Primer group, detection method and kit for identifying radix achyranthis bidentatae by 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 achyranthes roots by an LMTIA technology and application. The invention discloses an LMTIA primer group and a probe for detecting an achyranthes root source component. The LMTIA primer group comprises an HNiuX-F primer, an HNiuX-B primer, an HNiuX-LF primer and an HNiuX-probe. The LMTIA primer group and the probe can be used for detecting the radix achyranthis bidentatae source component, are high in speed, sensitivity and specificity, can effectively avoid a false positive detection result, can quickly, efficiently and accurately identify the radix achyranthis bidentatae source component in the traditional Chinese medicinal material, can be used for quickly and accurately detecting the quality of the traditional Chinese medicinal material, and can be used for detecting the radix achyranthis bidentatae source component in the traditional Chinese medicinal material. 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 Achyranthes bidentata using LMTIA technology. Background Art

[0002] Huai Niuxi is a commonly used Chinese medicine, which comes from the dried root of Huai Niuxi, a perennial herb of the Amaranthaceae family. It is mainly produced in Jiaozuo, Henan (ancient Huaiqing Prefecture), and is one of the "Four Huai Medicines" in Henan. It has a long history of cultivation, excellent quality, and large output. It is considered to be a top-grade product in the "Shennong Bencao Jing". Huai Niuxi is one of the major medicinal materials in my country, and the market demand is large. Huai Niuxi is often used to nourish the liver and kidneys, strengthen the tendons and bones. For liver and kidney deficiency, soreness of the waist and knees, and weakness of the lower limbs, Huai Niuxi often achieves better clinical effects. Sichuan Niuxi is often used to promote blood circulation and remove blood stasis. For blood stasis, Sichuan Niuxi has better clinical effects. In short, the effects of both can promote blood circulation and remove blood stasis, nourish the liver and kidneys, strengthen the tendons and bones, promote diuresis and promote defecation, occult blood and fire, and guide the downward movement of medicine. Huai Niuxi focuses on nourishing, while Sichuan Niuxi focuses on clearing. This is the most essential difference between the two.

[0003] From the perspective of the clinical drug safety of Achyranthes bidentata, there are few fake Achyranthes bidentata in the market, but there are many easily confused products. In addition to the same family of Achyranthes bidentata, some rhizome medicinal materials such as Codonopsis pilosula, Platycodon grandiflorum, and Glehnia littoralis are similar to Achyranthes bidentata in appearance and morphology, and are easily misused as Achyranthes bidentata, resulting in medication confusion and inappropriate efficacy. Therefore, choosing a scientific identification method to quickly and accurately identify the authentic Henan Achyranthes bidentata 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 adulteration identification, medicinal material origin tracing, and other research. In this regard, a method for detecting the presence of Achyranthes bidentata in drugs and tracing their origins was 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 advantages of non-thermal cycle and non-enzymatic single-stranded template production, which only requires a pair of primers to complete amplification within 20 minutes. LMTIA technology solves the false positive problems caused by non-specific 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 Achyranthes bidentata-derived components within 20 minutes. There is no report at home and abroad on methods and kits that can quickly, simply, specifically and sensitively detect Achyranthes bidentata-derived components 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 Rhizoma Cynoglossi, so as to rapidly detect components derived from Rhizoma Cynoglossi in traditional Chinese medicines. Summary of the invention

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

[0012] HNiuX-F:5`-ATATCACGCCGGGTTTTCTTGATTGGTGGTGCTGCC-3`

[0013] HNiuX-B:5`-CCCGGCGTGATATTTTTACACTCTCATTTTTGTTCCTTGGC-3`

[0014] HNiuX-LF:5`-GTTCGTTATTGTGTTG-3`

[0015] HNiuX-probe: 5`-BHQ2-GTTCGTTATTGTGTTA-3`6-FAM

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

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

[0018] The fourth object of the present invention is to provide a method for detecting the components of Achyranthes bidentata, and the specific technical scheme is: comprising 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 Achyranthes bidentata-derived components; if no amplification curve appears in the amplification result, the sample does not contain Achyranthes bidentata-derived components.

[0019] Preferably, the temperature of the isothermal amplification is 54° C., and the time of the isothermal amplification is 20 minutes.

