A SYBR Green fluorescent quantitative PCR method for identifying cyperus esculentus ingredients based on Ce44 gene and application thereof

Abstract

The application discloses a SYBR Green fluorescent quantitative PCR method for identifying Cyperus esculentus components based on a Ce44 gene and application thereof, and establishes a specific and high-sensitivity Cyperus esculentus component detection method by using a single copy gene of a Cyperus esculentus plant genome as a marker gene based on a SYBR Green real-time fluorescent quantitative technology.The method can accurately distinguish the Cyperus esculentus from four Cyperaceae plants and other 12 representative plants, and can accurately determine whether the six food samples on the market contain the Cyperus esculentus component.The specific real-time fluorescent quantitative PCR detection method for the Cyperus esculentus established by the application can provide a technical reference for qualitative and quantitative detection of the Cyperus esculentus component in Cyperus esculentus products.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a SYBR Green real-time PCR method for identifying tiger nuts based on the Ce44 gene and its application. Background Technology

[0002] Tiger nuts (scientific name: *Cyperus esculentus* L.) have become a research hotspot worldwide, particularly for the comprehensive development of edible products from their tubers. Countries like India, Algeria, France, and Spain started their research into the edible value of tiger nuts earlier, and have already produced various products such as pastries, candies, and beverages for sale. However, my country's development and utilization of tiger nuts lags behind, with research mainly focusing on cultivation techniques, nutritional analysis, and oil extraction. Research into edible products other than oil extraction from tiger nuts is still in its early stages.

[0003] Traditional pressing equipment and processes result in low oil yields, and the lack of advanced refining technology and equipment for tiger nut oil leads to high production costs. Furthermore, tiger nut has high nutritional value, and many unscrupulous vendors in the market mix or pass off low-value products as high-value ones to obtain higher profits. Products marketed as tiger nut are often difficult to distinguish from genuine tiger nut products by appearance after processing; for example, soybean flour may be used to imitate tiger nut flour, making it impossible to tell the difference visually. However, its nutritional value is greatly reduced, deceiving consumers who do not receive the necessary nutritional benefits.

[0004] Therefore, establishing a method that can specifically detect tiger nuts would help in the identification of tiger nut components, prevent counterfeiting, and protect consumer interests and health. Summary of the Invention

[0005] To address the problems in existing technologies, the inventors obtained DNA molecular markers that represent the genome copy number and cell number of tiger nuts through repeated experiments and explorations. They designed amplification primers with high sensitivity and specificity and explored a suitable amplification system and procedure for these primers, establishing the SYBR Green real-time PCR method that can accurately identify and quantify tiger nut components in products.

[0006] One aspect of this invention provides a primer composition for identifying tiger nuts, characterized in that the primers are an upstream primer Ce44-F40 and a downstream primer Ce44-R147, the sequence of the upstream primer Ce44-F40 is as shown in SEQ ID NO.1, and the sequence of the downstream primer Ce44-R147 is as shown in SEQ ID NO.2. The specific sequences of the primers and probe are as follows:

[0007] SEQ ID NO.1: GCCAT TTTAC TTGTAGTGGT GTTCA 25,

[0008] SEQ ID NO. 2: TCGGC TCGTC ATTTC TTCG 19.

[0009] Another aspect of the present invention provides a SYBR Green fluorescent PCR method for identifying tiger nuts, wherein the SYBR Green fluorescent quantitative PCR method uses the above-mentioned primer composition and SYBR Green amplification system for amplification.

[0010] Furthermore, the SYBR Green fluorescent PCR method specifically includes the following steps:

[0011] S1) Obtain the genomic DNA of the sample to be identified;

[0012] S2) The genomic DNA extracted in step 1) was amplified using a real-time fluorescence quantitative PCR reaction system containing the above primer composition;

[0013] S3) If a sample shows specific amplification, it is confirmed that the sample to be identified contains tiger nuts.

[0014] Furthermore, the real-time quantitative PCR reaction system in S2 also contains SYBR Green.

[0015] Further, the reaction system in step S2) is as follows: 1 μL DNA template, 10 μL qPCR SYBR Green MasterMix, 0.4 μL each of 10 μM forward and reverse primers, and ddH2O added to make up to 20 μL.

[0016] Furthermore, the Ct value amplified in S2) is less than 40, for example less than 38 or 36.

