LMTIA technology-based rapid identification method for radix dipsaci

Abstract

This invention belongs to the field of molecular identification technology of traditional Chinese medicine, specifically a rapid identification method for Dipsacus asperoides based on LMTIA technology. The invention discloses an LMTIA primer set for detecting Dipsacus asperoides, including XD-F1, XD-B1, and a loop primer XD-LB. This LMTIA primer set can be used for gene detection of Dipsacus asperoides components, offering fast detection speed, high sensitivity, and strong specificity. It effectively avoids false positive results and can rapidly, efficiently, and accurately identify components in Dipsacus asperoides. It is suitable for rapid on-site identification of the authenticity of components in Dipsacus asperoides and provides a technical means for the identification of authentic traditional Chinese medicine. By simplifying the genomic DNA extraction process, the medicinal material is ground into powder and extracted using conventional reagent kits. The dosage and amplification parameters of each component in the reaction system are clearly defined, eliminating the need for complex pretreatment steps and high-end professional equipment, significantly reducing the technical requirements for operators, and simultaneously controlling the dosage of reaction reagents and detection costs.

Description

Technical Field

[0001] This invention belongs to the field of molecular identification technology of Chinese medicinal materials, specifically a rapid identification method for Dipsacus asperoides based on LMTIA technology. Background Technology

[0002] Dipsacus root (also known as Dipsacus asperoides) is the dried root of the plant *Dipsacus asperoides*, belonging to the Dipsacaceae family. It was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and included in the 2020 edition of the *Chinese Pharmacopoeia (Part I)*. It is used to tonify the liver and kidneys, strengthen tendons and bones, promote healing of fractures, and stop metrorrhagia. Dipsacus root mainly contains triterpenoid saponins and iridoids. Pharmacological studies have shown that it has anti-fracture, anti-osteoporosis, and cardioprotective effects, making it a commonly used traditional Chinese medicine.

[0003] Currently, the market demand for Dipsacus asperoides mainly relies on wild resources, which have a wide natural distribution range. With the increasing demand for traditional Chinese medicine preparations using Dipsacus asperoides as a raw material, the quality of the medicinal material on the market varies greatly. Adulterated products are similar in appearance to genuine products, making accurate identification difficult through morphology alone, seriously affecting the safety and efficacy of clinical medication. Existing identification methods for Dipsacus asperoides include DNA barcoding technology and high-performance liquid chromatography, but these methods suffer from problems such as complex operation, long processing time, high requirements for professional personnel, and a tendency to produce false positives.

[0004] Melting temperature gradient isothermal amplification (LMTIA) is a novel rapid molecular detection technique that effectively overcomes the problems of nonspecific amplification, aerosol contamination, and false positives caused by primer dimers. It has the advantages of low cost, high specificity, and high sensitivity, and has been applied in food adulteration detection, detection of animal and plant-derived components, and detection of some traditional Chinese medicines. However, it has not yet been used for the identification of Dipsacus asperoides. Based on this, a rapid identification method for Dipsacus asperoides based on LMTIA technology is proposed to solve the above problems. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0006] This invention employs the latest LMTIA technology, the technical principle of which is detailed in CN202011105405.3 or PCT / CN2020 / 133584. LMTIA primers are designed to establish a rapid detection method for the components of *Dipsacus asper* medicinal materials. The rapid identification method for *Dipsacus asper* medicinal materials based on LMTIA technology described in this invention includes the following steps: Step 1: Select the ITS2 sequence of Dipsacus as the identification target. The ITS2 sequence of Dipsacus as the target is 309 bp in length and contains 298 conserved sites and 11 variable sites. Step 2: The ITS2 sequence of Dipsacus asperoides was analyzed using Oligo7 software to screen for specific target sequences with a trapezoidal melting temperature curve. Step 3: Design LMTIA primers using Primer3Plus online software based on the specific target sequence; Step 4: Extract genomic DNA from Dipsacus asperoides and the sample to be tested; Step 5: Configure the LMTIA reaction system and carry out the LMTIA amplification reaction at the optimized reaction temperature; Step 6: Determine whether the sample to be tested is Dipsacus asper based on the amplification curve and melting curve results.

