and / or ddH2O.

[0024] The PCR reaction premix includes reagents commonly used in PCR reactions. Specific examples of reaction reagents include enzymes, salts, buffer substances, buffers, dNTPs, and stabilizers for PCR reactions, and the present invention does not limit these reagents. By providing positive and negative controls, false negative or false positive results can be effectively avoided, further ensuring the accuracy of the test.

[0025] The present invention also provides the use of the primer-probe combination, reagent or kit in detecting mint components.

[0026] In addition, the present invention also provides a method for detecting mint components, comprising using the genomic nucleic acid of the sample to be tested as a template, using the above-mentioned primer probe combination, reagent or kit to perform a fluorescent quantitative PCR reaction; collecting the fluorescent signal of the fluorescent quantitative PCR reaction, and using the fluorescent signal for result analysis.

[0027] The method for detecting mint components provided by the present invention performs PCR detection on a sample to be tested using the primer combination or product provided by the present invention, and achieves accurate molecular identification of mint components based on different test results. The method has the characteristics of simple operation, good result specificity, high sensitivity, etc., and has important application value.

[0028] The result analysis includes, but is not limited to, qualitative, semi-quantitative, or quantitative analysis. In an alternative embodiment, the presence of mint components in the sample to be tested is qualitatively determined based on whether an amplification curve appears in the sample to be tested. In an alternative embodiment, the number of mint component copies in the sample to be tested is semi-quantitatively determined based on the Ct value of the sample to be tested, using conventional relative fluorescence quantitative calculation methods known in the art. In an alternative embodiment, the number of mint component copies in the sample to be tested is calculated based on the fluorescence signal by constructing a standard curve.

[0029] Preferably, when the Ct value of the sample detected by the two sets of primer probes is less than 40, and the curve shows an "S"-shaped amplification curve, it can be determined that the sample contains mint components.

[0030] Furthermore, the fluorescence quantitative PCR reaction program includes: a decontamination program of 50°C for 5 minutes, 1 cycle; a holding stage program of 95°C for 5 minutes, 1 cycle; a cycling stage of 95°C for 15 seconds, 60°C for 30 seconds, 40 cycles, and collecting fluorescence signals at 60°C for 30 seconds.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention targets the rpl16 and psbA genes of mint and designs a primer and probe set capable of specifically detecting mint components. Using the primer and probe set of the present invention, mint components can be specifically detected with a sensitivity of 0.01 fg / μL. The detection method of the present invention exhibits strong specificity and reproducibility, providing an effective technical means for detecting mint components in flavoring assays. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 This is the amplification result of the mint component rpl16 primer probe set 1 in the embodiment of the present invention.

[0035] Figure 2 This is the amplification result of the mint component rpl16 primer probe set 2 in the embodiment of the present invention.

[0036] Figure 3 This is the amplification result of the primer probe set 1 for the mint component psbA in the embodiment of the present invention.

[0037] Figure 4 This is the amplification result of the mint component psbA primer probe set 2 in the embodiment of the present invention.

[0038] Figure 5 This is the specific detection of the mint primer rpl16 probe group 1 in the embodiment of the present invention, and the negative controls are: star anise, ground maple bark, large star anise, motherwort, perilla leaf, Eclipta prostrata, and ddH2O.

[0039] Figure 6 This is the specific detection of the mint primer psbA probe group 1 in the embodiment of the present invention, and the negative controls are: star anise, ground maple bark, large star anise, motherwort, perilla leaf, Eclipta prostrata, and ddH2O.

[0040] Figure 7 This is the sensitivity test result of the mint component primer rpl16 probe group 1 in the embodiment of the present invention.

[0041] Figure 8 This is the sensitivity test result of the mint component primer psbA probe group 1 in the embodiment of the present invention.

[0042] Figure 9This is the test result of mint components in real spice samples in the examples of the present invention.

[0043] Figure 10 This is the test result of mint components in real spice samples in the examples of the present invention. DETAILED DESCRIPTION

[0044] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0045] Generally, the nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein and its technology are those well-known and commonly used in this area.Unless otherwise indicated, the methods and techniques of the present invention are generally according to those well-known in the art, and are carried out as described in various general and more specific references, which are cited and discussed throughout this specification.Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in this area, or as described herein.The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein and its laboratory procedures and technology are those well-known and commonly used in this area.

