Nucleic acid detection method based on triple amplification and application of nucleic acid detection method in ginseng identification

Abstract

The invention belongs to the technical field of detection, and particularly relates to a nucleic acid detection method based on triple amplification and application of the nucleic acid detection method in ginseng identification. Mixing the liquid to be detected with the hairpin DNA, and assembling to form DNA enzyme; a hairpin substrate is added, enzyme digestion reaction is completed, and a trigger chain is generated; adding a circular DNA probe, carrying out rolling circle amplification, and collecting generated long single-stranded DNA; a G quadruplex structure in the long single-stranded DNA is utilized to catalyze a chromogenic reaction, and the concentration of miRNA in the to-be-detected liquid is analyzed through the RGB value of the color. According to the method disclosed by the invention, the low detection limit of the 651 fM is realized. The analysis of ginseng samples and related products verifies the effectiveness of the triple amplification platform, and a portable and reliable solution is provided for food safety supervision and quality control.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a nucleic acid detection method based on triple amplification and its application in ginseng identification. Background Technology

[0002] Ginseng (Panax ginseng CA Mey.), derived from the dried root and rhizome of the Panax genus in the Araliaceae family, has become a globally recognized tonic with significant health benefits, including enhancing physical strength, restoring cardiovascular function, and strengthening the spleen and lungs. With the increasing consumer demand for natural and functional nutrients, ginseng has been widely used in dual-purpose products for both medicinal and dietary purposes, driving significant expansion of its global market. The ginseng market was valued at $8.9 billion in 2023 and is projected to reach $17.7 billion by 2030. However, the high profitability of the ginseng market and the significant price differences between ginseng and related species have led to frequent instances of deliberate adulteration. Furthermore, the morphological similarities between ginseng and related species, and the loss of visible ginseng characteristics during processing, pose a serious challenge to the accurate identification of ginseng products.

[0003] With the continuous development of analytical instruments, various identification techniques based on component specificity have emerged, including liquid chromatography-mass spectrometry (LC-MS) detection, microscopic examination, and gas chromatography detection. Based on the gene expression specificity of different species, molecular identification techniques have gradually emerged as a tool with strong universality and high specificity in recent years. MicroRNAs (miRNAs), as an important type of nucleic acid regulating gene expression and various physiological processes, have unique advantages in identification markers. The expression level of miRNAs is not only closely related to the growth environment of plants and animals but also helps in the identification of species and even subspecies, and has been widely used in quality analysis and origin traceability. However, the low abundance and inherent instability of miRNAs place higher demands on the sensitivity of detection systems. Furthermore, existing detection methods rely on large instruments, making portable on-site detection difficult and hindering their practical application and promotion in market supervision. Therefore, there is an urgent need to develop a highly sensitive portable detection platform for on-site identification of ginseng.

[0004] Point-of-care testing (POCT) is a rapid diagnostic technology performed at the patient's location or during a consultation, eliminating the need for complex equipment and procedures found in traditional laboratories. Developing POCT platforms offers unique advantages for food and drug analysis, enabling point-of-care testing in a small footprint and facilitating market penetration at a relatively low cost. POCT systems are typically made using nanomaterials or microfluidic chips and employ various methods to generate multiple signal types, such as colorimetry, fluorescence, electrochemical methods, chemiluminescence, and magnetic methods.

[0005] With the rapid development of DNA nanotechnology, DNA has gradually become a key specific recognition molecule. DNA-based point-of-care testing (POCT) platforms, utilizing fluorescence, electrochemical, and colorimetric signal responses, have been used to detect various analytes, including metal ions, small molecules, DNA, RNA, proteins, and cells. Among these, colorimetric sensors offer a convenient on-site detection method, offering advantages such as visual observation and no need for specialized instruments, enabling convenient detection of DNA or miRNA. However, colorimetric methods typically have low sensitivity and require larger sample volumes, limiting their application in detecting low-abundance targets. Therefore, there is an urgent need to design a highly sensitive colorimetric platform for miRNA detection to achieve portable identification of ginseng. Summary of the Invention

[0006] This invention primarily provides a triple-amplification-based nucleic acid detection method for ginseng that can be detected instantly and has a low detection limit, and its application in ginseng detection. This addresses the difficulties in accurately identifying ginseng products, detecting low-abundance targets, and achieving portable on-site detection in existing technologies. The technical solution is as follows: A nucleic acid detection kit based on triple amplification includes a detection solution a containing hairpin DNA, a detection solution b containing hairpin substrate, a detection solution c containing circular DNA probe and DNA polymerase, and an enzyme-catalyzed colorimetric reaction solution.