[0007] As a preferred technical solution of this application, in step two, the nucleotide sequence of the specific target sequence is: GGTTGAACAAGCCTTCTTATCGAGTCGTGCGCCTCCCCGTCGCCAGGGAGACTGTTAGACCCTGACGCGTCGTCCTCGGACGTCGCTCCGACCGCGACCCCAGGTCAGGCGGATCACCCGCTGAGTTTAAGCATATCAATAAG.

[0008] As a preferred technical solution of this application, in step three, the LMTIA primers include XD-F1, XD-B1, and the loop primer XD-LB, and the nucleotide sequence of primer XD-F1 is as follows: The nucleotide sequence of primer XD-B1 is 5'-GTCCGAGGACGACGCGTTTTTCGCCAGGGAGACTGTTAGA-3', and the nucleotide sequence of loop primer XD-LB is 5'-AGGTCAGGCGGGA-3'.

[0009] As a preferred technical solution of this application, in step four, the method for extracting genomic DNA is as follows: the medicinal material is ground into a fine powder, 100mg of powder is weighed, extracted using a plant genomic DNA extraction kit, the DNA concentration and purity are detected by a micro-nucleic acid protein analyzer, and the extracted DNA is stored at -20℃.

[0010] As a preferred technical solution of this application, in step five, the total volume of the LMTIA reaction system is 10 μL, including 4.4 μL of premixed solution, 10.13 μL of 100 μM primer XD-F, 10.13 μL of 100 μM primer XD-B, 0.065 μL of 100 μM loop primer XD-LB, 3.275 μL of enzyme-free water and 2 μL of template DNA.

[0011] As a preferred technical solution of this application, in step five, the optimized reaction temperature is 58℃, and the LMTIA amplification reaction is performed using a real-time fluorescence quantitative PCR instrument. Fluorescence signals are collected once every 90 seconds for each cycle, and a total of 40 cycles are performed.

[0012] As a preferred technical solution of this application, in step six, the judgment criteria are as follows: if the sample to be tested shows a specific amplification curve and the peak shape of the melting curve is consistent with the positive control of Dipsacus asperoides, it is determined to be Dipsacus asperoides; if no amplification curve appears or the peak shape of the melting curve is inconsistent with the positive control, it is determined to be non-Dipsacus asperoides.

[0013] As a preferred technical solution of this application, the method is characterized by having a sensitivity of 1 pg / μL and a detection limit of 0.1%, and can effectively distinguish between Dipsacus asperoides and adulterants such as Arctium lappa root, Gynostemma pentaphyllum, Pinellia ternata, Angelica dahurica, and Fritillaria thunbergii.

[0014] As a preferred technical solution of this application, the feature is that, in step five, the premixed solution and primer set in the LMTIA reaction system can still maintain a stable amplification effect after at least four freeze-thaw treatments, and the freeze-thaw treatment conditions are alternating between freezing at -20°C and thawing at room temperature.

[0015] The beneficial effects of this invention are as follows: 1. This invention uses the unique ITS2 sequence of Dipsacus as the identification target, combined with Oligo7 software to screen specific target sequences with a ladder-shaped melting temperature curve, and Primer3Plus online software to design targeted LMTIA primers, to accurately lock the molecular characteristics of Dipsacus, effectively eliminating interference from adulterants such as Arctium lappa root, Ganoderma lucidum, and Pinellia ternata, ultimately achieving a highly specific identification effect, completely avoiding false positives caused by similar components, and ensuring the accuracy of Dipsacus asper identification.

[0016] 2. This invention optimizes the construction of a 10 μL LMTIA reaction system and determines 58℃ as the optimal reaction temperature. Combined with the high-efficiency amplification mode of a real-time quantitative PCR instrument: 40 cycles, with fluorescence signals collected for 90 seconds per cycle. Relying on the high sensitivity of 1 pg / μL and the low detection limit of 0.1%, even if the content of Dipsacus asperoides component in the sample is extremely low or there is trace adulteration, it can be detected quickly and accurately. Ultimately, it achieves the goal of high-efficiency and sensitive identification, meeting the comprehensive detection needs of Dipsacus asperoides medicinal materials from pure products to mixed samples.

