LMTIA primer group, detection method and kit for detecting subprostrate sophora component and application of LMTIA primer group, detection method and kit

Abstract

The invention discloses an LMTIA primer group for detecting a subprostrate sophora component, a detection method, a kit and application of the LMTIA primer group, and belongs to the technical field of molecular biology nucleic acid detection. The invention designs an LMTIA primer group for identifying the components of the subprostrate sophora, the LMTIA primer group comprises primers SdouG-1F1, SdouG-1B1, SdouG-1LF and SdouG-1LB, and a detection method for identifying the components of the subprostrate sophora is constructed on the basis of an LMTIA technology. The method is simple to operate, short in reaction time, high in sensitivity and strong in specificity. The minimum detection concentration of the vietnamese sophora root can reach 1pg / mu L, and the minimum detection limit content of the vietnamese sophora root is 0.1% for a mixed system containing other sample gene DNA (Deoxyribose Nucleic Acid). The detection method not only can specifically identify the true and false characteristics of the subprostrate sophora product, but also can effectively detect the subprostrate sophora component in the medicine matrix. The method is suitable for the fields of food and drug quality control, Chinese herbal medicine identification and market supervision.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nucleic acid detection in molecular biology, and particularly relates to a LMTIA primer set for detecting components of Sophorae tonkinensis, a detection method, a kit and application thereof. BACKGROUND

[0002] According to the "Dictionary of Identification of Chinese Herbal Medicine", Sophorae tonkinensis is also known as Ku-shu- gen, Guang-dou-gen, which is the dried root and rhizome of Sophora tonkinensis Gapnep of Leguminosae. The root is mostly long cylindrical, with a diameter of 0.7 to 1.5 cm, and is hard and not easy to break. It often has branches of different lengths, with a brown surface and irregular longitudinal lines, and transverse protruding pores. Sophorae tonkinensis is cold in nature and bitter in taste, and mainly contains active ingredients such as sophoramine and oxysophoramine. Sophorae tonkinensis has the pharmacological effect of inhibiting bacteria, and the main effects are clearing heat and resolving toxicity, reducing swelling and inflammation. It is widely used in clinical practice, and can be used for treating sore throat, lung heat cough, and cancer, and can be used for treating tinea and insect bites. The latest toxicology research shows that Sophorae tonkinensis can cause multiple system toxic reactions of the digestive system, nervous system and cardiovascular system. If used improperly, patients may have symptoms such as chest tightness, cold hands and feet, cold sweat all over the body, headache and dizziness, and lower abdominal pain. In order to avoid misuse and misapplication, it is particularly important to accurately identify Sophorae tonkinensis. At present, there are many counterfeit products of Sophorae tonkinensis on the market, such as North Sophora, Hu-zhi-zi and Baise Sophora. Under the background of the continuous rise of the price of medicinal materials due to the surge in market demand, the number of misuse poisoning incidents caused by confusion of varieties has increased significantly. In order to ensure the safety and effectiveness of Sophorae tonkinensis in clinical practice and avoid poisoning incidents caused by confusion of varieties, accurate identification of Sophorae tonkinensis has become a key quality control link.

[0003] At present, the identification methods of Sophorae tonkinensis mainly include physical methods such as identification of characteristics, spectral identification, microscopic identification, chromatographic identification and fingerprint identification. The physical methods such as identification of characteristics and microscopic identification mainly distinguish the shape and texture of the rhizome of medicinal materials. However, Sophorae tonkinensis, North Sophora and Hu-zhi-zi are not easy to distinguish in appearance, and are easily affected by personal cognitive bias, so it is difficult to accurately distinguish Sophorae tonkinensis; the chromatographic and spectral identification methods can effectively distinguish Sophorae tonkinensis and some of its counterfeit products based on the thin layer of chemical components, and have the advantages of convenient operation and fast detection speed. However, when dealing with a large sample size, these two detection techniques are difficult to achieve efficient identification, and there are also certain limitations in identifying adulteration and counterfeiting of traditional Chinese medicine. The fingerprint method is widely used to determine the liquid chromatogram of medicinal material samples to determine the authenticity of the medicinal material samples. This method has the advantages of low cost and strong practicability. However, it takes a long time, and only reflux extraction takes 1-2 hours, and the steps are more and the operation is complex, which also limits the detection efficiency, making it difficult to preliminarily screen a large number of samples in a short time. SUMMARY

[0004] The application aims to provide a LMTIA primer set for detecting a component of Sophorae tonkinensis Pierre, a detection method, a kit and application thereof, so as to solve the problems in the prior art.

[0005] To achieve the above-mentioned purpose, the application provides the following solutions.

[0006] The application provides a primer set for identifying a component of Sophorae tonkinensis Pierre by LMTIA technology, comprising the following primers.

[0007] SdouG-1F1: 5'-ATACACGTGTCGCAACTTTTCCCATCGGCCCGGAGA-3';

[0008] SdouG-1B1: 5'-GTATCCAAAAGACTCTTTTTTTCGCTACGTTCTTCATCGA-3';

[0009] SdouG-1LF: 5'-ACCACGCAAGCACCG-3';

[0010] SdouG-1LB: 5'-CGGATATCTCGGCTC-3'.

[0011] The application further provides a kit for identifying a component of Sophorae tonkinensis Pierre by LMTIA technology, comprising the primer set.