[0007] Furthermore, the hairpin DNA includes the sequence H1 as shown in SEQ ID NO:1: CTG CTC AGC GATGGTT TCA TCA GCA AAC AGT ACG T TGT TTG CTG ATG ATC ATC TAA, The sequence H2 is shown in SEQ ID NO:2: ACG TAC TGT TTG CTG ATG A GCT TGG TCA TCAGCA AAC A, The sequence H3, as shown in SEQ ID NO:3, is: TCA TCA GCA AAC ATG GGC TTT TGT TTG CTG ATGACC AAG C AACC CAC CCA TGT AGT CAA. And the sequence H4 as shown in SEQ ID NO:4: TCA TCA GCA AAC ATG GGC TTT TGT TTG CTGATG ACC AAG C AACC CAC CCA TGT AGT CAA.

[0008] Furthermore, the sequence of the DNase shown is as indicated in SEQ ID NO:5: CTG CTC AGC GAT GGTT TTTTTTAACC CAC CCA TGT AGT CAA; The sequence of the hairpin substrate is shown in SEQ ID NO:6: Cy3-TGG ACT TCT GGC ATA T TTT TTTTTT TTT TTT TTT TTG ACT TrAG GAG CAG TTT TTT TTT TTT A TAT GCC AGA AGT CCA-BHQ2; The sequence of the miRNA is shown in SEQ ID NO:7: UUA GAU GAU CAU CAG CAA ACA.

[0009] Furthermore, the detection solution a includes 100-1200 nM hairpin DNA; the detection solution b includes 2-10 μM hairpin substrate; the detection solution c includes 1-5 μM circular DNA probe, 0.1-5 U / μL DNA polymerase, 0.02-0.5 mM deoxyribonucleotide triphosphate, and 0.1-1 μg / μL bovine serum albumin; the enzymatic colorimetric reaction solution contains 5-20 mM hydrogen peroxide and chromogen reagent; and also includes a heme solution.

[0010] A nucleic acid detection method based on triple amplification, comprising the following steps: preparing a test solution; mixing the test solution with hairpin DNA to prepare long double-stranded DNA and DNase; adding hairpin substrate to complete the enzymatic digestion reaction and generate a trigger strand; adding a circular DNA probe to perform rolling circle amplification and collecting the generated long single-stranded DNA; performing enzymatic color development on the long single-stranded DNA and analyzing the miRNA concentration in the test solution by the RGB values ​​of the color.

[0011] Furthermore, the RGB value refers to the chromaticity value of the blue channel; the relationship between the RGB value and the miRNA concentration in the test solution is: 255 - RGB value = a × log (miRNA concentration) + b; where a is 9.5~10; and b is 5.5~6.

[0012] Furthermore, the preparation of the circular DNA probe includes the following steps: mixing phosphorylated linear DNA with ligation DNA, completely denaturing it into a single-stranded linear structure, and then cooling it to room temperature within 2-4 hours; incubating with T4 DNA ligase and then inactivating the enzyme; adding exonuclease for digestion and inactivating the enzyme; the sequence of the phosphorylated linear DNA is shown in SEQ ID NO:8: P-TTT TTTTCC CAA CCC GCC CTA CCC TGG ACT TCT GTG TTT T; the sequence of the ligation DNA is shown in SEQ ID NO:9: GGG AAA AAA AAA AAC ATC AG.

[0013] Furthermore, the preparation of the long single-stranded DNA includes the following steps: adding a circular DNA probe, DNA polymerase, deoxyribonucleotide triphosphate and bovine serum albumin to the reaction solution that generates the trigger chain, and incubating to obtain long single-stranded DNA.