[0017] 3. This invention simplifies the genomic DNA extraction process by grinding medicinal materials into powder and then extracting the DNA using a conventional reagent kit. The powder can be stored at -20°C. The amount of each component in the reaction system and the amplification parameters are clearly defined. There is no need for complex pretreatment steps and high-end professional equipment, which greatly reduces the technical requirements for operators. At the same time, it controls the amount of reaction reagents and the detection cost, ultimately achieving a convenient and low-cost identification effect for Dipsacus asperoides. It is suitable for the rapid detection needs of various scenarios such as grassroots testing units, pharmacies, and pharmaceutical companies, and helps to widely implement the quality control and market supervision of Dipsacus asperoides. Attached Figure Description

[0018] The invention will now be further described with reference to the accompanying drawings.

[0019] Figure 1 This is a schematic diagram of the method flow of the present invention; Figure 2 This is a schematic diagram of the sequence alignment results of the present invention; Figure 3 This is a schematic diagram of the temperature-optimized amplification curve of the present invention; Figure 4 This is a schematic diagram of the specific amplification curve of the present invention; Figure 5 This is a schematic diagram of the specific melting curve of the present invention; Figure 6 This is a schematic diagram of the sensitivity amplification curve of the present invention; Figure 7 This is a schematic diagram of the sensitivity melting curve of the present invention; Figure 8 This is a schematic diagram of the detection limit amplification curve of the present invention; Figure 9 This is a schematic diagram of the melting curve at the detection limit of the present invention; Figure 10 This is a schematic diagram of the stability amplification curve of the present invention; Figure 11 This is a schematic diagram of the market testing amplification curve of the present invention; Figure 12 This is a schematic diagram of the melting curve for market testing according to the present invention. Detailed Implementation