[0020] Preferably, the molar ratio of HNiuX-F:HNiuX-B:HNiuX-LF:HNiuX-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 components derived from Achyranthes bidentata in traditional Chinese medicines, which can be used to detect components derived from Achyranthes bidentata in traditional Chinese medicines.

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

[0023] The LMTIA primer set and probe of the present invention have better specificity at 54°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 Achyranthes bidentata source of the present invention is simple and rapid, and the detection result can be obtained after a constant temperature reaction for 20 minutes.

[0026] The LMTIA primer set and probe of the present invention can be used to detect components derived from Achyranthes bidentata. 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 Achyranthes bidentata LMTIA primer set and probe temperature of the present invention; the amplification curve of the Achyranthes bidentata sample DNA is above the baseline, and the blank control (ddH2O) is below the baseline.

[0029] Figure 2 It is the specific result of detecting Achyranthes bidentata by the Achyranthes bidentata LMTIA primer set and probe of the present invention at 54°C; wherein the amplification curve of Achyranthes bidentata sample DNA is above the baseline, and the DNA of six samples including Achyranthes bidentata, burdock root, Platycodon, Codonopsis pilosula, Glehnia littoralis, and Achyranthes bidentata and a blank control (ddH2O) are below the baseline; 1 represents Achyranthes bidentata DNA, and 2 represents the DNA of six samples including Achyranthes bidentata, burdock root, Platycodon, Codonopsis pilosula, Glehnia littoralis, and Achyranthes bidentata and a blank control (ddH2O).

[0030] Figure 3 The present invention is an amplification curve of the absolute sensitivity test of the Achyranthes bidentata LMTIA primer set and probe for detecting the Achyranthes bidentata at 54°C; wherein the amplification curve of the Achyranthes bidentata 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 a blank control (ddH2O).

[0031] Figure 4 The present invention is amplification curve of the relative sensitivity test of Achyranthes bidentata LMTIA primer set and probe for detecting Achyranthes bidentata at 54°C; wherein the amplification curve above the baseline is the amplification curve of the Achyranthes bidentata sample DNA, and 1, 2, 3, 4, 5, 6, 7 and 8 respectively represent the mass fraction of Achyranthes bidentata powder in the mixed sample of Achyranthes bidentata powder and Achyranthes bidentata powder of 100%, 20%, 10%, 5%, 2%, 1%, 0.1% and blank control (ddH2O).

[0032] Figure 5 The present invention is a polysaccharide LMTIA primer set and probe for detecting the amplification curve of real polysaccharide samples sold on the market at 54°C; wherein the amplification curve above the baseline is the amplification curve of the polysaccharide sample DNA, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 represent polysaccharide No. 1, polysaccharide No. 2, polysaccharide No. 3, polysaccharide No. 4, polysaccharide No. 5, polysaccharide No. 6, polysaccharide No. 7, polysaccharide No. 8, polysaccharide No. 9, polysaccharide No. 10 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 internal transcribed spacer (ITS) of the rRNA gene of Achyranthes bidentata was used as the target sequence. The sequence was analyzed using the Oligo 7 software, and the fragments with ladder-shaped melting temperatures were selected. Then, the Primer 3 Plus online software (http: / / www.primer3plus.com) was used to design LMTIA primers. The HNiuX-probe was designed based on the loop primer HNiuX-LF. The fluorescent group was labeled on the mismatched base at the 3' end, and the quenching group was labeled on the 5' end of the base. The LMTIA primer sequence of Achyranthes bidentata and the Proofman HNiuX-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 of Achyranthes bidentata LMTIA

[0040]

[0041] (2) DNA extraction:

[0042] The DNA of Achyranthes bidentata 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 Achyranthes bidentata 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 Achyranthes bidentata DNA to the eight-tube strip, add 2μL of ddH2O, do three replicates for each sample, add 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 Achyranthes bidentata 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 54℃, the DNA of Achyranthes bidentata 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, 54℃ was selected as the optimal amplification temperature of Achyranthes bidentata 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 Achyranthes bidentata, Platycodon grandiflorum, Glehnia littoralis, Codonopsis pilosula, Arctium lappa, Achyranthes bidentata, 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 Cyathula officinalis DNA and 2 μL Achyranthes bidentata DNA in order, and repeat three times for each sample. Finally, add paraffin oil to each well, cover the lid, centrifuge, and place in the Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature is set to 54°C, and 40 cycles are set, each cycle is 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 54℃, only the DNA of Achyranthes bidentata 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 LMTIA primer probe designed in this experiment had strong specificity for amplifying DNA of Achyranthes bidentata only.