[0014] Furthermore, the enzymatic color development includes the following steps: adding a heme stock solution to long single-stranded DNA and incubating to construct a G4 / heme DNAase; adding a chromogen reagent containing hydrogen peroxide to initiate the catalytic reaction.

[0015] Application of the above-mentioned nucleic acid detection method based on triple amplification in ginseng identification.

[0016] By adopting the above scheme, the method of the present invention has the following advantages: This invention integrates a triple signal amplification strategy to construct a highly sensitive visual inspection platform for on-site ginseng identification. By digitizing apparent color data using any device capable of recognizing RGB color data, including mobile phones, this invention enables on-site, low-barrier-to-entry identification operations.

[0017] The triple signal amplification process of this invention includes hybridization chain reaction, DNase cleavage and rolling circle amplification reaction; the sequences of the four hairpin probes are rationally designed so that they can coexist in a metastable state in the absence of a target and prevent cross-opening through a kinetic mechanism.

[0018] This invention designs a circular padlock probe that binds to the initiating strand, wherein a portion of the padlock probe is a complementary sequence to the G-quadruplex domain. After enzymatic digestion to generate a large number of initiating strands, a rolling circle amplification reaction occurs in the presence of phi29 polymerase and dNTPs, forming a long single-stranded DNA containing numerous G-quadruplex structures, achieving third-level signal amplification. The formation of these G-quadruplexes alters the local conformation of the DNA, promoting the establishment of a specific hydrogen bond network.

[0019] The RGB values ​​output by the method of this invention show a strong linear relationship with the concentration of the target miRNA. The detection limit of the target analyte is 651 fM, and R² = 0.9915. The fit to the actual data is very good. The method of this invention can accurately provide response detection results.

[0020] The method of this invention can directly identify ginseng as well as American ginseng and platycodon, which are similar to ginseng. It can be used to identify related species that have significant similarities to ginseng in macroscopic morphology.

[0021] The method of the present invention can identify the ginseng component in drugs of different dosage forms, such as interstitial preparations and granules, whose original morphological characteristics may have been altered or destroyed. By accurately quantifying the ginseng content in the sample, adulterants can be identified. Attached Figure Description

[0022] Figure 1 This is a triple enlarged schematic diagram of the present invention; Figure 2 It is a polyacrylamide gel electrophoresis analysis of target-triggered HCR; Figure 3 The fluorescence spectra are those of samples incubated with and without DNase. Figure 4 It is a polyacrylamide gel electrophoresis analysis of rolling circle amplification reaction; Figure 5 The circular dichroism spectrum of the rolling ring amplification product obtained with or without a target is shown. Figure 6 These are photographs after incubation with different miRNA concentrations, and calibration curves between B1 values ​​and miRNA concentrations. Figure 7 This is a schematic diagram of zero amplification, single amplification, and dual amplification strategies; Figure 8 These are photographs of the colorimetric reaction results, with or without a target. Figure 9 (A) is a comparison of the morphology of the roots of ginseng, American ginseng, and platycodon, and the corresponding decoction in (B); (C) and (F) are UV-Vis absorption spectra; (D) and (G) are B1 values ​​measured by a smartphone; and (E) and (H) are qPCR analysis results of target miRNAs after incubation with PGS, PQ, or PGF samples or decoctions; error bars represent mean ± standard deviation (n=3). Figure 10The images show (A) tablets and (B) granules of ginseng, American ginseng, and platycodon grandiflorum; (C) and (F) are the UV-Vis absorption spectra of the tablets and granules, respectively; (D) and (G) are the B1 values ​​measured by smartphones for the tablets and granules; (E) and (H) are the qPCR analysis results of the target miRNA after incubation with PGS, PQ, or PGF tablets or granules, respectively; the error bars represent the mean ± standard deviation (n=3). Detailed Implementation

