A kit for rapid identification of ginseng by one-step method and a detection method thereof

Abstract

This invention relates to the field of gene detection technology, and discloses a kit and detection method for rapid one-step identification of ginseng. The kit integrates the transcription of the T7 / crDNA hybrid strand, RPA amplification, transcription of the DNA product, and CRISPR / Cas13a trans-cutting reaction into a single reaction tube for simultaneous execution, constructing an integrated one-step rapid detection system. This kit can achieve specific identification of ginseng within 30 minutes, eliminating the need for chemical synthesis of crRNA, significantly reducing reagent costs and operation time, simplifying the experimental procedure, and effectively avoiding the risk of cross-contamination by eliminating the need to open the lid throughout the process. The detection sensitivity reaches 10. ‑4 The assay showed a concentration of ng / μL and no cross-reactivity with 11 common adulterants such as American ginseng and Panax notoginseng. In actual sample testing, the results were highly consistent with Sanger sequencing results (100% concordance rate), indicating promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a kit and detection method for rapid one-step identification of ginseng. Background Technology

[0002] Ginseng (Panax ginseng) is a perennial herb belonging to the genus Panax in the family Araliaceae. Its dried root and rhizome are traditional and precious Chinese medicinal materials, possessing effects such as greatly replenishing vital energy, restoring pulse and consolidating the body, tonifying the spleen and lungs, promoting body fluid production and nourishing blood, and calming the mind and improving intelligence. Due to the high economic value and market demand for ginseng, it is common for ginseng to be counterfeited by closely related species of the genus Panax, such as American ginseng, Panax notoginseng, and Panax japonicus, or other morphologically similar plants, seriously damaging consumer rights and the order of the Chinese medicinal materials market. Therefore, establishing a rapid, accurate, and convenient method for identifying ginseng is of significant practical importance.

[0003] Currently, the main methods for identifying ginseng include morphological identification, microscopic identification, physicochemical identification, and molecular identification. Traditional morphological and microscopic identification methods rely on the experience of the identifyers, are highly subjective, and are difficult to effectively identify powdered, processed, or deeply processed products. Physicochemical identification mainly involves qualitative or quantitative analysis of chemical components such as ginsenosides, but the specificity of these chemical components is limited and is easily affected by factors such as origin, growth period, and processing method.

[0004] In recent years, molecular identification technology has received widespread attention due to its ability to directly analyze genetic material and avoid interference from environmental factors. Commonly used molecular identification methods include DNA barcoding, random amplified polymorphic DNA (RAPD), sequence characteristic amplification region (SCAR) labeling, and PCR-RFLP. Among these, conventional PCR methods require large-scale equipment such as thermal cyclers, are complex to operate, have long testing cycles, and demand high levels of technical expertise from operators, making them difficult to meet the needs of rapid on-site testing at the grassroots level. While real-time quantitative PCR (qPCR) offers high sensitivity and specificity, it still relies on specialized equipment and trained personnel, hindering its widespread adoption.

[0005] In recent years, CRISPR / Cas technology has shown promising applications in pathogen diagnosis and molecular detection. Combining isothermal amplification techniques (such as recombinase polymerase amplification (RPA)) with the CRISPR / Cas system enables rapid nucleic acid detection under isothermal conditions, meeting the needs of on-site point-of-care testing. In the CRISPR / Cas13 system, CRISPR RNA (crRNA) is the core guiding element and activation switch; however, RNA is highly susceptible to degradation and difficult to preserve long-term in the reaction system, which is a major challenge and pain point in the practical application of CRISPR / Cas technology. Furthermore, traditional CRISPR detection typically requires nucleic acid amplification followed by transferring the amplified product to a tube containing the CRISPR detection system, increasing the risk of cross-contamination and operational complexity through multiple steps. Therefore, there is an urgent need for a simple, stable, reliable, highly sensitive, and rapid ginseng identification method that does not require large-scale equipment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a kit and detection method for rapid one-step identification of ginseng, solving the technical problems of complex operation, reliance on large instruments, long detection cycles, easy degradation of crRNA, and cross-contamination risks in CRISPR detection. This method integrates RPA isothermal amplification of target nucleic acid, in vitro transcription of crRNA, and Cas13a-mediated fluorescence signal detection into a single sealed tube, eliminating the need for separate synthesis and addition of crRNA. This effectively avoids the storage and handling limitations caused by the easy degradation of RNA molecules, while significantly simplifying the detection process.

