Primer group, detection method and kit for identifying radix cyathulae through LMTIA technology and application of primer group, detection method and kit

Abstract

The invention belongs to the technical field of biomolecule detection, and particularly relates to a primer group, a detection method and a kit for identifying radix cyathulae through an LMTIA technology and application. The invention discloses an LMTIA primer group and a probe for detecting source components of radix cyathulae. The LMTIA primer group and the probe comprise a CNiuX-F primer, a CNiuX-B primer, a CNiuX-LF primer and a CNiuX-probe. The LMTIA primer group and the probe can be used for detecting the radix cyathulae source component, are high in speed, sensitivity and specificity, can effectively avoid false positive detection results, can quickly, efficiently and accurately identify the radix cyathulae source component in traditional Chinese medicinal materials, can be used for quickly and accurately detecting the quality of the traditional Chinese medicinal materials, and can be widely applied to detection of radix cyathulae source components in traditional Chinese medicinal materials. The urgent need of the country for authenticity detection of traditional Chinese medicinal materials and the like is met.

Description

Technical Field

[0001] The invention belongs to the technical field of biomolecule detection, and specifically relates to a primer set, a detection method, a kit and application for identifying Cyathula officinalis using LMTIA technology. Background Art

[0002] Sichuan Cyathula is the dried root of the Amaranthaceae plant Sichuan Cyathula, which is included in the 1990 edition of the Pharmacopoeia of the People's Republic of China and is a famous local medicinal material in Sichuan Province. Sichuan Cyathula is nearly cylindrical, slightly twisted, slightly thinner or with a few branches, 30-60cm long and 0.5-3cm in diameter. The surface is yellow-brown or gray-brown, with longitudinal wrinkles, root traces and most transverse protruding lenticels. It is tough and not easy to break. The cross section is light yellow or brownish yellow, and the vascular bundles are dotted and arranged in several concentric rings. The smell is slight and the taste is sweet. It is better to use thick strips, flexible texture, few branches and light yellow cross section. Sichuan Cyathula is often used to promote blood circulation and remove blood stasis. For blood stasis, Sichuan Cyathula will have better clinical efficacy. Huai Achyranthes is often used to nourish the liver and kidneys and strengthen the tendons and bones. For liver and kidney deficiency, waist and knee pain, and lower limb weakness, Huai Achyranthes often achieves better clinical effects. In short, both can promote blood circulation and remove blood stasis, nourish the liver and kidneys, strengthen the tendons and bones, promote diuresis and defecate, draw fire from hidden blood, and guide drugs downward. Huai Niuxi focuses on nourishing, while Sichuan Niuxi focuses on clearing, which is the most essential difference between the two.

[0003] From the perspective of the clinical drug safety of Achyranthes bidentata, there are few counterfeit products of Sichuan Achyranthes bidentata on the market, but there are many easily confused products. In addition to the same family of Achyranthes bidentata, some rhizome medicinal materials such as Platycodon grandiflorum, Glehnia littoralis, and Achyranthes bidentata are similar to Sichuan Achyranthes bidentata in appearance and morphology, and are easily misused as Sichuan Achyranthes bidentata, resulting in medication confusion and inappropriate efficacy. Therefore, choosing a scientific identification method to quickly and accurately identify Sichuan Achyranthes bidentata, a local medicinal material in Sichuan, is of great significance for safe and rational use of drugs.

[0004] At present, the methods for identifying Achyranthes bidentata from different origins include microscopic identification, physical and chemical identification, analysis methods combining liquid chromatography-mass spectrometry technology with multivariate statistical analysis technology, liquid chromatography fingerprinting, infrared spectroscopy combined with machine learning methods and multiple site-specific PCR identification methods. Among the above established methods, microscopic identification and physical and chemical identification are not accurate enough; chromatography-mass spectrometry technology equipment is expensive and requires high professional skills of operators; near-infrared spectroscopy needs to be combined with other methods, the amount of spectral data is large, and it is not easy to achieve rapid detection.

[0005] Modern molecular biotechnology, represented by PCR, is convenient, fast, and accurate. It does not require large instruments and complex operating systems. It analyzes the sources and characteristics of raw materials and products at the genetic level and has been widely used in food and drug authenticity identification research such as food type identification and origin tracing. In this regard, a method for detecting the presence of Sichuan Cyathula components in traditional Chinese medicines is established from a genetic perspective. This genetic detection method has the characteristics of short time, strong specificity, and high sensitivity, and is of great research value and significance.