[0023] Example 1: (1) 4.5 μL of phosphorylated linear DNA (50 μM) and 4.5 μL of 50 μM ligation DNA were mixed in 1 μL of 10×rCutSmart™ buffer, heated at 95°C for 5 minutes, and then slowly cooled to room temperature for more than two hours. Then, 1 μL of T4 DNA ligase (400 U / μL), 1 μL of 10×rCutSmart™ buffer and 8 μL of ultrapure water were added, mixed and incubated at 25°C for 2 hours. The T4 DNA ligase was inactivated by heating at 65°C for 10 minutes. Then, 2 μL of ExoI (20 U / μL) and 2 μL of ExoIII (100 U / μL) were added and the reaction system was incubated at 37°C overnight. Then, the product was heated at 80°C for 15 minutes to obtain a circular padlock probe; (2) Long single-stranded DNA was generated by incubating with phi29 DNA polymerase (0.2 U / μL) at 37°C for 1.5 hours. The reaction system included 6 μL of circular padlock probe, 0.4 μg / μL of bovine serum albumin and 0.1 mM dNTPs. The resulting long single-stranded DNA was incubated at 65°C for 10 minutes and purified by ultrafiltration (10k molecular weight cutoff membrane, Millipore).

[0024] (3) The target miRNA (100 nM) and four hairpin DNAs (H1, H2, H3, H4, 200 nM each) were mixed in equal amounts to a final volume of 10 μL for hybridization chain reaction (HCR) and incubated at 37°C for 90 minutes to form long double-stranded DNA (dsDNA) and DNA enzyme. (4) Subsequently, 10 μL of 4 μM HP hairpin substrate was added, and the enzyme digestion reaction was completed at 28°C for 30 minutes to generate a large number of trigger chains; (5) Add 6 μL of the prepared circular DNA probe and phi29 DNA polymerase (0.2 U·μL). -1 ), dNTP (0.1 mM) and BSA (0.4μg·μL -1 The mixture was incubated at 37°C for 90 minutes to obtain long single-stranded DNA (ssDNA). The product was then purified three times using ultrafiltration tubes. (6) Add 5 μM heme stock solution to 20 μL of RCA product and incubate the mixture at 37 °C for 30 min to construct G4 / heme DNase. Finally, add TMB solution containing 10 mM H2O2 to initiate the catalytic reaction. The absorbance signal of the obtained product was recorded using a multi-functional microplate reader. The RGB value of the product was obtained, and the difference between the RGB value and 255 was recorded as the B1 value, which was used for data interpretation and calculation.

[0025] Example 2: The difference from Example 1 is: (3) The target miRNA (10 nM) and four hairpin DNAs (H1, H2, H3, H4, 200 nM each) were mixed in equal amounts, with a final volume of 10 μL.

[0026] Example 3: The difference from Example 1 is: (3) The target miRNA (1 nM) and four hairpin DNAs (H1, H2, H3, H4, 200 nM each) were mixed in equal amounts, with a final volume of 10 μL.

[0027] Example 4: The difference from Example 1 is: (3) The target miRNA (100pM) and four hairpin DNAs (H1, H2, H3, H4, 200nM each) were mixed in equal amounts, with a final volume of 10μL.

[0028] Example 5: The difference from Example 1 is: (3) The target miRNA (10pM) and four hairpin DNAs (H1, H2, H3, H4, 200nM each) were mixed in equal amounts, with a final volume of 10μL.

[0029] Example 6: The difference from Example 1 is: (3) Take 0.6 g of ginseng root tissue and add it to a 2 mL centrifuge tube containing 1 mL of Trizol reagent. Mix thoroughly and then cool for 5 min. After centrifuging at 12,000 rpm for 10 min at 4 °C, add the supernatant to 200 μL of chloroform and shake the mixture vigorously until a milky white solution is produced. After cooling for 10 min, centrifuge the mixture at 12,000 rpm for 15 min at 4 °C. Transfer the supernatant to an equal volume of isopropanol, vortex and mix. Centrifuge at 4 °C for 10 min to obtain RNA. Dissolve the extracted RNA in 20 μL of DEPC water and store it in a low-temperature freezer at -80 °C. (4) The extracted RNA was added to four hairpin DNAs (H1, H2, H3, H4, 200 nM each) and mixed in equal amounts, with a final volume of 10 μL.