[0017] Furthermore, the concentration of genomic DNA in S1) was adjusted to the range of 0.032 ng / μL-20 ng / μL.

[0018] Further, step S2) uses quantitative real-time PCR for amplification. The reaction conditions for quantitative real-time PCR are: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, for 40 cycles.

[0019] Another aspect of the present invention provides a SYBR Green real-time PCR method for identifying tiger nuts, the SYBR Green real-time PCR method specifically comprising the following steps:

[0020] S1) Obtain the genomic DNA of the sample to be identified; and perform quantitative gradient dilution;

[0021] S2) Amplify genomic DNA of different concentrations extracted and diluted in step 1) using a real-time fluorescence quantitative PCR reaction system containing the above primer composition;

[0022] S3) Amplify DNA samples of known concentrations using the reaction system of S2), and plot a standard curve based on fluorescence intensity;

[0023] S4) The fluorescence intensity values ​​of genomic DNA obtained in step S2) are used to perform regression calculations on the standard curve obtained in step S3) to calculate the genomic DNA content in the sample.

[0024] Further, the reaction system in step S2) is as follows: 1 μL DNA template, 10 μL qPCR SYBR Green MasterMix, 0.4 μL each of 10 μM forward and reverse primers, and ddH2O added to make up to 20 μL.

[0025] Furthermore, the Ct value amplified in S2) is less than 40, for example less than 38 or 36.

[0026] Furthermore, the concentration of genomic DNA in S1) was adjusted to the range of 0.032 ng / μL-20 ng / μL.

[0027] Further, step S2) uses quantitative real-time PCR for amplification. The reaction conditions for quantitative real-time PCR are: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, for 40 cycles.

[0028] In another aspect, the present invention provides a molecular marker for identifying tiger nuts, said molecular marker being amplified by the primer composition described above.

[0029] In another aspect, the present invention provides the use of a molecular marker for identifying tiger nuts in a sample, said molecular marker being amplified by the primer composition described above.

[0030] In another aspect, the present invention provides the use of a primer composition for identifying tiger nuts in a sample.

[0031] Furthermore, the sample may be a plant specimen, plant seed, plant or animal sample particles or powder, or a liquid containing plant or animal samples.

[0032] In another aspect, the present invention provides a kit comprising the primer composition described above for identifying tiger nuts, wherein the sequence of the upstream primer Ce44-F40 is as shown in SEQ ID NO.1 and the sequence of the downstream primer Ce44-R147 is as shown in SEQ ID NO.2.

[0033] Furthermore, the kit also contains SYBR Green.

[0034] In another aspect, the present invention provides the use of a molecular marker for identifying tiger nuts in the preparation of a kit for identifying tiger nuts in a sample.

[0035] In another aspect, the present invention provides the use of primers for identifying tiger nuts in the preparation of a kit for identifying tiger nut components in a sample.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] 1. A nucleic acid-based detection method was established. Compared to protein detection, DNA is not affected by high temperatures and complex processing conditions, nor by mixed refined samples. It is also easier to obtain the same detection results from the same plant and tissue, making it more reliable.

[0038] 2. The detection target is a single-copy gene in the nuclear genome. A single-copy gene exists singly in the cell nucleus of the animal. This gene can represent the genome copy number and cell number of tiger nuts, which is more quantitative than many current species detection methods based on mitochondrial genes.

[0039] 3. The established quantitative detection method only requires observation of the amplification curve and Ct value, eliminating the need for cumbersome gel electrophoresis experiments and avoiding contamination during the opening of PCR products. It is simple to operate, has low time costs, and is particularly suitable for relevant testing departments to detect product components and conduct random sampling of mixed refined samples from the market.

[0040] 4. The fluorescence quantitative method based on SYBR Green established in this study has higher specificity than the dye method.

[0041] 5. The species-specific PCR detection method for tiger nuts established in this study can be used not only for DNA samples extracted in plant form, but also for commonly used processed tiger nut products on the market, which is of great significance for maintaining the food market.

[0042] 6. This invention provides a foundation for further in-depth research and exploration of the relationship between Ct value and the exact content of tiger nuts in more complex mixed processed foods.