[0046] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0047] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0048] Example 1 Effect of primer sets on detection of mint components

[0049] 1. Experimental Methods

[0050] 1. Plasmid DNA Synthesis

[0051] The gene sequence designed with primers for mint components was connected to the pUC57 vector to construct a plasmid.

[0052] 2. Primer Design

[0053] The whole genome sequence of mint components was searched from Genbank, and the BLAST analysis and comparison were performed. The rpl16 gene was used as the target gene. According to the real-time fluorescence PCR primer probe design principles, specific primers and probes were designed using PrimerExpress primer design software.

[0054] Peppermint rpl16 primer probe set 1 (rpl16-Y1):

[0055] Forward primer ME-1-F: 5′-TGGATCTAAAGAACCAGTCAAGATATGT-3′ (SEQ ID NO: 1);

[0056] Reverse primer ME-1-R: 5′-CCTTTCTTCTTATCCACACCTTTCTT-3′ (SEQ ID NO: 2);

[0057] Fluorescent probe ME-1-P: 5′-AATCAGTCATGTCTTTGTAGCAA-3′ (SEQ ID NO: 3);

[0058] The 5' end of the fluorescent probe ME-1-P is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group MGB.

[0059] Peppermint rpl16 primer probe 2 (rpl16-Y2):

[0060] Forward primer ME-2-F: 5′-TCAGTCATGTCTTTGTAGCAACTGAA-3′ (SEQ ID NO: 4);

[0061] Reverse primer ME-2-R: 5′-TCCTTCCATTTATCCACACCTTTCT-3′ (SEQ ID NO: 5);

[0062] Fluorescent probe ME-2-P: 5′-TTATCCACACCTTTCTTCTGTAA-3′ (SEQ ID NO: 6);

[0063] The 5' end of the fluorescent probe ME-2-P is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group MGB.

[0064] The whole genome sequence of mint components was searched from Genbank, and the sequences were compared by BLAST analysis. The psbA gene was used as the target gene. According to the design principles of real-time fluorescence PCR primers and probes, specific primers and probes were designed using PrimerExpress primer design software.

[0065] Peppermint component psbA primer probe set 1 (psbA-Y1):

[0066] Forward primer ME-3-F: 5′-AGAAAAAGGCTCCTAATTGAAAAGAA-3′ (SEQ ID NO: 7);

[0067] Reverse primer ME-3-R: 5′-AGGAGCAAGAAACCCTTTTTGA-3′ (SEQ ID NO: 8);

[0068] Fluorescent probe ME-3-P: 5′-AGGAGCAAGAAACCCTT-3′ (SEQ ID NO: 9);

[0069] The 5' end of the fluorescent probe ME-3-P is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group MGB.

[0070] Peppermint component psbA primer detection set 2 (psbA-Y2):

[0071] Forward primer ME-4-F: 5′-GAAAAAGGCTCCTAATTGAAAAGAAA-3′ (SEQ ID NO: 10);

[0072] Reverse primer ME-4-R: 5′-AATAGACTAGATAAATATAAGGAGCAAGAAACC-3′ (SEQ ID NO: 11);

[0073] Fluorescent probe ME-4-P: 5′-ACCCTTTCTTGTTTTATCA-3′ (SEQ ID NO: 12);

[0074] The 5' end of the fluorescent probe ME-4-P is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group MGB.

[0075] 3. Perform PCR Reaction

[0076] The reaction system is shown in Table 1.

[0077] Table 1 Real-time fluorescence PCR reaction system

[0078] Reagent name Working fluid concentration Sample volume Final concentration Reaction premix 2× 12.5μL 1× Upstream primer F 10 μM 1 μL 0.4μM Downstream primer R 10 μM 1 μL 0.4 μM Probe P 10 μM 0.5μL 0.2μM DNA template 10–100 ng / μL 5μL - <![CDATA[ddH2O]]> - 5μL -

[0079] The reaction program was as follows: decontamination program at 50°C for 5 min, 1 cycle; holding stage program at 95°C for 5 min, 1 cycle; cycling stage program at 95°C for 15 s, 60°C for 30 s, 40 cycles, collecting fluorescence signals at 60°C for 30 s, and observing the reaction results using a fluorescence PCR instrument.