[0020] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0021] like Figure 1-12 As shown, the rapid identification method for Dipsacus asperoides based on LMTIA technology of the present invention specifically includes the following steps: Step 1: Target Sequence Screening and Primer Design: The ITS2 fragment was selected as the target. This sequence is 309 bp in length and contains 298 conserved sites and 11 variable sites. Oligo7 software was used to screen for specific target sequences with a gradient-shaped melting temperature curve. The nucleotide sequence is: GGTTGAACAAGCCTTCTTATCGAGTCGTGCGCCTCCCCGTCGCCAGGGAGACTGTTAGACCCTGACGCGTCGTCCTCGGACGTCGCTCCGACCGCGACCCCAGGTCAG GCGGGATCACCCGCTGAGTTTAAGCATATCAATAAG; LMTIA primers were designed using Primer3Plus online software, including primer XD-F1: 5'-GTCCGAGGACGACGCGTTTTTCGCCAGGGAGACTGTTAGA-3', primer XD-B1: 5'-CCGACCGCGACCCCTTTTTGCTTAAACTCAGCGGGTGA-3', and loop primer XD-LB: 5'-AGGTCAGGCGGGA-3'; Step 2: Genomic DNA extraction: Grind the Dipsacus asperoides and the sample to be tested (such as burdock root, Ganoderma lucidum and other adulterants) into a fine powder. Weigh 100mg of the powder and extract DNA using a plant genomic DNA extraction kit. Detect the DNA concentration (ng / μL) and purity (A260 / A280) using a micro-nucleic acid protein analyzer. Store at -20℃ for later use. Step 3: Optimization of LMTIA reaction system and conditions: The total volume of the LMTIA reaction system was 10 μL, containing 4.4 μL of premixed solution, 0.13 μL of 100 μM XD-F, 0.13 μL of 100 μM XD-B, 0.065 μL of 100 μM XD-LB, 3.275 μL of enzyme-free water, and 2 μL of template DNA. The reaction temperature was optimized by setting four temperature gradients: 57℃, 58℃, 59℃, and 60℃. The results showed that the discontinuous DNA amplification efficiency was highest at 58℃ (18 cycles) with good repeatability, thus 58℃ was determined to be the optimal reaction temperature. The amplification reaction was performed using a real-time quantitative PCR instrument, with fluorescence signals collected once every 90 seconds for a total of 40 cycles. Method performance verification: Specificity verification: Using Dipsacus as a positive control, enzyme-free water as a negative control, and DNA from Arctium lappa root, Ganoderma lucidum, Pinellia ternata, Angelica dahurica, and Fritillaria thunbergii as adulterant controls, an LMTIA reaction was performed at 58℃; the results are as follows: Figure 4 and Figure 5 As shown, only the dissected DNA showed specific amplification and melting curves, while the other controls did not, indicating that the method has high specificity. Sensitivity verification: The fragmented DNA was serially diluted with TE buffer to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, and 10 fg / μL, and then subjected to LMTIA reaction; Figure 6 and Figure 7 As shown, amplification curves were observed for disseminated DNA at concentrations of 10 ng / μL to 1 pg / μL, and the melting curve peaks were uniform. No amplification was observed at concentrations of 100 fg / μL and below, and for the negative control. The sensitivity of the determination method was 1 pg / μL. Detection limit validation: Mixed samples were prepared by combining *Dipsacus asper* and *Fritillaria thunbergii* DNA, with *Dipsacus asper* DNA volume percentages of 50%, 20%, 10%, 5%, 2%, 1%, 0.1%, and 0.01%, and subjected to LMTIA reaction; Figure 8 and Figure 9 It can be seen that samples with a DNA content of 0.1% or higher all showed amplification curves and a uniform melting peak, while samples with 0.01% DNA and negative controls showed no amplification, thus the detection limit was determined to be 0.1%. Stability verification: The reaction premix and primer set were subjected to 1-4 freeze-thaw cycles (-20°C freeze / room temperature thaw) before the LMTIA reaction; Figure 10 As shown, the dissected DNA after four freeze-thaw cycles still showed amplification curves, with a detection rate of 100%. The negative control showed no amplification, indicating that the method has good stability. Market sample testing: DNA extraction and LMTIA testing were performed on three samples of Dipsacus asper purchased from different channels, with Dipsacus asper DNA as a positive control and enzyme-free water as a negative control; Figure 11 and Figure 12 As shown, all three market samples exhibited amplification and melting curves consistent with the positive control, while the negative control showed no amplification, indicating that this method can accurately identify market samples. Example 1: Identification of Dipsacus asperoides (a medicinal herb): Sample preparation: Take the Dipsacus asperoides material to be identified, grind it into fine powder using a high-speed pulverizer, and set aside; DNA extraction: Weigh 100mg of the herbal powder and extract genomic DNA according to the operating procedure of the plant genomic DNA extraction kit. Use a micro-volume nucleic acid and protein analyzer to detect the DNA concentration and purity, ensuring that the A260 / A280 is between 1.8 and 2.0, and store at -20℃. LMTIA reaction system preparation: Add 4.4 μL of premixed solution, 0.13 μL of 100 μM D-F1, 0.13 μL of 100 μM D-B1, 0.065 μL of 100 μM D-LB, 3.275 μL of enzyme-free water and 2 μL of extracted DNA template to a centrifuge tube in sequence, mix gently, and centrifuge for a few moments; LMTIA amplification reaction: Place the centrifuge tube into the real-time fluorescence quantitative PCR instrument, set the reaction temperature to 58℃, collect the fluorescence signal once every 90 seconds for a total of 40 cycles, and start the reaction. Result Interpretation: After the reaction is complete, observe the amplification curve and melting curve; if a specific amplification curve appears and the peak shape of the melting curve is consistent with that of the continuation positive control, refer to the above results. Figure 5 , Figure 7 , Figure 9 , Figure 12 If the amplification curve or melting curve peak shape is inconsistent with the positive control, the sample to be identified is determined to be Dipsacus asperoides; if no amplification curve or melting curve peak shape is found, it is determined to be non-Dipsacus asperoides. Example 2: Differentiation and identification of Dipsacus asperoides and adulterants: Sample preparation: Take the following medicinal materials separately: Dipsacus asper, Arctium lappa root, Gynostemma pentaphyllum, Pinellia ternata, Angelica dahurica, and Fritillaria thunbergii, and grind them into fine powder. DNA extraction: Genomic DNA was extracted from the above six medicinal materials according to the method in Example 1; LMTIA reaction: Using DNA from six medicinal materials as templates, LMTIA amplification was performed according to the reaction system and conditions of Example 1, with enzyme-free water as a negative control. Result judgment: such as Figure 4 and Figure 5 As shown, only the DNA of Dipsacus asper showed specific amplification and melting curves, while those of Arctium lappa root, Ganoderma lucidum, Pinellia ternata, Angelica dahurica, Fritillaria thunbergii and the negative control did not, successfully distinguishing Dipsacus asper from adulterants; The terms "front," "back," "left," "right," "top," and "bottom" all refer to the figures in the accompanying drawings. Figure 1 Based on the perspective of the observer, the side of the device facing the observer is defined as the front, the left side of the observer is defined as the left, and so on.

[0022] In the description of this invention, it should be understood that the terms "center", "longitudinal", "lateral", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the scope of protection of this invention.