[0043] 7. This invention conducted a cross-sectional specificity study by validating four plants of the same family as tiger nuts, two model organisms, seven oilseed crops, and three other crops. The primer combination used in this invention can specifically identify tiger nuts with high specificity. Furthermore, the detection results for 16 different tiger nut species demonstrate the intraspecific stability of the primer pair combination of this invention.

[0044] 8. This invention has found that although the amplification of the target gene can be achieved by using 3 upstream primers and 2 downstream primers, and the amplified parts overlap, the parallelism of amplification varies greatly depending on the combination. The primer pairs of this invention can achieve a significant improvement in amplification parallelism. Attached Figure Description

[0045] Figure 1 The results of real-time quantitative PCR amplification of genomic DNA from 16 plant samples using 18S rDNA primers, and the results of real-time quantitative PCR amplification of 4 Cyperaceae plants (Cyperus rotundus, water chestnut, Cyperus difformis, and Cyperus rotundus), 2 model organisms (Arabidopsis thaliana and tobacco), 7 oil crops (rapeseed, maize, soybean, peanut, sunflower, oil palm, and safflower), and 3 other crops (rice, wheat, and potato) using 18S rDNA primers.

[0046] Figure 2 The image shows the results of BLAST analysis of sequences from the NCBI Genome Database.

[0047] Figure 3 Results of real-time quantitative PCR amplification of tiger genomic DNA using 6 primer pairs.

[0048] Figure 4 Real-time quantitative PCR amplification results of genomic DNA from 16 tiger nut varieties using the Ce44-F40 / Ce44-R147 primer pair.

[0049] Figure 5 Real-time quantitative PCR amplification results of 16 plants (4 sedges: Cyperus rotundus, water chestnut, Cyperus difformis, and Cyperus rotundus; 12 other plants: Arabidopsis thaliana, tobacco, rapeseed, maize, soybean, peanut, sunflower, oil palm, safflower, rice, wheat, and potato) using the Ce44-F40 / Ce44-R147 primer pair.

[0050] Figure 6 Real-time quantitative PCR amplification curves of tiger pea genomic DNA concentration gradient dilution.

[0051] Figure 7 Standard curves were plotted using real-time quantitative PCR amplification of tiger genomic DNA at gradient dilution concentrations.

[0052] Figure 8The real-time fluorescence quantitative amplification results of six food samples using the Ce44-F40 / Ce44-R147 primer pair are shown in the figure. Samples 1-6 correspond to commercially available tiger nut powder, commercially available tiger nut flour, commercially available refined tiger nuts, commercially available tiger nut powder, supermarket lotus root powder, and supermarket black sesame paste powder, respectively. Detailed Implementation

[0053] To better understand the technical solution of the present invention, the technical solution provided by the present invention will be described in detail below with reference to embodiments.

[0054] Example 1: DNA Extraction and Amplifiability Experiment

[0055] 1. Experimental Materials

[0056] 1.1 Plant materials

[0057] The tiger nut (Cyperus esculentus L.) used in the experiment. The other 16 different plant materials included: nutgrass (Cyperus rotundus), water chestnut (Eleocharis dulcis), windmill grass (Cyperus involucratus), rice grass (Cyperus iria), Arabidopsis thaliana, tobacco (Nicotiana tabacum), rapeseed (Brassica napus), corn (Zea mays), soybean (Glycine max), peanut (Arachis hypogaea), sunflower (Helianthus annuus), oil palm (Elaeis guineensis), safflower (Carthamus tinctorius), rice (Oryza sativa), wheat (Triticum aestivum), and potato (Solanum tuberosum).

[0058] 1.2 Enzymes and Reagents

[0059] The HieffUNICON Universal Blue qPCR SYBR Green Master Mix (real-time quantitative PCR amplification premix solution) was purchased from Shanghai Yisheng Biotechnology Co., Ltd., catalog number 11184ES08.

[0060] Chloroform, isoamyl alcohol, ethanol, and NaCl were domestically produced analytical grade reagents, all purchased from Sinopharm Chemical Reagent Co., Ltd., while Tris, EDTA, CTAB, SDS, etc., were purchased from Sigma-Aldrich.

[0061] The primers required for this experiment were synthesized by the Wuhan Synthesis Department of Beijing Qingke Biotechnology Co., Ltd.

[0062] 1.3 Experimental Apparatus

[0063] Real-time quantitative PCR instrument: CFX Connect (BIO-RAD);

[0064] NanoPhotometer-N80 (Implen) Ultraviolet Spectrophotometer;

[0065] Other instruments include: constant temperature water bath, centrifuge, vortex mixer, ultrapure water system, etc.