[0007] The technical solution of this invention is as follows: A detection system for ginseng identification, the system comprising a T7 / crDNA hybrid strand capable of transcribing corresponding crRNA under the action of T7 RNA polymerase, and an RPA primer pair capable of specifically amplifying the ginseng 18S rRNA target sequence; the downstream primer of the RPA primer pair has a T7 promoter recognition sequence fused to its 5' end.

[0008] This system targets a ginseng-specific conserved region in the ginseng genome 18S rRNA sequence. It includes an RPA amplification primer pair for specifically amplifying the target and a T7 / crDNA hybrid strand for transcribing crRNA. The crRNA can specifically recognize the characteristic sequence on the ginseng target RNA and activate the trans-cleavage activity of the Cas13a protein.

[0009] The T7 / crDNA hybrid strand is obtained by mixing T7 promoter primers (sequence shown in SEQ ID NO.7) and crDNA templates (sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6) in a 1:1 molar ratio, followed by denaturation and annealing. This hybrid strand can be efficiently transcribed into mature crRNA by T7 RNA polymerase, without the need for prior chemical synthesis.

[0010] The RPA amplification primer pair includes an upstream primer RPA-F and a downstream primer RPA-R. The nucleotide sequence of the upstream primer RPA-F is shown in SEQ ID NO.8, SEQ ID NO.9, or SEQ ID NO.10; the nucleotide sequence of the downstream primer RPA-R is shown in SEQ ID NO.11, SEQ ID NO.12, or SEQ ID NO.13; the downstream primer RPA-R introduces a T7 promoter complementary sequence at its 5' end, enabling the RPA amplification product to be directly used as a transcription template for T7 RNA polymerase, transing the double-stranded DNA product into an RNA target while the amplification reaction is underway.

[0011] As a preferred technical solution, the T7 / crDNA hybrid chain is formed by mixing the T7 primer shown in SEQ ID NO.7 with any one of the crDNA templates in SEQ ID NO.1 to 6 in equal molar amounts and then annealing; the upstream primer of the RPA primer pair is selected from any one of SEQ ID NO.8 to 10, and the downstream primer is selected from any one of SEQ ID NO.11 to 13.

[0012] As a preferred technical solution, the crDNA template is SEQ ID NO.2, the upstream primer is SEQ ID NO.10, and the downstream primer is SEQ ID NO.11. This primer combination exhibits optimal amplification efficiency and recognition sensitivity for the ginseng target sequence.

[0013] This invention also discloses a ginseng identification kit for the above-mentioned detection system. The kit further comprises a fluorescent reporter probe U5 (sequence SEQ ID NO.14), with a FAM group attached to its 5' end and a BHQ1 group attached to its 3' end. When Cas13a is activated, it can be trans-cleaved, releasing a fluorescent signal that can be monitored in real time. The concentration range of each component in the kit is as follows: RPA primer 0.1–0.8 μM, probe 0.5 μM, T7 RNA polymerase 0.25–2 U / μL, Cas13a protein 12.5–100 nM, T7 / crDNA hybrid strand 0.375–3 nM, rNTP 2 mM, RNase inhibitor 1 U / μL, supplemented with 1×RPABasic reaction buffer and 14 mM MgOAc.

[0014] As a preferred technical solution, the concentrations of each component are as follows: RPA upstream primer 0.2 μM, downstream primer 0.2 μM, probe 0.5 μM, T7 RNA polymerase 1 U / μL, Cas13a protein 25 nM, T7 / crDNA hybrid strand 1.5 nM, rNTP 2 mM, RNase inhibitor 1 U / μL, 1×RPA Basic E-mix and 14 mM MgOAc. This ratio achieves the fastest signal response speed while ensuring detection sensitivity.