[0006] Since the ladder-shape melting temperature isothermal amplification (LMTIA) technology was reported in 2021, it has become another fast and convenient new nucleic acid amplification technology after PCR and LAMP technology because of its non-thermal and non-enzymatic single-stranded template production advantages. Only one pair of primers is needed to complete amplification within 20 minutes. LMTIA technology solves the false positive problems caused by nonspecific amplification, aerosol contamination, and primer dimers in LAMP technology, and has the advantages of low cost, strong specificity, and high sensitivity.

[0007] At present, there is no method that uses the Proofman fluorescent probe combined with the LMTIA system to complete the rapid detection of components derived from Sichuan Cyathula within 20 minutes. There is no method or kit reported at home and abroad that can quickly, simply, specifically and sensitively detect components derived from Sichuan Cyathula in samples such as Chinese herbal medicines.

[0008] Therefore, there is a need in the art for a rapid, specific, and sensitive method for detecting components derived from Cyathulae Bidentatae for rapid detection of components derived from Cyathulae Bidentatae in traditional Chinese medicines. Summary of the invention

[0009] The present invention intends to adopt the latest LMTIA technology (for technical principles, see CN202011105405.3), design LMTIA primers and probes, establish a rapid detection method for components derived from Cyathulae, and provide technical support for the supervision of adding Cyathulae-derived components to Chinese herbal medicines. One purpose of the present invention is to provide a primer set, detection method, and kit for identifying Cyathulae-derived components using LMTIA technology, which are used to detect components derived from Cyathulae-derived components in Chinese herbal medicines and improve the safety supervision and detection technology of Chinese herbal medicines.

[0010] The present invention is achieved through the following technical solutions:

[0011] The sequences of LMTIA primers, probes, etc. used to detect components from Cyathula officinalis are as follows:

[0012] CNiuX-F:5`-TTAACACGCCGGGTTTTTTGTTTGGGGGTGCTGCTT-3`

[0013] CNiuX-B: 5`-CCGGCGTGTTAAGCTTTTCACTCTTGTGTATGTTCCTTGGC-3`

[0014] CNiuX-LF:5`-GTTCGTTGTTGTGCC-3`

[0015] CNiuX-probe: 5`-BHQ2-GTTCGTTGTTGTGCT-3`6-FAM

[0016] The second objective of the present invention is to provide a reagent for detecting components derived from Cyathula capitata, and the specific technical scheme is: comprising a universal LMTIA premix, Bst polymerase, LMTIA primers and CNiuX-probe.

[0017] The third object of the present invention is to provide a kit for detecting components derived from Cyathula capitata, and the specific technical scheme is: comprising the LMTIA primer probe for detecting components of Cyathula capitata as described above or the reagent for detecting components of Cyathula capitata as described above.

[0018] The fourth object of the present invention is to provide a method for detecting the components of Cyathula officinalis, and the specific technical scheme is: comprising the following steps: (1) extracting sample DNA; (2) preparing the LMTIA reaction system using the LMTIA primer set and probe as claimed in claim 1 or the reagent as claimed in claim 3 or the kit as claimed in claim 4; (3) placing the prepared LMTIA reaction system in a real-time fluorescence PCR instrument for constant temperature amplification; (4) observing the amplification curve after 20 minutes of amplification. (5) If an exponential curve appears in the amplification result graph, the sample contains the components derived from Cyathula officinalis; if no amplification curve appears in the amplification result, the sample does not contain the components derived from Cyathula officinalis.

[0019] Preferably, the temperature of the isothermal amplification is 58° C., and the time of the isothermal amplification is 20 minutes.

[0020] Preferably, the molar ratio of CNiuX-F:CNiuX-B:CNiuX-LF:CNiuX-probe in the LMTIA reaction system is 4:4:1:1.

[0021] The present invention provides the use of the LMTIA primer set, probe, reagent, and kit for detecting the Cyathula-derived components in traditional Chinese medicines, which can be used to detect the Cyathula-derived components in traditional Chinese medicines.

[0022] The beneficial effects of the present invention are as follows: the LMTIA primer set and probe for detecting the Cyathula-derived components of the present invention have good repeatability, specificity and sensitivity, fast detection speed, and can detect the Cyathula-derived components in the sample under constant temperature conditions.

[0023] The LMTIA primer set and probe of the present invention have better specificity at 58°C.

[0024] The absolute sensitivity of the LMTIA primer set and probe of the present invention can reach 50pg / μL, and the relative sensitivity can reach 0.1%.

[0025] The method for detecting the components of Cyathula officinalis of the present invention is simple and rapid, and the detection result can be obtained after 20 minutes of constant temperature reaction.