[0080] 2. Experimental Results

[0081] The results are as follows Figure 1-4 As shown, all primers for rpl16 were able to amplify. Primer set 1 had a small Ct value and good reproducibility, with no nonspecific amplification in negative results. Primer set 2 exhibited negative tailing, confirming that primer probe set 1 was the optimal primer probe set for rpl16. All primers for psbA were able to amplify. Primer set 1 had a small Ct value and good reproducibility, with no nonspecific amplification in negative results. Primer probe set 1 was confirmed as the optimal primer probe set for psbA.

[0082] Example 2 A kit for detecting mint components in spices

[0083] 1. Composition

[0084] 1. Primer probe set 1 for detecting mint components

[0085] Forward primer ME-1-F: 5′-TGGATCTAAAGAACCAGTCAAGATATGT-3′ (SEQ ID NO: 1);

[0086] Reverse primer ME-1-R: 5′-CCTTTCTTCTTATCCACACCTTTCTT-3′ (SEQ ID NO: 2);

[0087] Fluorescent probe ME-1-P: 5′-AATCAGTCATGTCTTTGTAGCAA-3′ (SEQ ID NO: 3);

[0088] The 5' end of the fluorescent probe ME-1-P is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group MGB.

[0089] 2. Primer probe set 2 for detecting mint components

[0090] Forward primer ME-3-F: 5′-AGAAAAAGGCTCCTAATTGAAAAGAA-3′ (SEQ ID NO: 7);

[0091] Reverse primer ME-3-R: 5′-AGGAGCAAGAAACCCTTTTTGA-3′ (SEQ ID NO: 8);

[0092] Fluorescent probe ME-3-P: 5′-AGGAGCAAGAAACCCTT-3′ (SEQ ID NO: 9);

[0093] The 5' end of the fluorescent probe ME-3-P is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group MGB.

[0094] 3. PCR Reagents

[0095] PCR reaction premix, positive control reference, negative control reference;

[0096] The positive control reference material is a plasmid containing the mint component rpl16 gene sequence and the mint component psbA gene sequence; the negative control reference material is ddH2O.

[0097] 2. Usage

[0098] Two sets of mint component primer probe sets were used to perform PCR reaction on the DNA of the test samples. The reaction system is shown in Table 2.

[0099] Table 2 Real-time fluorescence PCR reaction system

[0100] Reagent name Working fluid concentration Sample volume Final concentration Reaction premix 2× 12.5μL 1× Upstream primer F 10 μM 1 μL 0.4μM Downstream primer R 10 μM 1 μL 0.4μM Probe P 10 μM 0.5μL 0.2μM DNA template 10–100 ng / μL 5μL - <![CDATA[ddH2O]]> - 5μL -

[0101] The reaction program was as follows: decontamination program at 50°C for 5 min, 1 cycle; holding stage program at 95°C for 5 min, 1 cycle; cycling stage program at 95°C for 15 s, 60°C for 30 s, 40 cycles, collecting fluorescence signals at 60°C for 30 s, and observing the reaction results using a fluorescence PCR instrument.

[0102] 3. Interpretation Method

[0103] When the Ct value of one or more primer-probe sets is ≥40 or no Ct value is found, and the curve is a straight line or a slightly slanted line with no "S"-shaped amplification curve, it can be determined that the sample does not contain mint components or the content is below the detection limit;

[0104] When the Ct value of the sample detection of the two sets of primer probe groups is less than 40, the curve shows an "S"-shaped amplification curve, which can be determined that the sample contains mint components.

[0105] Example 3 Specificity Experiment

[0106] 1. Experimental Methods

[0107] DNA was extracted from each sample using a commercially available DNA extraction kit, and the purity and concentration of the DNA were measured using a full-wavelength micro-spectrophotometer and stored at -20°C for future use.

[0108] Samples include: star anise, ground maple bark, large star anise, motherwort, perilla leaves, and Eclipta prostrata.

[0109] PCR amplification was performed using the primer and probe set for detecting mint components in the kit of Example 2.

[0110] 2. Experimental Results

[0111] The specificity test results of the primer probe set for mint components showed that primer probe set 1 did not detect any amplified fluorescent signals except for the DNA samples of Perilla leaves and Eclipta prostrata ( Figure 5 ), primer probe set 2 did not detect any amplified fluorescence signal except for the sample DNA of Leonurus japonicus ( Figure 6 ), indicating that the established real-time fluorescence PCR detection method has good specificity.