[0015] The detection components in this kit are provided in lyophilized particle form to enhance reagent stability and facilitate room temperature transportation and field use. The lyophilized particles are prepared as follows: a one-step premix containing no MgOAc and template is mixed with a lyophilization protection buffer containing 10% trehalose, 0.5% mannitol, and 0.5% polyethylene glycol 20000. The mixture is aliquoted into reaction tubes and lyophilized for 10–12 hours. The resulting lyophilized particles can be stored at -20°C or room temperature for extended periods, with no significant decrease in activity at room temperature for at least two months. When using the lyophilized formulation, a reconstitution solution is required before detection. This reconstitution solution consists of 5 µL RPA reconstitution buffer, 3 µL water, and 1 µL 140 mM MgOAc. Adding 9 µL of this reconstitution solution and 1 µL of DNA template to each tube of lyophilized particles initiates the reaction, eliminating the cumbersome step of preparing the premix on-site.

[0016] As a preferred technical solution, regarding the content of reagent components, the concentration range of each key component in the detection kit is as follows: upstream primer RPA-F 0.1–0.8 μM, probe 0.5 μM, T7 RNA polymerase 0.25–2 U / μL, Cas13a protein 12.5–100 nM, T7 / crDNA hybrid strand 0.375–3 nM, rNTP 2 mM, RNase inhibitor 1 U / μL, supplemented with 1×RPA Basic reaction buffer and 14 mM MgOAc.

[0017] This invention also provides a detection method for identifying ginseng using the above-mentioned kit, comprising the following steps: extracting total DNA from the sample to be tested, adding 1-2 μL of DNA template to a reaction tube containing all components of the kit, mixing well, and incubating at 37 °C for 30 min, during which the fluorescence intensity is recorded at a wavelength of 465-510 nm every 30-60 s; if the fluorescence signal in the reaction tube shows a significant upward trend over time, and the endpoint fluorescence value is significantly higher than that of the negative control (containing no template or containing non-ginseng DNA template), it is determined to be positive (containing ginseng), otherwise it is negative.

[0018] As a preferred technical solution, the following steps are included: (1) Using the genomic DNA of the sample to be tested as a template, add the DNA template to the reaction tube containing the detection kit (if it is a lyophilized form, add the above reconstitution solution at the same time for reconstitution), mix well and then centrifuge briefly; (2) Place the reaction tube in a constant temperature device and incubate at 37 °C for 30 min to perform an integrated reaction of isothermal amplification and transcription detection; (3) During the reaction, the fluorescence detection device is used to collect the FAM fluorescence signal every 30-60 s at a wavelength of 465-510 nm. The dynamic change of the fluorescence signal is used to determine whether the sample contains ginseng.

[0019] The criteria for determining the results are as follows: if the reaction tube produces obvious green fluorescence during incubation, and the fluorescence increment is statistically significantly different from that of the negative control (without template or non-ginseng DNA template) (P<0.05), then the sample is determined to contain ginseng DNA; if the reaction tube does not produce obvious fluorescence, and the fluorescence intensity is not significantly different from that of the negative control, then the sample is determined to not contain ginseng DNA.

[0020] As a preferred technical solution, the sample to be tested includes raw ginseng, slices, powder, ginseng seeds, processed ginseng slices, extracts, traditional Chinese medicine preparations containing ginseng, food containing ginseng, and health products containing ginseng.

[0021] The present invention also discloses a T7 / crDNA hybrid chain, wherein the hybrid chain is formed by mixing the T7 primer shown in SEQ ID NO.7 with any one of the crDNA templates in SEQ ID NO.1 to 6 in equal molar amounts and then annealing; the hybrid chain can be transcribed into crRNA for recognizing target RNA under the action of T7 RNA polymerase.