[0066] 2 Experimental Methods

[0067] 2.1 Extraction of plant genomic DNA

[0068] Plant genomic DNA was extracted using Magen's RaPure Plant DNA Kit (catalog number D3187), and the specific procedures are as follows:

[0069] 1. Grind the plant sample into powder using liquid nitrogen, and transfer 50-100 mg of fresh / frozen sample or 15-30 mg of dried sample into a 2 mL centrifuge tube.

[0070] 2. Immediately add 700 μL of preheated 65°C Buffer PAL to the sample, vortex vigorously to fully disperse the sample, and incubate in a 65°C water bath for 20 minutes, mixing 2-3 times during the process.

[0071] 3. Add 700 μL of chloroform to the sample and vortex mix for 15 seconds.

[0072] 4. Centrifuge at 12,000×g for 5 minutes at room temperature, and carefully transfer the supernatant to a new centrifuge tube. (To completely remove RNA, add 10 μL of RNase A to the supernatant, invert to mix, and incubate at room temperature for 5–10 minutes.)

[0073] 5. Add 700 μL of Buffer GWP to the supernatant and mix by inverting 10 to 15 times.

[0074] 6. Load the DNA column into the collection tube and transfer half the volume of the mixture into the column. Centrifuge at 12,000×g for 60 seconds.

[0075] 7. Discard the filtrate, reassemble the column into the collection tube, and transfer the remaining mixture into the column. Centrifuge at 12,000 × g for 60 seconds.

[0076] 8. Discard the filtrate, reattach the column to the collection tube, and add 500 μL of Buffer PW1 to the column. Centrifuge at 12,000 × g for 60 seconds.

[0077] 9. Discard the filtrate, reassemble the column into the collection tube, and add 500 μL of Buffer PW2 to the column. Centrifuge at 12,000 × g for 60 seconds.

[0078] 10. Discard the filtrate, reassemble the column into the collection tube, and add 500 μL of Buffer PW2 to the column. Centrifuge at 12,000 × g for 60 seconds.

[0079] 11. Discard the filtrate and reassemble the column into the collection tube. Centrifuge at 12,000 × g for 2 minutes to remove any residual ethanol from the column.

[0080] 12. Transfer the column to a new 1.5 mL centrifuge tube, add 40–75 μL of preheated 65 °C Buffer AE to the center of the membrane in the column. Let stand at room temperature for 2 minutes, then centrifuge at 12,000 × g for 1 minute.

[0081] 13. (Optional) Add 40–75 μL of preheated Buffer AE (65°C) to the center of the membrane in the column. Let stand for 2 minutes. Centrifuge at 12,000 × g for 1 minute.

[0082] 14. Discard the DNA binding column and store the DNA at -20°C.

[0083] 2.2 Real-time quantitative PCR amplification

[0084] Using the genomic DNA of the plant material described in 1.1 as a template, the plant genomic DNA was amplified by real-time quantitative PCR using 18S rDNA primers. The amplifiability of the extracted genomic DNA was determined based on the amplification curve and melting point curve. Primer information and amplified fragment sizes are shown in Table 1.

[0085] Using the genomic DNA of the plant material described in 1.1 as a template, the plant genomic DNA was amplified by real-time quantitative PCR using 18S rDNA primers. The amplifiability of the extracted genomic DNA was determined based on the amplification curve and melting point curve. Primer information and amplified fragment sizes are shown in Table 1.

[0086] Table 1. 18S rDNA primer information and amplified fragment size

[0087]

[0088] The real-time quantitative PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, 40 cycles; plate reading every 0.5℃ from 65 to 95℃, and incubation at 16℃.

[0089] The real-time quantitative PCR reaction system was as follows: 1 μL DNA template, 10 μL Hieff UNICON Universal Blue qPCRSYBR Green Master Mix, 0.4 μL each of 10 μM forward and reverse primers, and ddH2O to a final volume of 20 μL.

[0090] 3 Experimental Results

[0091] 3.1 Plant genomic DNA concentration

[0092] The concentration and purity of the extracted genomic DNA were determined using an ultra-micro UV spectrophotometer. The results are shown in Table 2. As can be seen from the table, the concentration and purity of the extracted genomic DNA both meet the amplification requirements.