[0112] Example 4 Sensitivity and Repeatability Experiment

[0113] 1. Experimental Methods

[0114] DNA was extracted from mint using a commercially available DNA extraction kit, and the DNA was quantified on a spectrophotometer, yielding a concentration of 100 ng / μL. The DNA was serially diluted, using seven concentrations of DNA as template: 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, 1 fg / μL, 0.1 fg / μL, and 0.01 fg / μL. Detection was performed using the primer and probe set for detecting mint components in the kit described in Example 2.

[0115] 2. Experimental Results

[0116] The results showed that both primer probe set 1 and primer probe set 2 showed typical amplification curves, and the detection sensitivity of the plasmid containing the rpl16 gene and the psbA gene of the mint component reached 0.01 fg / μL ( Figure 7 、 Figure 8 The results of two parallel experiments for each of the seven dilution gradients showed that the coefficient of variation for primer probe set 1 was between 0.03% and 1.25%, and the coefficient of variation for primer probe set 2 was between 0.02% and 1.14%, indicating that the established real-time fluorescence PCR detection method has good reproducibility (Tables 3 and 4).

[0117] Table 3 Repeatability experiment of rpl16 primer probe set of mint components

[0118]

[0119] Table 4 Repeatability experiment of the psbA primer probe set of mint components

[0120]

[0121] Example 5 Detection of mint components in spice samples

[0122] 1. Experimental Methods

[0123] DNA was extracted from 12 spice samples and the kit of Example 2 was used to detect whether mint components were present.

[0124] 2. Experimental Results

[0125] The DNA of 12 spice samples extracted was tested using the kit of Example 2. The results are as follows: Figure 9 as well as Figure 10 As shown. The results of primer probe set 1 showed that 4 DNA samples tested positive for mint components; 8 spice samples tested negative for mint components, with positive samples 1, 3, 8, and 11. The results of probe set 2 showed that 3 DNA samples tested positive for mint components; 9 spice samples tested negative for mint components, with positive samples 1, 3, and 8. The combined results showed that 3 of the tested samples tested positive for mint components; 9 spice samples tested negative for mint components.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer-probe combination for detecting mint components, characterized in that: Includes at least one of the following primer pairs: Primer pair for rpl16 detection: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NOs. 1 and 2, respectively, and the nucleotide sequence of the probe P1 is shown in SEQ ID NO. 3; Primer pair for psbA detection: the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NOs. 7 and 8, respectively, and the nucleotide sequence of the probe P2 is shown in SEQ ID NO.

9.

2. The primer-probe combination according to claim 1, wherein Each probe is independently modified with a luminescent group and a quenching group.

3. The primer-probe combination according to claim 2, characterized in that The 5' end of the fluorescent probe P1 is labeled with a fluorescent group FAM, and the 3' end is labeled with a quencher group MGB; the 5' end of the fluorescent probe P2 is labeled with a fluorescent group FAM, and the 3' end is labeled with a quencher group MGB.

4. The primer-probe combination according to any one of claims 1 to 3, characterized in that The working concentrations of the rpl16 detection primer pair and the psbA detection primer pair are independently 8 to 12 μM, preferably 10 μM.

5. A reagent for detecting mint components, characterized in that: The reagent comprises the primer-probe combination according to any one of claims 1 to 4.

6. A kit for detecting mint components, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 4 or the reagent according to claim 5.

7. The kit according to claim 6, characterized in that The kit further comprises at least one of a PCR reaction premix, a positive control reference substance, a negative control reference substance and / or ddH2O.

8. Use of the primer-probe combination according to any one of claims 1 to 4, the reagent according to claim 5, or the kit according to claim 6 in detecting mint components.

9. A method for detecting mint components, characterized in that: The method comprises performing a fluorescent quantitative PCR reaction using the genomic nucleic acid of the sample to be tested as a template, using the primer-probe combination according to any one of claims 1 to 4, the reagent according to claim 5, or the kit according to claim 6; collecting a fluorescent signal from the fluorescent quantitative PCR reaction, and using the fluorescent signal for result analysis; Preferably, when the Ct value of the sample detected by the two sets of primer probes is less than 40, and the curve shows an "S"-shaped amplification curve, it can be determined that the sample contains mint components.

10. The method for detecting mint components according to claim 9, characterized in that: The fluorescence quantitative PCR reaction program includes: a decontamination program of 50°C for 5 minutes, 1 cycle; a holding stage program of 95°C for 5 minutes, 1 cycle; a cycling stage of 95°C for 15 seconds, 60°C for 30 seconds, 40 cycles, and collecting fluorescence signals at 60°C for 30 seconds.

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