[0022] The present invention also discloses an RPA primer pair, which is used to specifically amplify the ginseng 18S rRNA target sequence, wherein the 5' end of the downstream primer is fused with a T7 promoter recognition sequence; the upstream primer is selected from any one of SEQ ID NO. 8 to 10, and the downstream primer is selected from any one of SEQ ID NO. 11 to 13.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention is the first to combine molecular identification of ginseng with a one-step CRISPR / Cas13a detection system to construct a specific detection scheme for the ginseng 18S rRNA target sequence. This method not only retains the advantages of high specificity and high sensitivity of the CRISPR / Cas system, but also eliminates the dependence on PCR thermal cyclers through RPA isothermal amplification, making it suitable for grassroots laboratories and rapid on-site detection scenarios.

[0024] (2) This invention employs a strategy of in situ transcription of T7 / crDNA hybrid strands to generate crRNA in the reaction system. On the one hand, this avoids the high cost and cumbersome operation of chemically synthesizing crRNA separately. On the other hand, it solves the industry pain point that crRNA is easily degraded during transportation and storage. At the same time, all reaction components are pre-mixed and completed in one tube. The operator only needs to add the nucleic acid template to be tested, which significantly reduces human error.

[0025] (3) The present invention adopts a closed tube detection mode. From the addition of nucleic acid to the completion of result interpretation, no opening operation is required. This effectively avoids the aerosol diffusion of amplification products and eliminates the risk of cross-contamination in the laboratory. It is especially suitable for the daily large-scale sample screening needs of Chinese herbal medicine testing institutions.

[0026] (4) The detection limit of the kit of the present invention for ginseng genomic DNA can reach 10. -4 With a sensitivity comparable to qPCR, this method achieves high specificity by detecting ng / μL of common adulterants such as American ginseng, Panax notoginseng, Sophora flavescens, Codonopsis pilosula, and Platycodon grandiflorus without cross-signal generation. Verification of commercially available ginseng materials showed that the detection results were completely consistent with Sanger sequencing (100% concordance rate), demonstrating promising prospects for clinical application and market regulation.

[0027] (5) The present invention further prepares the detection reagent into a lyophilized form. The lyophilization protection buffer (containing trehalose, mannitol and PEG-20000) used can effectively maintain the activity of key enzymes such as Cas13a protein and T7 RNA polymerase. The lyophilized particles can be stably stored at room temperature for at least two months without the need for cold chain transportation, which greatly reduces storage and transportation costs and operational difficulty. When using, only the reconstitution solution needs to be added to reconstitute the reagent, eliminating the step of preparing multi-component premixed solutions on-site. This allows even untrained personnel to easily complete the detection, significantly improving the practicality of this method in rapid on-site testing of Chinese medicinal materials and its promotion at the grassroots level. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the principle of the one-step rapid identification of ginseng according to the present invention; Figure 2 This is a graph showing the crDNA screening results of ginseng gene detection in Example 1 of the present invention; Figure 3 This is a diagram showing the RPA primer screening results for detecting ginseng genes in Example 2 of the present invention; Figure 4 This is a diagram showing the optimized reaction system results for detecting ginseng genes in Example 3 of the present invention; Figure 5 This is a graph showing the sensitivity results of the kit for detecting ginseng genes in Example 4 of the present invention; Figure 6 This is a graph showing the specificity test results of the kit used in Example 5 of this invention for detecting ginseng genes; Figure 7 This is a graph showing the stability test results of the lyophilized reagent kit in Example 6 of the present invention; Figure 8 The figure shows the actual test results of the reagent kit of Example 7 of the present invention for detecting commercially available ginseng and other similar medicinal materials. Detailed Implementation

[0029] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature or according to the product instructions.

[0031] Sources of main materials and reagents used in the examples: The Cas13a protein was purchased from Tolo Harbour Company, catalog number 32117-01.

[0032] T7 RNA polymerase was purchased from New England Biolabs, catalog number M0251.

[0033] The rNTP mix was purchased from New England Biolabs, product number N0466L.

[0034] The recombinant RNase inhibitor was purchased from Wuhan Saiweier Biotechnology Co., Ltd., catalog number G3414.