[0026] The LMTIA primer set and probe of the present invention can be used to detect components derived from Cyathula capitata. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 It is the result of the optimization of the Cyathulae LMTIA primer set and probe temperature of the present invention; the amplification curve of the Cyathulae sample DNA is above the baseline, and the blank control (ddH2O) is below the baseline.

[0029] Figure 2 The specific results of the Cyathulae LMTIA primer set and probe of the present invention for detecting Cyathulae at 58°C; wherein the amplification curve of the Cyathulae sample DNA is above the baseline, and the six sample DNAs of Achyranthes bidentata, burdock root, Platycodon grandiflorum, Codonopsis pilosula, Glehnia littoralis, and Achyranthes bidentata and a blank control (ddH2O) are below the baseline; 1 represents the Cyathulae DNA, and 2 represents the six sample DNAs of Achyranthes bidentata, burdock root, Platycodon grandiflorum, Codonopsis pilosula, Glehnia littoralis, and Achyranthes bidentata and a blank control (ddH2O)

[0030] Figure 3The present invention is an amplification curve of the absolute sensitivity test of Cyathulae Bidentatae using the Cyathulae Bidentatae LMTIA primer set and probe at 58°C; wherein the amplification curve of the Cyathulae Bidentatae sample DNA is above the baseline, 1 indicates that the concentration of the DNA template is 5 ng / μL; 2 indicates that the concentration of the DNA template is 1 ng / μL, 3 indicates that the concentration of the DNA template is 100 pg / μL, 4 indicates that the concentration of the DNA template is 50 pg / μL, 5 indicates that the concentration of the DNA template is 10 pg / μL, and 6 indicates a blank control (ddH2O).

[0031] Figure 4 The present invention is a primer set and probe for LMTIA of the Cyathulae Bidentata of the present invention at 58°C, and an amplification curve of the relative sensitivity test of the Cyathulae Bidentata is detected; wherein the area above the baseline is an amplification curve of the Cyathulae Bidentata sample DNA, and 1, 2, 3, 4, 5, 6, 7, and 8 respectively represent the mass fractions of the Cyathulae Bidentata powder in the mixed sample of the Cyathulae Bidentata and the Huai River Cyathulae Bidentata powder, and the mass fractions of the Cyathulae Bidentata powder are 100%, 20%, 10%, 5%, 2%, 1%, 0.1%, and a blank control (ddH2O), respectively.

[0032] Figure 5 The present invention is a Cnidium monnieri LMTIA primer set and probe at 58°C, which are used to detect the amplification curve of real Cnidium monnieri samples sold on the market; wherein the amplification curve above the baseline is the amplification curve of the Cnidium monnieri sample DNA, and 1, 2, 3, 4, 5, 6 and 7 represent Cnidium monnieri No. 2, Cnidium monnieri No. 3, Cnidium monnieri No. 4, Cnidium monnieri No. 5, Cnidium monnieri No. 7, Cnidium monnieri No. 8 and a blank control (ddH2O), respectively. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The sources of the reagents used in the present invention are shown in Table 1 below.

[0035] Table 1 Sources of reagents

[0036]

[0037] Example 1 LMTIA primer set, probe design and temperature optimization

[0038] (1) Design of LMTIA primers and probes: The target sequence was the internal transcribed spacer (ITS) of the rRNA gene of Cyathula capitata. The sequence was analyzed using the Oligo 7 software, and the fragments with ladder-shaped melting temperatures were selected. Then, the LMTIA primers were designed using the Primer 3 Plus online software (http: / / www.primer3plus.com). The CNiuX-probe was designed based on the loop primer CNiuX-LF. The fluorescent group was labeled on the mismatched base at the 3' end, and the quenching group was labeled on the 5' end. The LMTIA primer sequence of Cyathula capitata and the Proofman CNiuX-probe were synthesized by General Biosystems (Anhui) Co., Ltd. The specific sequences are shown in Table 2 below.

[0039] Table 2 Primer set and probe design for Cyathula officinalis LMTIA

[0040]

[0041] (2) DNA extraction:

[0042] The DNA of Cyathula obovata was extracted using a plant genomic DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The specific operation steps are shown in the instruction manual of the kit.

[0043] (3) Temperature optimization test of the Cyathulae LMTIA primer set and probe of the present invention

[0044] According to the LMTIA reaction system in Table 3, take the PCR eight-tube strip, add the reaction system prepared as above to the first three and last three wells of each eight-tube strip (the system does not include template DNA, 8μL in each tube), add 2μL of Cyathulae Radix DNA to the eight-tube strip, add 2μL of ddH2O, drop paraffin oil to each well, cover the lid and centrifuge, and put it into the Gentier 96E fully automatic medical PCR instrument for amplification reaction. The insulation temperatures of each group were 52℃, 54℃, 56℃, and 58℃, respectively, and 40 cycles were set, each cycle was 30s.