[0093] Table 2. Results of DNA concentration determination for different samples

[0094]

[0095]

[0096] 3.2 Plant genomic DNA amplification

[0097] Real-time quantitative PCR amplification of extracted plant genomic DNA was performed using primers 18S rDNA F and 18S rDNA R. The melting temperature in the melting curve and the Ct value in the amplification curve are shown below. Figure 1 Table 3 shows that the genomic DNA extracted from the four Cyperaceae plants and 12 other plants was amplified by primers 18S rDNA F and 18S rDNA R. In addition, the Ct values ​​were all less than 36, indicating that the concentration and purity of the genomic DNA in all samples met the requirements for real-time quantitative PCR amplification and were amplifiable.

[0098] Table 3. Results of real-time quantitative PCR amplification of 16 plant genomic DNA samples.

[0099]

[0100]

[0101] Example 2: Primer screening for specific amplification of tiger nut components

[0102] 1. Experimental Materials

[0103] 1.1 Tiger bean genomic DNA

[0104] 1.2 Enzymes and Reagents

[0105] Molecular biology reagents, Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (real-time quantitative PCR amplification premix solution), were purchased from Shanghai Yisheng Biotechnology Co., Ltd., catalog number 11184ES08.

[0106] PCR primers were synthesized by the Wuhan Synthesis Department of Beijing Qingke Biotechnology Co., Ltd.

[0107] 1.3 Experimental Apparatus

[0108] Real-time quantitative PCR instrument: CFX Connect (BIO-RAD);

[0109] NanoPhotometer-N80 (Implen) Ultraviolet Spectrophotometer;

[0110] Other instruments include: constant temperature water bath, centrifuge, vortex mixer, ultrapure water system, etc.

[0111] 2 Experimental Methods

[0112] 2.1 Primer Design

[0113] The sequences were obtained from our laboratory's genome sequencing database. Figure 2 A BLAST analysis of this sequence against the NCBI Genome Database revealed no significantly similar sequences. The BLAST results also indicated that this gene sequence had not been discovered or uploaded to the NCBI Genome Database by other researchers. Primers were designed using this sequence as the amplification target.

[0114] The primers are shown in Table 4, involving a total of 6 primer combinations: Ce44-F38 / Ce44-R132, Ce44-F38 / Ce44-R147, Ce44-F38 / Ce44-R151, Ce44-F40 / Ce44-R132, Ce44-F40 / Ce44-R147, and Ce44-F40 / Ce44-R151.

[0115] Table 4 shows the designed primer sequences and amplified fragment lengths.

[0116]

[0117] 2.2 Real-time quantitative PCR

[0118] The genomic DNA of tiger nuts was amplified by real-time quantitative PCR using 5 different primer combinations. The method of real-time quantitative PCR amplification was the same as that in Example 1.

[0119] 3 Experimental Results

[0120] As shown in Table 5, Figure 3 As shown, primers Ce44-F38 / Ce44-R132 and Ce44-F40 / Ce44-R132 exhibited poor parallelism in their amplification curves, while Ce44-F38 / Ce44-R151 and Ce44-F40 / Ce44-R151 showed high Ct values ​​and poor parallelism. Primer Ce44-F38 / Ce44-R147 showed inconsistent melting temperatures. The amplification results of tiger pea genomic DNA using primers Ce44-F40 / Ce44-R147 showed typical amplification curves, good parallelism, and strong signal, with sharp and single melting point peaks. Therefore, primers Ce44-F40 / Ce44-R147 are the optimal primer combination. The optimal primer combination was screened using the SYBR Green method.

[0121] Table 5 shows the real-time fluorescence quantitative PCR amplification results of the six primer pairs designed.

[0122]

[0123] Example 3: Intraspecific and interspecific characteristics analysis of primers Ce44-F40 / Ce44-R147 for specifically amplifying tiger nut components.