[0035] RPA lyophilized powder, RPA resuspension (RPA Basic E-mix), and 280 mM magnesium acetate (MgOAc) were purchased from TwistDx™, catalog number TABAS03KIT.

[0036] The U5 probe, RPA primers, and crDNA were synthesized by Jiangsu Saisofi Company.

[0037] Preparation of T7 / crDNA hybrid strands: The 10 μL annealing reaction system contained 1 μL of chemically synthesized crDNA (100 μM), 1 μL of T7 primer (100 μM), 1 μL of Standard Taq buffer (10×) (purchased from New England Biolabs, catalog number B9014), and 7 μL of enzyme-free sterile water (purchased from Solarbio, catalog number R1600). The reaction system was denatured at 95°C for 5 min on an ABI PCR instrument (Thermo, model: Veriti 96), and then annealed to 4°C at a cooling rate of 0.1°C / s.

[0038] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0039] The following examples use GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. Two-tailed Student's t test was used, and p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), p < 0.05 (*).

[0040] Table 1. crDNA sequence and T7 primer sequence

[0041] Note: The straight line represents the repeating sequence, and the wavy line represents the complementary sequence of the T7 promoter.

[0042] Table 2 RPA primer sequences and U5 probe sequences

[0043] Note: The wavy line represents the complementary sequence of the T7 initiator.

[0044] I. Design and synthesis of crDNA The 18S rRNA gene sequence of ginseng (P. ginseng) was obtained from GenBank (GenBank number: MK408780). Detection sites were selected from the conserved 18S rRNA gene sequence, and multiple crDNAs of Cas13a protein were designed, as shown in Table 1.

[0045] II. Design and synthesis of RPA amplification primers Based on the aforementioned ginseng 18S rRNA gene sequence, upstream primer RPA-F and downstream primer RPA-R were designed for RPA amplification. A T7 promoter complementary sequence was added to the 5' end of the downstream primer to generate an ssRNA sequence that can be recognized and cleaved by Cas13a. The upstream primer RPA-F corresponds to SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10 in Sequence Listing 2; the downstream primer RPA-R corresponds to SEQ ID NO. 11, SEQ ID NO. 12, and SEQ ID NO. 13 in Sequence Listing 2; and the U5 probe corresponds to SEQ ID NO. 14 in Sequence Listing 2.

[0046] III. Rapid DNA Extraction Genomic DNA of ginseng was extracted using a rapid DNA extraction and detection kit (Tiangen, catalog number: KG203-02) following the kit instructions. The extracted DNA was stored at -20°C.

[0047] IV. One-step reaction The reaction system for detecting ginseng 18S rRNA gene in 10 μL consisted of: 0.2 μM upstream primer RPA-F, 0.2 μM downstream primer RPA-R, 0.5 μM U5 probe, 1 U / μL T7 RNA polymerase, 50 nM Cas13a, 1.5 nM T7 / crDNA hybrid strand, 2 mM rNTP mix, 1 U / μL recombinant RNase inhibitor, 1×RPA Basic E-mix, and 14 mM MgOAc. Subsequently, 1 μL of DNA template was added to the detection kit, and the reaction was carried out at 37°C for 30 min using an ABI 7500 Fast real-time PCR instrument or a Gene-8C thermostat from Hangzhou Ausen PCR Technology Co., Ltd. FAM fluorescence signals were collected every 1 min at wavelengths of 465–510 nm.

[0048] V. Preparation of Lyophilized Detection Reagents The one-step premix (excluding MgOAc and template) was prepared in lyophilization protection buffer (containing 10% trehalose, 0.5% mannitol, and 0.5% polyethylene glycol 20000, Shanghai Sangon Biotech), aliquoted, and lyophilized for 10 to 12 hours using a freeze dryer (Shanghai Hefan Instrument Co., Ltd.). The lyophilized particles were stored at -20°C and room temperature for stability studies. Before use, each tube of lyophilized particles was reconstituted with 9 μL of reconstitution solution (containing 5 μL RPA reconstitution buffer, 3 μL water, 1 μL 140 mM MgOAc) and 1 μL of DNA template.