[0030] Example 7: The difference from Example 6 is: Preparation of ginseng decoction: Accurately weigh 40g of chopped fresh ginseng, add 160mL of double-distilled water (8 times the volume), soak for 30min, decoct at 85℃ for 2h, and filter the decoction for later use; add 160mL of double-distilled water (8 times the volume) to the dregs, decoct for 2h in the same way, and filter, combine the two filtrates; use low temperature and high pressure method for rotary evaporation, and finally concentrate the total volume to 20mL; take 0.5mL of the decoction for subsequent RNA extraction and reaction.

[0031] Example 8: The difference from Example 6 is: (3) Take 0.6g of ginseng slices for subsequent RNA extraction and reaction.

[0032] Example 9: The difference from Example 6 is: (3) Take 0.5 g of ginseng formula granules for subsequent RNA extraction and reaction.

[0033] Comparative Example 1: Unlike Example 6, in that: American ginseng was used instead of ginseng root tissue.

[0034] Comparative Example 2: Unlike Example 6, in that: Platycodon grandiflorus was used instead of ginseng root tissue.

[0035] Comparative Example 3: Unlike Example 7, in that: American ginseng was used instead of ginseng to prepare a decoction.

[0036] Comparative Example 4: Unlike Example 7, in that: Platycodon grandiflorus was used instead of ginseng to prepare a decoction.

[0037] Comparative Example 5: Unlike Example 8, in that: American ginseng was used instead of ginseng slices.

[0038] Comparative Example 6: Unlike Example 8, in that: Platycodon grandiflorus was used instead of ginseng slices.

[0039] Comparative Example 7: Unlike Example 9, in that: American ginseng was used instead of ginseng in the formula granules.

[0040] Comparative Example 8: Unlike Example 9, in that: Platycodon grandiflorus was used as a substitute for ginseng in the formula granules.

[0041] Comparative Example 9: Unlike Example 1, in that: Zero-amplification strategy: The target miRNA (100 nM) and the S0-lock0 assembly were mixed in equal volumes to a final volume of 10 μL. After incubation at 37°C for 90 minutes, the colorimetric reaction was performed directly.

[0042]

[0043] Comparative Example 10: Unlike Example 1, in that: Single amplification strategy: only includes rolling circle amplification reaction.

[0044] Comparative Example 11: Unlike Example 1, in that: The dual amplification strategy includes only enzyme digestion and rolling circle amplification reactions.

[0045] Example and comparative sample testing: Feature Description: During the experiments in Example 1, corresponding tests and verifications were performed. The triple signal amplification process includes hybridization chain reaction (HCR), DNase cleavage, and rolling circle amplification reaction (RCA). Figure 1 ). Figure 2 Lanes 1-7 represent: target sequence, H1 hairpin, H2 hairpin, H3 hairpin, H4 hairpin, a mixture of target sequence and four hairpins, and a mixture of four hairpins, respectively. Figure 2 It can be seen that the sequences of the four hairpin probes, after reasonable design, can coexist in a metastable state in the absence of a target and prevent cross-opening through a dynamic mechanism. Figure 2 (line 6). After incubation with the target, PAGE showed the generation of a new, slowly moving band ( Figure 2 (line 7), and when there is no target ( Figure 2 The significant difference compared to (line 6) indicates that the target miRNA successfully activated the HCR to achieve initial signal amplification.

[0046] This invention verifies the cleavage activity of DNase using a self-quenching hairpin substrate strand (HP) labeled with a fluorescent group Cy3 and a quencher BHQ2 at the 5' and 3' ends, respectively. In the absence of DNase, the fluorescence signal is weak due to the proximity of Cy3 and BHQ2. Figure 3 The addition of DNase cleavage of the substrate strand leads to the spatial separation of Cy3 and BHQ2, thereby achieving fluorescence recovery. Figure 3 This observation indicates that the activated DNase successfully cleaved the substrate, releasing the trigger strand to initiate a tertiary signal amplification reaction.