[0045] Table 3 Cyathula LMTIA reaction system (10 μL)

[0046]

[0047] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 1 As shown in the figure, at 58℃, the DNA of Cyathulae Radix can be amplified in large quantities under the action of primers, with the least number of cycles, good curve reproducibility and the highest efficiency. Therefore, 58℃ was selected as the optimal temperature for Cyathulae Radix LMTIA primers.

[0048] Example 2 Specificity of the LMTIA primer set and probe of the present invention

[0049] (1) DNA extraction of samples: DNA of Cyathula officinalis, Platycodon grandiflorum, Glehnia littoralis, Codonopsis pilosula, Arctium lappa root, Cyathula officinalis, and Cyathula officinalis were extracted using a plant genomic DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0050] (2) According to the LMTIA reaction system in Table 3, take the PCR eight-tube strip and add the prepared reaction system to the wells of the eight-tube strip (the system does not include template DNA, 8 μL in each well). Add 2 μL ddH2O, 2 μL Platycodon grandiflorum DNA, 2 μL Adenophora scabra DNA, 2 μL Codonopsis pilosula DNA, 2 μL Burdock root DNA, 2 μL Achyranthes bidentata DNA, 2 μL Achyranthes bidentata DNA and 2 μL Cyathulae bidentata DNA in order. Finally, add paraffin oil to each well, cover the lid and centrifuge, then put it into the Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature was set to 58°C, and 40 cycles were set, with each cycle lasting 30 seconds.

[0051] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 2 As shown in the figure, at 58℃, only the Cyathula capitata DNA was amplified in large quantities under the action of LMTIA primers, while no amplification curves appeared in the six samples including Achyranthes bidentata and the blank control (ddH2O), which fully demonstrated that the primer probe designed in this experiment had strong specificity for the amplification of Cyathula capitata DNA only.

[0052] Example 3 Absolute sensitivity of the LMTIA primer set and probe of the present invention

[0053] The extracted Cyathulae Radix DNA was gradiently diluted to 5ng / μL, 1ng / μL, 100pg / μL, 50pg / μL and 10pg / μL. According to the LMTIA reaction system in Table 3, eight PCR tubes were taken, and the reaction system prepared as above was added to the eight tubes (the system did not include template DNA, 8μL in each tube), and 2μL ddH2O, 2μL 5ng / μL Cyathulae Radix DNA, 2μL 1ng / μL Cyathulae Radix DNA, 2μL 100pg / μL Cyathulae Radix DNA, 2μL 50pg / μL Cyathulae Radix DNA, 2μL 10pg / μL Cyathulae Radix DNA were added in sequence, and three replicates were made for each sample, and finally paraffin oil was added to each well, the lid was covered and centrifuged, and then the amplification reaction was carried out in a Gentier 96E fully automatic medical PCR instrument. The temperature was set to 58°C, with 40 cycles of 30 s per cycle.

[0054] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 3 As shown in the figure, there is obvious amplification when the concentration of Cyathulae Radix is ​​5ng / μL, 1ng / μL, 100pg / μL, and 50pg / μL, but the amplification is unstable when the DNA concentration is 10pg / μL, because the concentration exceeds the sensitivity of the method. Therefore, the absolute sensitivity of this method can reach 50pg / μL. In a 10μL reaction system, the Cyathulae Radix DNA extracted from the sample can be detected when it reaches 50pg.

[0055] Example 4 Relative sensitivity of the LMTIA primer set and probe of the present invention

[0056] (1) DNA extraction: Sichuan Cyathula powder and Chinese Cyathula powder were premixed, wherein the mass fraction of Sichuan Cyathula powder was 100%, 20%, 10%, 5%, 2%, 1% and 0.1%, and the DNA of the mixed sample of Sichuan Cyathula powder and Chinese Cyathula powder was extracted using the plant genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. as template DNA.

[0057] (2) According to the LMTIA reaction system in Table 3, take eight PCR tubes, add the reaction system prepared as above (the system does not include template DNA, 8 μL in each tube) and 2 μL of template DNA of mixed samples with 100%, 20%, 10%, 5%, 2%, 1% and 0.1% of Cyathula officinalis and blank control (ddH2O) into 8 tubes respectively, and perform three replicates for each sample; finally, add a drop of paraffin oil to each well, cover the lid, centrifuge and place in Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature is set to 58°C, 40 cycles, and each cycle is 30 seconds.