[0049] VI. Result Interpretation The type of sample to be tested is determined by observing the FAM fluorescence. If there is FAM fluorescence and it is significantly different from the negative control, the sample is judged to contain ginseng (P. ginseng) DNA. If there is no fluorescence and the fluorescence value is not significantly different from the negative control, the sample is judged not to contain ginseng DNA.

[0050] Example 1: Screening of crDNA The ginseng 18S rRNA RPA amplification primers F3, R1 and T7 / crDNA 1-6 prepared above were used to screen crDNA (sequences are shown in Table 1 and Table 2). Figure 2 The image shows the results of crDNA screening for the ginseng 18S rRNA gene. Figure 2 A through 2F are fluorescence curves obtained from the detection of ginseng-positive samples using RPA amplification primer pairs F3 / R1 and crDNA combinations 1 through 6. Figure 2 G to 2L represent the endpoint fluorescence values ​​(30 min) obtained from detecting ginseng-positive samples using RPA amplification primer pairs F3 / R1 and T7 / crDNA 1 to 6 combinations. Figure 2 As shown in Figure H, the endpoint fluorescence value obtained by the RPA amplification primer pair F3 / R1 and T7 / crDNA 2 in detecting ginseng positive samples had the highest signal-to-noise ratio (27.66 times) compared to the endpoint fluorescence value obtained in detecting American ginseng (P. quinquefolius), while negative samples showed no obvious fluorescence signal. Therefore, crDNA 2 (SEQ ID NO.2) was selected for the one-step reaction of ginseng 18S rRNA.

[0051] Example 2: Screening of RPA amplification primers The RPA amplification primers F1-F3 and R1-R3 of the ginseng 18S rRNA prepared above were screened with T7 / crDNA 2 (sequences are shown in Table 1 and Table 2). Figure 3This image shows the RPA primer screening results for detecting the ginseng 18S rRNA gene; F1~F3 are abbreviations for RPA-F1~F3, and R1~R3 are abbreviations for RPA-R1~R3. Figure 3 As shown, the T7 / crDNA 2 primer pair F3 / R1 combination yielded the highest endpoint fluorescence value (30 min) in ginseng positive samples, while no obvious fluorescence signal was observed in American ginseng and negative samples. Therefore, primer pair F3 / R1 (SEQ ID NO.10 / SEQ ID NO.11) was selected for the one-step reaction of ginseng 18S rRNA.

[0052] Example 3: Optimization of the reaction system The concentrations of key components in the reaction system, including LwaCas13a, T7 RNA polymerase, T7 / crDNA, and RPA primers, were gradually optimized. Figure 4 Figures A through 4D show the results of optimizing the concentrations of reaction components LwaCas13a, T7 RNA polymerase, T7 / crDNA, and RPA primers. Figure 4 As shown in A to 4D, the optimal component concentrations for this system are: LwaCas13a 25 nM, T7 RNA polymerase 1 U / μL, T7 / crDNA 1.5 nM, and RPA primers 0.2 μM each.

[0053] Example 4: Sensitivity Test of One-Step Detection Kit To determine the sensitivity of the one-step reaction for ginseng 18S rRNA, ginseng-positive sample DNA was sequentially diluted to different concentrations and detected according to the optimized detection system. The results are as follows: Figure 5 As shown, for the detection of ginseng 18S rRNA, at a concentration of 10... -4 The fluorescence signal after amplification of templates at concentrations of ng / μL or higher for 30 min was significantly higher than that of the negative control group (NTC), indicating that the one-step ginseng detection system established in this invention can detect 10 ng / μL ginseng. -4 Samples with concentrations above ng / μL exhibit high sensitivity.