[0047] This invention designs a circular padlock probe that can bind to the priming strand, wherein a portion of the padlock probe is a complementary sequence to the G-quadruplex domain. After enzymatic digestion to generate a large number of priming strands, a rolling circle amplification (RCA) reaction occurs in the presence of phi29 polymerase and dNTPs, forming a long ssDNA containing numerous G-quadruplex structures, achieving third-level signal amplification. The padlock probe strands are ligated using T4 DNA ligase, and the unreacted DNA strands are digested with exonuclease I and exonuclease III. Figure 4 In the PAGE analysis, lanes 1-5 represent: padlock probe, ligation sequence, circular template, padlock probe + ligation sequence, and RCA product, respectively. Figure 4 A migration speed was observed that correlated with the linear DNA strand ( Figure 4 The first band is equivalent to the band ( Figure 4 The third band confirmed the successful synthesis of the circular padlock probe. PAGE analysis confirmed the successful synthesis of the long single-stranded DNA (ssDNA), which showed a band with a significantly slower migration rate. Figure 4 (Band 5). Circular pentatonic (CD) spectroscopy analysis revealed that the rolling circle amplification (RCA) reaction, performed in the presence of the initiating strand, circular DNA, phi29 DNA polymerase, and dNTPs, produced G-tetramer structures. The formation of these G-tetramers altered the local conformation of the DNA, promoting the establishment of a specific hydrogen bond network. This structural change resulted in a significant negative peak near 245 nm and a significant positive peak near 265 nm in the CD spectrum.

[0048] Accuracy verification: The B1 value was calculated according to the methods of the above examples and comparative examples. The relationship between the B1 value and concentration in Examples 1-6 is as follows: Figure 6 As shown. Figure 6 It can be seen that the B1 value exhibits a strong linear relationship with the target miRNA concentration in the range of 100 pM to 105 pM (R²=0.9915), and the detection limit of the target analyte is 651 fM. This indicates that data analysis based on the B1 value is also a reliable way to provide response detection results.

[0049] according to Figure 7 The amplification strategies are not shown in the diagram. Zero amplification (Comparative Example 9), single amplification (Comparative Example 10), and dual amplification (Comparative Example 11) are used as controls. In the zero amplification system, the target miRNA hybridizes with the lock strand (Lock0) and releases the G4 sequence, thereby triggering the colorimetric reaction. In the single amplification system, the target miRNA triggers the RCA reaction and generates repeating G4 sequences to enhance color difference. In the dual amplification system, a DNase structure is assembled in the presence of the target miRNA to generate multiple trigger strands for the RCA reaction and G4 sequence generation. The results of the colorimetric reactions with and without the target are shown below. Figure 8 As shown, compared to the triple signal amplification strategy employed in Example 1 of the present invention, these methods employing zero amplification, single amplification, and dual amplification strategies exhibit lighter colors, and after incubation with the target miRNA, the B1 value detected by a smartphone shows only a slight difference compared to the target-free blank group; conversely, the triple amplification system of the present invention exhibits a distinct blue color and a high colorimetric signal compared to the target-free blank group. The comparison of the results of Example 1 with Comparative Examples 9-11 demonstrates that the method of the present invention has excellent amplification performance, and the effects are not simply additive.