[0124] 1. Experimental Materials

[0125] Sixteen different varieties of tiger nuts (Cyperus esculentus L.) (RY4, RY5, RY7, RY8, RY9, RY10, RY11, RY17, RY19, RY20, RY22, RY24, RY25, RY30, RY27, GY2). The other 16 different plant materials are: Cyperus rotundus, water chestnut (Eleocharis dulcis), windmill grass (Cyperus involucratus), sedge (Cyperus iria), Arabidopsis thaliana, tobacco (Nicotiana tabacum), rapeseed (Brassica napus), corn (Zea mays), soybean (Glycine max), peanut (Arachis hypogaea), sunflower (Helianthus annuus), oil palm (Elaeis guineensis), safflower (Carthamus tinctorius), rice (Oryza sativa), wheat (Triticum aestivum), and potato (Solanum tuberosum).

[0126] 2 Experimental Methods

[0127] 2.1 Extraction of plant genomic DNA

[0128] The extraction method is the same as that in Example 1.

[0129] 2.2 Real-time quantitative PCR

[0130] The primer combination used for real-time quantitative PCR was Ce44-F40 / Ce44-R147.

[0131] The real-time quantitative PCR reaction system is as follows: 1 μL DNA template, 10 μL qPCR SYBR Green Master Mix, 0.4 μL each of 10 μM forward and reverse primers, and ddH2O to a final volume of 20 μL.

[0132] The real-time quantitative PCR reaction conditions were: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, for 40 cycles.

[0133] 3 Experimental Results

[0134] like Figure 4 As shown in Table 6, real-time quantitative PCR amplification of genomic DNA from 16 tiger nuts was performed using the primer combinations of this invention, and amplification was observed in all samples. This indicates that the gene fragments and detection methods disclosed in this invention have good intraspecific stability in tiger nuts.

[0135] like Figure 5 As shown, 16 other plant species were selected, and real-time quantitative PCR amplification of these 16 genomic DNA samples was performed using the primer combination of the present invention. No amplification was observed in any of them, indicating that the gene fragments and detection methods disclosed in this invention have good inter-species specificity.

[0136] Table 6. Amplification of Ce44-F40 / Ce44-R147 genomic DNA in tiger nuts.

[0137]

[0138]

[0139] Example 4: Sensitivity analysis of primers Ce44-F40 / Ce44-R147 for specific amplification of tiger nut components.

[0140] 1 Experimental Methods

[0141] 1.1 Extraction of plant genomic DNA

[0142] The extraction method is the same as that in Example 1.

[0143] 1.2 Serial dilution of genomic DNA and real-time quantitative PCR amplification

[0144] Tiger genomic DNA was serially diluted with water to 20 ng / μL, 4 ng / μL, 0.8 ng / μL, 0.16 ng / μL, and 0.032 ng / μL. Real-time quantitative PCR amplification was performed using the primer combination of the present invention. The real-time quantitative PCR amplification primers used were Ce44-F40 / Ce44-R147. The amplification method and conditions were the same as those in Example 3.

[0145] 2 Experimental Results

[0146] DNA amplification curves after gradient dilution are shown below. Figure 6 As shown in Table 7, the amplification Ct values ​​were obtained, and the detectable template DNA concentration was as low as 0.032 ng / μL, indicating that the method has good sensitivity. Based on the amplification curves obtained from gradient dilutions (…), Figure 6 A) Plot the standard curve ( Figure 6 B) In the real-time PCR reaction system constructed with the primer combination Ce44-F40 / Ce44-R147, the template DNA concentration showed a good linear relationship in the range of 0.032 ng / μL to 20 ng / μL (y = -3.329x + 28.544, R...). 2 =0.999), indicating that this method can be used for accurate quantitative detection.

[0147] Table 7 Results of Real-Time Quantitative PCR Amplification of Tiger Pea Genomic DNA Concentration Gradient Dilution

[0148]

[0149] Example 5: Primers Ce44-F40 / Ce44-R147 specifically amplify tiger nut components for the detection of tiger nut components in actual samples.

[0150] 1. Experimental Materials

[0151] 1.1 Food Samples

[0152] Commercially available tiger nut powder, commercially available tiger nut flour, commercially available refined tiger nut powder, commercially available tiger nut powder, supermarket lotus root powder, and supermarket black sesame paste powder. Tiger nut is another name for tiger nut, and some commercially available products are also called tiger nut.

[0153] 1.2 Enzymes and Reagents

[0154] The Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (real-time quantitative PCR amplification premix solution) was purchased from Shanghai Yisheng Biotechnology Co., Ltd., catalog number 11184ES08.