[0054] Example 5: Specificity test of the one-step detection kit To determine the specificity of the one-step reaction for detecting ginseng, a ginseng 18S rRNA detection reagent was prepared using the optimal formula established in this study. Specificity was then assessed against 11 similar traditional Chinese medicines: *R. adenophora*, *R. glehniae*, *P. grandiflorus*, *R. isatidis*, *A. macrocephalae rhizoma*, *A. membranaceus*, *E. senticosus*, *A. sinensis*, *P. notoginseng*, *C. pilosula*, and *P. quinquefolius*. The results are as follows: Figure 6 As shown, the method established in this study did not produce positive reactions in other Chinese medicinal materials, indicating that the method has good specificity and will not produce false positive fluorescence signals.

[0055] Example 6: Preparation and stability verification of a one-step lyophilized detection kit To improve the field applicability of the detection method, a lyophilized reagent formulation was developed. The preparation method of the lyophilized detection reagent is as follows: the above-mentioned one-step premixed solution (excluding MgOAc and template) was prepared in lyophilization protection buffer (containing 10% trehalose, 0.5% mannitol, and 0.5% polyethylene glycol 20000, Shanghai Sangon Biotech), aliquoted, and then lyophilized for 10 to 12 hours using a freeze dryer (Shanghai Hefan Instrument Co., Ltd.). The lyophilized particles were stored at -20°C and room temperature for stability studies. When using, each tube of lyophilized particles was reconstituted with 9 μL of reconstitution solution (containing 5 μL RPA reconstitution buffer, 3 μL water, 1 μL 140 mM MgOAc) and 1 μL of DNA template.

[0056] This system only requires reconstitution of the lyophilized reagent and addition of the DNA sample to be tested to achieve amplification and detection, eliminating the tedious system preparation steps before each test. Figure 7 As shown, the lyophilized reagent was stored at -20°C and room temperature for 60 days, respectively. Tests showed that the lyophilized reagent exhibited excellent stability, with no significant decrease in activity after storage at room temperature for up to two months. The lyophilized kit effectively reduces the workload of operators without sacrificing analytical performance; at the same time, its good durability facilitates long-term storage and transportation without a cold chain. These features allow even untrained personnel to easily obtain and perform on-site testing.

[0057] Example 7: Field verification of the one-step detection kit for commercially available ginseng and similar medicinal materials To explore the market applicability of this method, 40 market samples were collected. First, 12 ginseng and 12 non-ginseng samples were tested to determine the threshold for a positive signal, which was calculated as the average fluorescence intensity of the non-ginseng samples plus three standard deviations. The results are as follows: Figure 8 As shown in A, the threshold was determined to be 38485. Then, one-step detection and Sanger sequencing were performed on 40 market samples. The results are shown in Table 3. Figure 8 As shown in B, the results indicate that all ginseng samples were accurately identified. The detection results of the method described in this application for market ginseng samples are completely consistent with those of the Sanger sequencing method (100% consistency rate), indicating that the method can not only meet the market testing needs, but also has the advantages of short detection time and convenient operation.

[0058] The Sanger sequencing method used in this study to detect ginseng 18S rRNA served as a control for the one-step detection method. Sanger sequencing of 40 samples was commissioned to Shanghai Sangon Biotech Co., Ltd.

[0059] Table 3. Validation results of clinical samples detected by Sanger sequencing and the one-step method of this application for detecting P. ginseng.

[0060] Sequence list description: This application involves a total of 14 nucleotide sequences, the sequence numbers and names of which are as follows: SEQ ID NO. 1–6: sequences of crDNA 1–6, used for annealing with T7 primers to form T7 / crDNA hybrid chains; SEQ ID NO. 7: T7 primer sequence; SEQ ID NO. 8–10: sequences of upstream RPA primers RPA-F1–F3; SEQ ID NO. 11–13: sequences of downstream RPA primers RPA-R1–R3 (with a T7 promoter complementary sequence fused to their 5' ends); SEQ ID NO. 14: sequence of fluorescent reporter probe U5 (5' end labeled FAM, 3' end labeled BHQ1).