[0050] Verification of direct application of ginseng: Due to the significant similarity in macroscopic morphology between ginseng and related species, differentiation by naked-eye observation alone is difficult. To verify the feasibility of using the miRNA detection platform proposed in this study for naked-eye identification of traditional Chinese medicine, we selected three morphologically similar plants—ginseng, American ginseng, and platycodon grandiflorus—as examples. Ginseng and American ginseng belong to the same family, while platycodon grandiflorus belongs to a different family. Detection was performed according to the methods of Example 7, as well as Comparative Examples 1 and 2, and color changes were observed. Figure 9 As shown, only the solution treated with ginseng exhibited a significant color change, and the absorbance A450 value calculated by UV-Vis absorption spectroscopy was significantly increased. Figure 9 C, the red curve), while similar substances include American ginseng and platycodon ( Figure 9 C, blue and green curves) compared to the control group without samples and treated with probes ( Figure 9 Compared to the black curve (C), no distinguishable signal or color change was produced. To achieve portable detection and on-site analysis, a "color recognition" application on a smartphone was used to analyze the RGB values ​​of the colorimetric solution, and then the B1 value was calculated from it. The results are as follows: Figure 9 As shown in Figure D, the B1 value of the ginseng group increased significantly by 51.67, while the American ginseng and platycodon groups showed only slight changes compared to the control group. This is consistent with... Figure 9 The RT-qPCR results for E were consistent, indicating that significant miRNA overexpression was observed only in ginseng samples. These results validate the highly sensitive and visible detection capability of the triple amplification detection method of this invention for miRNAs in herbaceous plants, and also demonstrate its portable quantitative analysis and on-site identification capabilities.

[0051] Ginseng is typically prepared and used as a decoction. Analysis of the decoction components can be used to verify the authenticity of the medicinal material, thereby avoiding situations where the treatment effect is poor or the quality is substandard. The decoctions of ginseng, American ginseng, and platycodon were tested according to the methods of Example 7, Comparative Examples 3 and 4, and their color changes were observed to verify the feasibility of the method of the present invention for sensitive detection of trace amounts of miRNA in the decoction. Figure 9The UV-Vis spectrum of F showed that the absorbance of the ginseng group at A450 wavelength was significantly higher than that of its analogues. Furthermore, Figure 9 G observed colorimetric changes in ginseng samples and their similarities (American ginseng and platycodon). The ginseng group showed a distinct blue color, while the American ginseng and platycodon groups were almost indistinguishable from the blank group. Figure 9 Analysis using a smartphone-based color recognition application also confirmed that the B1 value of the ginseng group was higher than that of the other three groups, while the B1 values ​​of the American ginseng and platycodon groups were only slightly increased relative to the control group. This is consistent with... Figure 9 The RT-qPCR results for H were consistent. These findings indicate that the method of the present invention has the ability to identify traditional Chinese medicine decoctions by naked-eye inspection.

[0052] Validation of Ginseng Applications in Special Dosage Forms: In the commercial market, ginseng is mainly sold in the form of sliced ​​ginseng or granules, and its original morphological characteristics may have been altered or destroyed, which further increases the difficulty of its on-site visual identification. Colorimetric analysis and RGB analysis were performed on ginseng slices and their adulterants (American ginseng slices and Platycodon grandiflorus slices) according to Example 8 and Comparative Examples 5 and 6, respectively. Figure 10 The UV-Vis spectrum of C showed that the ginseng group exhibited a significant signal enhancement at 450 nm, while the other three groups showed no change or only weak signals. Figure 10 As shown in Figure D, the ginseng group exhibited a clear color change from colorless to blue, while the other three groups did not show significant changes. Further analysis of the RGB values ​​using a "color recognition" smartphone application revealed that the B1 value of the ginseng group was significantly higher than that of the adulterant group and the blank group. These results are consistent with... Figure 10 The results of conventional RT-qPCR experiments on E were consistent. These results indicate that the method of the present invention can be used for colorimetric identification of ginseng slices.

[0053] Traditional Chinese medicine (TCM) formula granules are granular products prepared from traditional Chinese medicinal herbs using modern processing techniques. Their production process mainly includes pretreatment of medicinal materials, extraction and separation, concentration and drying, and granulation. Each process unit has a significant impact on the product's composition. Here, we selected ginseng, American ginseng, and platycodon grandiflorus formula granules to verify the applicability of the method of this invention for the visible identification of formula granules. Following the methods of Example 9 and Comparative Examples 7 and 8, the ginseng sample and its similar substances (American ginseng and platycodon grandiflorus) granules were tested. Figure 10 The ginseng group (red curve) showed a significant color change, and UV-Vis spectroscopy analysis revealed that its absorbance at A450 wavelength was significantly higher than that of the American ginseng group (blue curve) and the platycodon group (green curve). Furthermore, Figure 10 G shows a clear color difference between the ginseng group and the other groups. At the same time, the B1 value of the ginseng group is significantly higher than that of the American ginseng group and the platycodon group. This is consistent with... Figure 10The results of the RT-qPCR experiment were consistent with those of H. The results indicate that the method of this invention can be used for the visual identification of traditional Chinese medicine formula granules.