[0155] Chloroform, isoamyl alcohol, ethanol, and NaCl were domestically produced analytical grade reagents, all purchased from Sinopharm Chemical Reagent Co., Ltd., while Tris, EDTA, CTAB, SDS, etc., were purchased from Sigma-Aldrich.

[0156] The primers required for this experiment were synthesized by the Wuhan Synthesis Department of Beijing Qingke Biotechnology Co., Ltd.

[0157] 1.3 Experimental Apparatus

[0158] Real-time PCR instrument: CFX Connect (BIO-RAD);

[0159] NanoPhotometer-N80 (Implen) Ultraviolet Spectrophotometer;

[0160] Other instruments include: constant temperature water bath, centrifuge, vortex mixer, ultrapure water system, etc.

[0161] 2. Experimental Methods

[0162] 2.1 Extraction of product genomic DNA

[0163] The extraction method is the same as that in Example 1.

[0164] 2.2 Real-time quantitative PCR amplification

[0165] Using the genomic DNA extracted in step 2.1 above as a template, quantitative real-time PCR was performed with 18S rDNA primers, and ddH2O was used as a blank control to demonstrate the amplifference of the extracted genomic DNA. The 18S rDNA primers are shown in Table 1.

[0166] The real-time quantitative PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, 40 cycles; plate reading every 0.5℃ from 65 to 95℃, and incubation at 16℃.

[0167] The real-time quantitative PCR reaction system was as follows: 1 μL DNA template, 10 μL Hieff UNICON Universal Blue qPCRSYBR Green Master Mix, 0.4 μL each of 10 μM forward and reverse primers, and ddH2O to a final volume of 20 μL.

[0168] Real-time quantitative PCR was performed using the tigernut-specific primer combination Ce44-F40 / Ce44-R147, with ddH2O as a blank control, to detect the presence of tigernut-like components in the samples. Primer information and amplified fragment sizes are shown in Table 8.

[0169] Table 8 Primer information and amplified fragment size

[0170]

[0171] The real-time quantitative PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, for 40 cycles.

[0172] The real-time quantitative PCR reaction system was as follows: 1 μL DNA template, 10 μL Hieff UNICON Universal Blue qPCRSYBR Green Master Mix, 0.4 μL each of 10 μM forward and reverse primers, and ddH2O to a final volume of 20 μL.

[0173] 3 Experimental Results

[0174] 3.1 Amplifiability of extracted genomic DNA

[0175] Table 9 shows the amplification results of six genomic DNA samples extracted from commercially available tiger nut powder, commercially available tiger nut flour, commercially available refined tiger nuts, commercially available tiger nut powder, supermarket lotus root powder, and supermarket black sesame paste powder using real-time quantitative PCR with 18S rDNA primers. Figure 7 As shown, amplification curves appeared in all samples. This indicates that genomic DNA suitable for quantitative real-time PCR amplification was extracted from these samples.

[0176] The four extracted genomic DNA samples were amplified by real-time quantitative PCR using the primer combination Ce44-F40 / Ce44-R147. The results are shown in Table 10. Figure 8 As shown, samples 1-6 correspond to commercially available tiger nut powder, commercially available tiger nut flour, commercially processed tiger nuts, commercially available tiger nut powder, supermarket lotus root powder, and supermarket black sesame paste powder, respectively. Except for supermarket lotus root powder and supermarket black sesame paste powder, all others amplified the primer combination Ce44-F40 / Ce44-R147, and the Ct value was less than 36, therefore they are considered to contain tiger nut components. The experimental results show that the primer combination based on the tiger nut specific sequence of the present invention can accurately identify whether commercially available coarsely processed samples contain tiger nut components.

[0177] Table 9. Real-time fluorescence quantitative PCR amplification results of six food samples.

[0178]

[0179] Table 10. Real-time fluorescence quantitative PCR amplification results of six food samples.

[0180]

[0181] The above description is merely a detailed description of specific embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made based on the design concept of the present invention should be included within the protection scope of the present invention.

[0182] The present invention has been described in detail above with general descriptions and specific embodiments. However, modifications or improvements can be made to the present invention, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A primer composition for identifying tiger nuts, characterized in that, The primer composition includes an upstream primer. Ce44-F40 and downstream primers Ce44-R147 The upstream primer Ce44-F40 The sequence is shown in SEQ ID NO.1, and the downstream primer... Ce44-R147 The sequence is shown in SEQ ID NO.