[0061] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A detection system for ginseng identification, characterized in that: The system includes a T7 / crDNA hybrid strand that can transcribe the corresponding crRNA under the action of T7 RNA polymerase, and an RPA primer pair that can specifically amplify the ginseng 18S rRNA target sequence; the downstream primer of the RPA primer pair has a T7 promoter recognition sequence fused to its 5' end.

2. The detection system according to claim 1, characterized in that: The T7 / crDNA hybrid strand is formed by mixing the T7 primer shown in SEQ ID NO.7 with any one of the crDNA templates in SEQ ID NO.1 to 6 in equal molar amounts and then annealing; the upstream primer of the RPA primer pair is selected from any one of SEQ ID NO.8 to 10, and the downstream primer is selected from any one of SEQ ID NO.11 to 13.

3. The detection system as described in claim 2, characterized in that: The crDNA template is SEQ ID NO.2, the upstream primer is SEQ ID NO.10, and the downstream primer is SEQ ID NO.

11.

4. A ginseng identification kit containing the detection system according to any one of claims 1 to 3, characterized in that: The kit also includes a fluorescent reporter probe, the sequence of which is SEQ ID NO.14, with a FAM group attached to its 5' end and a BHQ1 group attached to its 3' end. The concentration range of each component in the kit is as follows: RPA primer 0.1–0.8 μM, probe 0.5 μM, T7 RNA polymerase 0.25–2 U / μL, Cas13a protein 12.5–100 nM, T7 / crDNA hybrid strand 0.375–3 nM, rNTP 2 mM, RNase inhibitor 1 U / μL, supplemented with 1×RPA Basic reaction buffer and 14 mM MgOAc.

5. The reagent kit according to claim 4, characterized in that, The concentrations of each component were as follows: RPA upstream primer 0.2 μM, downstream primer 0.2 μM, probe 0.5 μM, T7 RNA polymerase 1 U / μL, Cas13a protein 25 nM, T7 / crDNA hybrid strand 1.5 nM, rNTP 2 mM, RNase inhibitor 1 U / μL, 1×RPA Basic E-mix and 14 mM MgOAc.

6. A detection method for identifying ginseng using the kit according to any one of claims 4 or 5, characterized in that, The procedure includes the following steps: extract total DNA from the sample to be tested, add 1–2 μL of DNA template to a reaction tube containing all components of the kit, mix well, and incubate at 37 °C for 30 min. During this period, record the fluorescence intensity at a wavelength of 465–510 nm every 30–60 s. If the fluorescence signal in the reaction tube shows a significant upward trend over time, and the endpoint fluorescence value is significantly higher than that of the negative control, it is considered positive; otherwise, it is considered negative.

7. The detection method according to claim 6, characterized in that: The reagents in the reaction tubes are provided in the form of lyophilized particles; the lyophilization protection buffer used in the preparation of the lyophilized particles contains 10% trehalose, 0.5% mannitol and 0.5% polyethylene glycol 20000; during detection, each tube of lyophilized particles is first reconstituted with 9 µL of reconstitution solution and 1 µL of DNA template, the reconstitution solution consisting of 5 µL RPA reconstitution buffer, 3 µL of water and 1 µL of 140 mM MgOAc.

8. The detection method according to claim 6, characterized in that: The samples to be tested include raw ginseng, slices, powder, ginseng seeds, processed ginseng slices, extracts, traditional Chinese medicine preparations containing ginseng, food containing ginseng, and health products containing ginseng.

9. A T7 / crDNA hybridization strand, characterized in that: The hybrid chain is formed by mixing the T7 primer shown in SEQ ID NO.7 with any one of the crDNA templates in SEQ ID NO.1 to 6 in equal molar amounts and then annealing; the hybrid chain can be transcribed into crRNA for recognizing target RNA under the action of T7 RNA polymerase.

10. An RPA primer pair, characterized in that, The primer pair is used to specifically amplify the ginseng 18S rRNA target sequence, and the 5' end of the downstream primer is fused with a T7 promoter recognition sequence; the upstream primer is selected from any one of SEQ ID NO. 8 to 10, and the downstream primer is selected from any one of SEQ ID NO. 11 to 13.

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