[0054] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A nucleic acid detection kit based on triple amplification, characterized in that, The test solution includes a test solution containing hairpin DNA (a), a test solution containing hairpin substrate (b), a test solution containing circular DNA probe and DNA polymerase (c), and an enzyme-catalyzed colorimetric reaction solution.

2. The nucleic acid detection kit based on triple amplification according to claim 1, characterized in that, The hairpin DNA includes H1 with the sequence shown in SEQ ID NO:1, H2 with the sequence shown in SEQ ID NO:2, H3 with the sequence shown in SEQ ID NO:3, and H4 with the sequence shown in SEQ ID NO:

4.

3. The nucleic acid detection kit based on triple amplification according to claim 1, characterized in that, The sequence of the DNA enzyme is shown in SEQ ID NO:5; the sequence of the hairpin substrate is shown in SEQ ID NO:6; and the sequence of the miRNA is shown in SEQ ID NO:

7.

4. The nucleic acid detection kit based on triple amplification according to claim 1, characterized in that, The detection solution a contains 100-1200 nM hairpin DNA; the detection solution b contains 2-10 μM hairpin substrate; the detection solution c contains 1-5 μM circular DNA probe, 0.1-5 U / μL DNA polymerase, 0.02-0.5 mM deoxyribonucleotide triphosphate, and 0.1-1 μg / μL bovine serum albumin; the enzymatic colorimetric reaction solution contains 5-20 mM hydrogen peroxide and chromogen reagent; and also includes a heme solution.

5. A nucleic acid detection method based on triple amplification as described in claim 1, characterized in that, Includes the following steps: Preparation of the test solution; The test solution was mixed with hairpin DNA to prepare long double-stranded DNA and DNA enzyme; Add hairpin substrate to complete the enzyme digestion reaction and generate the trigger strand; add circular DNA probe to perform rolling circle amplification and collect the generated long single-stranded DNA. Long single-stranded DNA was subjected to enzymatic color development, and the concentration of miRNA in the test solution was analyzed by the RGB values ​​of the color.

6. The nucleic acid detection method based on triple amplification according to claim 1, characterized in that, The RGB value refers to the chromaticity value of the blue channel; the relationship between the RGB value and the miRNA concentration in the test solution is: 255 - RGB value = a × log (miRNA concentration) + b; where a is 9.5~10; and b is 5.5~6.

7. The nucleic acid detection method based on triple amplification according to claim 1, characterized in that, The preparation of the circular DNA probe includes the following steps: Phosphorylated linear DNA was mixed with ligation DNA and allowed to completely denature into a single-stranded linear structure. The mixture was then cooled to room temperature over 2-4 hours. T4 DNA ligase was added for incubation, followed by enzyme inactivation. Exonuclease was added for digestion, and the enzyme was inactivated. The sequence of the phosphorylated linear DNA is shown in SEQ ID NO:

8. The sequence of the ligation DNA is shown in SEQ ID NO:

9.

8. The nucleic acid detection method based on triple amplification according to claim 1, characterized in that, The preparation of the long single-stranded DNA includes the following steps: adding a circular DNA probe, DNA polymerase, deoxyribonucleotide triphosphate and bovine serum albumin to the reaction solution that generates the trigger chain, and incubating to obtain long single-stranded DNA.

9. The nucleic acid detection method based on triple amplification according to claim 1, characterized in that, The enzymatic colorimetric reaction includes the following steps: adding a heme stock solution to long single-stranded DNA and incubating to construct a G4 / heme DNAase; adding a chromogen reagent containing hydrogen peroxide to initiate the catalytic reaction.

10. The application of the nucleic acid detection method based on triple amplification as described in any one of claims 1 to 8 in ginseng identification.

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