[0058] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). Figure 4 As shown, there was obvious amplification when the mixed sample contained 100%, 20%, 10%, 5%, 1% and 0.1% of Cyathula officinalis powder, and there was no amplification in the blank control (ddH2O), so the detection limit (relative sensitivity) of this method can reach at least 0.1%.

[0059] Example 5 Detection of commercially available samples of the LMTIA primer set and probe of the present invention

[0060] (1) DNA extraction from samples: 8 samples of commercially available samples (Chuanxiong No. 1, Chuanxiong No. 2, Chuanxiong No. 3, Chuanxiong No. 4, Chuanxiong No. 5, Chuanxiong No. 6, Chuanxiong No. 7, Chuanxiong No. 8, etc.) were collected. The flake samples were crushed and weighed. The powder samples were extracted using the Plant Genomic DNA Extraction Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. The DNA of the commercially available samples was extracted according to the steps in the kit instructions and numbered 1-8 respectively.

[0061] (2) The system was prepared according to Table 3, and was used to analyze the primer set and probe to detect whether the commercially available samples contained the components of Cyathulae Radix. The reaction system prepared as above (the system does not include template DNA, 8 μL in each tube) and 2 μL of DNA extracted from samples 1-8, 2 μL of ddH2O, and 2 μL of Cyathulae Radix DNA (positive control) were added to the eight PCR tubes respectively. Finally, a drop of paraffin oil was added to each well, the lid was closed and centrifuged, and then the well was placed in the Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature was set to 58°C, 40 cycles, and each cycle was 30 seconds.

[0062] The test results were analyzed using the Gentier 96E fully automated medical PCR analysis system (V1). The test results are shown in Table 4 (“-” represents no amplification, “+” represents amplification) and Figure 5 As shown, samples No. 2, 3, 4, 5, 7, and 8 all showed amplification of Cyathula capitata, indicating that commercially available Cyathula capitata medicines contain Cyathula capitata-derived ingredients; No. 1 and 6 were not amplified, indicating that their actual ingredients did not match those on the labels and they may contain other medicinal ingredients.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0064] Table 4 Amplification results of LMTIA Cyathulae Bidentatae primer set and probe for commercially available samples

[0065]

Claims

1. A LMTIA primer set and probe for detecting components derived from Cyathula officinalis, characterized in that: The sequence of the primer set is: CNiuX-F:5`-TTAACACGCCGGGTTTTTTGTTTGGGGGTGCTGCTT-3` CNiuX-B: 5`-CCGGCGTGTTAAGCTTTTCACTCTTGTGTATGTTCCTTGGC-3` CNiuX-LF:5`-GTTCGTTGTTGTGCC-3` CNiuX-probe: 5`-BHQ2-GTTCGTTGTTGTGCT-3`6-FAM.

2. The primer combination according to claim 1, wherein a fluorescence quenching group BHQ2 is connected to the 5' end of the Cyathula capitata CNiuX-probe, and a fluorescence reporter group 6-FAM is connected to the 3' end.

3. A reagent for detecting components derived from Cyathula officinalis, characterized in that: It comprises a universal LMTIA premix, Bst polymerase and the LMTIA primer set and probe as claimed in claim 1.

4. A kit for detecting components derived from Cyathula officinalis, characterized in that: It includes the LMTIA primer set and probe as described in claim 1 or the reagent for detecting components derived from Cyathula officinalis as described in claim 3.

5. A method for detecting components derived from Cyathula officinalis, characterized in that: The method comprises the following steps: (1) extracting sample DNA; (2) preparing an LMTIA reaction system using the LMTIA primer set and probe as claimed in claim 1 or the reagent as claimed in claim 3 or the kit as claimed in claim 4; (3) placing the prepared LMTIA reaction system in a real-time fluorescence PCR instrument for constant temperature amplification; (4) observing the amplification curve after 20 minutes of amplification. (5) If an exponential curve appears in the amplification result graph, the sample contains a component derived from Cyathula capitata; if no amplification curve appears in the amplification result, the sample does not contain a component derived from Cyathula capitata.

6. The method for detecting components derived from Cyathula officinalis according to claim 5, characterized in that: The temperature of the isothermal amplification is 58° C., and the time of the isothermal amplification is 20 minutes.

7. The method for detecting components derived from Cyathula officinalis according to claim 5, characterized in that: The molar ratio of CNiuX-F:CNiuX-B:CNiuX-LF:CNiuX-probe in the LMTIA reaction system is 4:4:1:

1.

8. The use of the LMTIA primer set and probe according to claim 1, characterized in that: Application in detecting components from Cyathula capitata.

9. The use of the LMTIA primer set and probe according to claim 8, characterized in that: Application in detecting ingredients derived from Cyathula capitata in traditional Chinese medicines.

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