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

[0053] The extracted Achyranthes bidentata 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 Achyranthes bidentata DNA, 2μL 1ng / μL Achyranthes bidentata DNA, 2μL 100pg / μL Achyranthes bidentata DNA, 2μL 50pg / μL Achyranthes bidentata DNA, 2μL 10pg / μL Achyranthes bidentata DNA, 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 54°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, when the concentration of Achyranthes bidentata DNA was 5ng / μL, 1ng / μL, 100pg / μL, and 50pg / μL, there was obvious amplification, but when the DNA concentration was 10pg / μL, the amplification was unstable. This is because the concentration exceeded the sensitivity of the method, making the amplification unstable. Therefore, the absolute sensitivity of this method can reach 50pg / μL. In a 10μL reaction system, the Achyranthes bidentata DNA extracted from the sample can reach 50pg and can be detected.

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

[0056] (1) DNA extraction: Achyranthes bidentata powder and Achyranthes bidentata powder were premixed, wherein the mass fractions of Achyranthes bidentata powder were 100%, 20%, 10%, 5%, 2%, 1% and 0.1%, respectively. The DNA of the mixed sample of Achyranthes bidentata powder and Achyranthes bidentata powder was extracted using the plant genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. and used 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 Achyranthes bidentata 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 54°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%, 2%, 1% and 0.1% of Achyranthes bidentata 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: 10 samples of commercially available samples (Achyranthes bidentata No. 1, Achyranthes bidentata No. 2, Achyranthes bidentata No. 3, Achyranthes bidentata No. 4, Achyranthes bidentata No. 5, Achyranthes bidentata No. 6, Achyranthes bidentata No. 7, Achyranthes bidentata No. 8, Achyranthes bidentata No. 9, Achyranthes bidentata No. 10, etc.) were used. 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 commercially available samples was extracted according to the steps in the kit instructions and numbered 1-10 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 Achyranthes bidentata components. 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 1-10, 2 μL of ddH2O, and 2 μL of Achyranthes bidentata DNA (positive control) were added to the 8-tube PCR tubes, and finally a drop of paraffin oil was added to each well. After the lid was closed and centrifuged, the tubes were placed in the Gentier 96E fully automatic medical PCR instrument for amplification reaction. The temperature was set to 54°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. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 all showed amplification of Polygonum multiflorum, indicating that commercially available Polygonum multiflorum medicines contain Polygonum multiflorum-derived ingredients, and their labeled ingredients are consistent with the actual 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 principle of the present invention should be included in the protection scope of the present invention.

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

[0065] .

Claims

1. A LMTIA primer set and probe for detecting components derived from Achyranthes bidentata, characterized in that: The sequence of the primer set is: HNiuX-F:5`-ATATCACGCCGGGTTTTCTTGATTGGTGGTGCTGCC-3` HNiuX-B:5`-CCCGGCGTGATATTTTTACACTCTCATTTTTGTTCCTTGGC-3` HNiuX-LF:5`-GTTCGTTATTGTGTTG-3` HNiuX-probe: 5`-BHQ2-GTTCGTTATTGTGTTA-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 Achyranthes bidentata HNiuX-probe, and a fluorescence reporter group 6-FAM is connected to the 3′ end.

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

5. A method for detecting components derived from Achyranthes bidentata, 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 Achyranthes bidentata; if no amplification curve appears in the amplification result, the sample does not contain a component derived from Achyranthes bidentata.

6. The method for detecting components derived from Achyranthes bidentata according to claim 5, characterized in that: The temperature of the isothermal amplification is 54° C., and the time of the isothermal amplification is 20 minutes.

7. The method for detecting components derived from Achyranthes bidentata according to claim 5, characterized in that: The molar ratio of HNiuX-F:HNiuX-B:HNiuX-LF:HNiuX-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 Achyranthes bidentata.

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

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