2.

2. A SYBR Green fluorescent PCR method for identifying tiger nuts, characterized in that, The SYBR Green fluorescent PCR method uses the primer composition described in claim 1 to amplify the sample in the SYBR Green amplification system.

3. The SYBR Green fluorescent PCR method according to claim 2, characterized in that, The SYBR Green fluorescent PCR method specifically includes the following steps: S1) Obtain the genomic DNA of the sample to be identified; S2) The genomic DNA extracted in step S1) is amplified using a real-time fluorescence PCR reaction system containing the primer composition of claim 1; S3) If a sample shows specific amplification, it is confirmed that the sample to be identified contains tiger nuts.

4. The SYBR Green fluorescent PCR method according to claim 3, characterized in that, The real-time fluorescence PCR reaction system in S2 also contains SYBR Green.

5. The SYBR Green fluorescent PCR method according to claim 3, characterized in that, The reaction system in step S2) is as follows: 1 μL DNA template, 10 μL qPCR SYBR Green Master Mix, 0.4 μL each of 10 μM forward and reverse primers, and ddH2O added to make up to 20 μL.

6. The SYBR Green fluorescent PCR method according to claim 3, characterized in that, The Ct value amplified in S2 is less than 40.

7. The SYBR Green fluorescent PCR method according to claim 3, characterized in that, The concentration of genomic DNA in S1 was adjusted to the range of 0.032 ng / µL - 20 ng / µL.

8. The SYBR Green fluorescent PCR method according to claim 3, characterized in that, Step S2) uses fluorescent PCR for amplification. The reaction conditions for fluorescent PCR are: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, for 40 cycles.

9. A SYBR Green real-time quantitative PCR method for identifying tiger nuts, characterized in that, The SYBR Green real-time PCR method specifically includes the following steps: S1) Obtain the genomic DNA of the sample to be identified; and perform quantitative gradient dilution; S2) Amplify genomic DNA of different concentrations extracted and diluted in step S1) using a real-time fluorescence quantitative PCR reaction system containing the primer composition of claim 1; S3) Amplify DNA samples of known concentrations using the reaction system of S2), and plot a standard curve based on fluorescence intensity; S4) The fluorescence intensity values ​​of genomic DNA obtained in step S2) are used to perform regression calculations on the standard curve obtained in step S3) to calculate the genomic DNA content in the sample.

10. The SYBR Green real-time PCR method according to claim 9, characterized in that, The reaction system in step S2) is as follows: 1 μL DNA template, 10 μL qPCR SYBR Green Master Mix, 0.4 μL each of 10 μM forward and reverse primers, and ddH2O added to make up to 20 μL.

11. The SYBR Green real-time PCR method according to claim 9, characterized in that, The Ct value amplified in S2 is less than 40.

12. The SYBR Green real-time PCR method according to claim 9, characterized in that, The concentration of genomic DNA in S1 was adjusted to the range of 0.032 ng / µL - 20 ng / µL.

13. The SYBR Green real-time PCR method according to claim 9, characterized in that, Step S2) uses quantitative real-time PCR for amplification. The reaction conditions for quantitative real-time PCR are: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, for 40 cycles.

14. A molecular marker for identifying tiger nuts, characterized in that, The molecular marker can be obtained by amplifying tiger nut DNA using the primer composition of claim 1.

15. Use of the primer composition for identifying tiger nuts as described in claim 1 in identifying tiger nuts in a sample.

16. The use according to claim 15, wherein the sample is a plant specimen, plant seed, plant or animal sample particles or powder.

17. The use according to claim 15, wherein the sample is a liquid containing an animal or plant sample.

18. A reagent kit, characterized in that, The kit comprises the primer composition for identifying tiger nuts as described in claim 1, wherein the upstream primer... Ce44-F40 The sequence is shown in SEQ ID NO.1, and the downstream primer... Ce44-R147 The sequence is shown in SEQ ID NO.

2.

19. The reagent kit according to claim 18, characterized in that, The kit also contains SYBR Green.

20. Use of the primer composition of claim 1 in the preparation of a kit for identifying tiger nuts in a sample.

21. The use according to claim 20, wherein the sample is a plant specimen, plant seed, plant or animal sample particles or powder.

22. The use according to claim 20, wherein the sample is a liquid containing an animal or plant sample.

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