[0023] 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 the present invention is defined by the appended claims and their equivalents.

Claims

1. A rapid identification method for Dipsacus asperoides medicinal materials based on LMTIA technology, characterized in that, Includes the following steps: Step 1: Select the ITS2 sequence of Dipsacus as the identification target. The ITS2 sequence of Dipsacus as the target is 309 bp in length and contains 298 conserved sites and 11 variable sites. Step 2: The ITS2 sequence of Dipsacus asperoides was analyzed using Oligo7 software to screen for specific target sequences with a trapezoidal melting temperature curve. Step 3: Design LMTIA primers using Primer3Plus online software based on the specific target sequence; Step 4: Extract genomic DNA from Dipsacus asperoides and the sample to be tested; Step 5: Configure the LMTIA reaction system and carry out the LMTIA amplification reaction at the optimized reaction temperature; Step 6: Determine whether the sample to be tested is Dipsacus asper based on the amplification curve and melting curve results.

2. The rapid identification method for Dipsacus asperoides based on LMTIA technology according to claim 1, characterized in that, In step two, the nucleotide sequence of the specific target sequence is: GGTTGAACAAGCCTTCTTATCGAGTCGTGCGCCTCCCCGTCGCCAGGGAGACTGTTAGACCCTGACGCGTCGTCCTCGGACGTCGCTCCGACCGCGACCCCAGGTCAGGCGGATCACCCGCTGAGTTTAAGCATATCAATAAG.

3. The rapid identification method for Dipsacus asperoides based on LMTIA technology according to claim 1, characterized in that, In step three, the LMTIA primers include XD-F1, XD-B1, and the loop primer XD-LB. The nucleotide sequence of primer XD-F1 is as follows: The nucleotide sequence of primer XD-B1 is 5'-GTCCGAGGACGACGCGTTTTTCGCCAGGGAGACTGTTAGA-3', and the nucleotide sequence of loop primer XD-LB is 5'-AGGTCAGGCGGGA-3'.

4. The rapid identification method for Dipsacus asperoides based on LMTIA technology according to claim 1, characterized in that, In step four, the method for extracting genomic DNA is as follows: grind the medicinal material into a fine powder, weigh 100mg of the powder, extract it using a plant genomic DNA extraction kit, detect the DNA concentration and purity using a micro-nucleic acid protein analyzer, and store the extracted DNA at -20℃.

5. The rapid identification method for Dipsacus asperoides based on LMTIA technology according to claim 1, characterized in that, In step five, the total volume of the LMTIA reaction system is 10 μL, including 4.4 μL of premixed solution, 0.13 μL of 100 μM primer XD-F, 0.13 μL of 100 μM primer XD-B, 0.065 μL of 100 μM circular primer XD-LB, 3.275 μL of enzyme-free water, and 2 μL of template DNA.

6. The rapid identification method for Dipsacus asperoides based on LMTIA technology according to claim 1, characterized in that, In step five, the optimized reaction temperature was 58℃. The LMTIA amplification reaction was performed using a real-time quantitative PCR instrument. Fluorescence signals were collected once every 90 seconds for each cycle, and a total of 40 cycles were performed.

7. The rapid identification method for Dipsacus asperoides based on LMTIA technology according to claim 1, characterized in that, In step six, the judgment criteria are as follows: if the sample to be tested shows a specific amplification curve and the peak shape of the melting curve is consistent with that of the positive control of Dipsacus asperoides, it is judged to be Dipsacus asperoides; if no amplification curve is shown or the peak shape of the melting curve is inconsistent with that of the positive control, it is judged to be non-Dipsacus asperoides.

8. The rapid identification method for Dipsacus asperoides based on LMTIA technology according to claim 1, characterized in that, The method has a sensitivity of 1 pg / μL and a detection limit of 0.1%, and can effectively distinguish Dipsacus asper from adulterants such as Arctium lappa root, Gynostemma pentaphyllum, Pinellia ternata, Angelica dahurica, and Fritillaria thunbergii.

9. The rapid identification method for Dipsacus asperoides based on LMTIA technology according to claim 1, characterized in that, In step five, the premixed solution and primer set in the LMTIA reaction system can still maintain stable amplification effect after at least four freeze-thaw cycles. The freeze-thaw conditions are alternating between freezing at -20°C and thawing at room temperature.

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