[0012] The application further provides application of the primer set or the kit in identifying a component of Sophorae tonkinensis Pierre in traditional Chinese medicine and health products.

[0013] The application further provides a detection method for identifying a component of Sophorae tonkinensis Pierre based on LMTIA technology, comprising the following steps: obtaining genomic DNA of a sample to be detected, taking the genomic DNA as a template, performing LMTIA reaction by using the primer set, and judging whether the sample to be detected contains a component of Sophorae tonkinensis Pierre according to whether an amplification curve exists.

[0014] The application further provides the detection method, and the system of the LMTIA reaction comprises: 0.39 μL of primers SdouG-1F1, SdouG-1B1, SdouG-1LF and SdouG-1LB, 4.4 μL of universal LMTIA reaction premix, 3.21 μL of DEPC water and 2 μL of DNA template.

[0015] Preferably, the molar ratio of SdouG-1F1, SdouG-1B1, SdouG-1LF and SdouG-1LB in the system of the LMTIA reaction is 2:2:1:1.

[0016] Preferably, the procedure of the LMTIA reaction is isothermal amplification at 58℃, 1 minute and 30 seconds for one cycle, and 40 cycles in total.

[0017] Preferably, the judging method is that if an amplification curve appears in the amplification result, the sample to be tested contains the Radix Sophorae Tonkinensis component; if no amplification curve appears in the amplification result, the sample to be tested does not contain the Radix Sophorae Tonkinensis component.

[0018] The present application discloses the following technical effects:

[0019] The present application discloses a method for detecting Radix Sophorae Tonkinensis by ladder-shape melting temperature isothermal amplification (LMTIA). The LMTIA is based on the difference between the primer and the target sequence melting temperature, and uses the ITS2 difference sequence as the target, and uses the Oligo 7 software to screen the target sequence with ladder-shape melting temperature curve, and designs the LMTIA reaction primer. The experiment determines that the optimum temperature of the LMTIA reaction is 58℃, compared with the traditional PCR, the LMTIA method does not need to use the thermal cycler in the operation process, can be applied to the shorter target sequence, the operation is simple, the reaction time is short, the sensitivity is high, the specificity is strong, the detection concentration of the Radix Sophorae Tonkinensis is as low as 1 pg / μL, and the minimum detection limit of the Radix Sophorae Tonkinensis in the mixed system containing other sample gene DNA is 0.1%. The detection method can not only specifically identify the authenticity of the Radix Sophorae Tonkinensis product, but also effectively detect the Radix Sophorae Tonkinensis component in the product matrix. It is suitable for food and drug quality control, Chinese herbal medicine identification and market supervision and other fields. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0021] Figure 1 PCR amplification diagram of the first set of system for Radix Sophorae Tonkinensis LMTIA primer; 1: Radix Sophorae Tonkinensis at 52℃; 2: Radix Sophorae Tonkinensis at 54℃; 3: Radix Sophorae Tonkinensis at 56℃; 4: Radix Sophorae Tonkinensis at 58℃; 5: DEPC water;

[0022] Figure 2PCR amplification chart for screening the second set of LMTIA primers for Radix Sophorae Tonkinensis; 1: Radix Sophorae Tonkinensis at 52℃; 2: Radix Sophorae Tonkinensis at 54℃; 3: Radix Sophorae Tonkinensis at 56℃; 4: Radix Sophorae Tonkinensis at 58℃; 5: DEPC water;

[0023] Figure 3 PCR amplification chart for screening the third set of LMTIA primers for Radix Sophorae Tonkinensis; 1: Radix Sophorae Tonkinensis at 52℃; 2: Radix Sophorae Tonkinensis at 54℃; 3: Radix Sophorae Tonkinensis at 56℃; 4: Radix Sophorae Tonkinensis at 58℃; 5: DEPC water;

[0024] Figure 4 Optimization result chart for LMTIA detection of Radix Sophorae Tonkinensis; 1: 57℃; 2: 58℃; 3: 59℃; 4: DEPC water;

[0025] Figure 5 Sensitivity result chart for LMTIA detection of Radix Sophorae Tonkinensis; 1: Radix Sophorae Tonkinensis 1 ng / μL; 2: Radix Sophorae Tonkinensis 100 pg / μL; 3: Radix Sophorae Tonkinensis 10 pg / μL; 4: Radix Sophorae Tonkinensis 1 pg / μL; 5: Radix Sophorae Tonkinensis 100 fg / μL, 10 fg / μL, DEPC water;

[0026] Figure 6 Detection limit result chart for LMTIA detection of Radix Sophorae Tonkinensis; 1: Radix Sophorae Tonkinensis 100%; 2: Radix Sophorae Tonkinensis 20%; 3: Radix Sophorae Tonkinensis 10%; 4: Radix Sophorae Tonkinensis 5%; 5: Radix Sophorae Tonkinensis 1%; 6: Radix Sophorae Tonkinensis 0.1%; 7: Radix Sophorae Tonkinensis 0.01%, DEPC water;

[0027] Figure 7 Specificity result chart for LMTIA detection of Radix Sophorae Tonkinensis; 1: Radix Sophorae Tonkinensis; 2: Radix Dioscoreae, Ophiopogon japonicus, Saposhnikovia divaricata, Pueraria lobata, DEPC water;

[0028] Figure 8 Real sample result chart for LMTIA detection of Radix Sophorae Tonkinensis; 1: Radix Sophorae Tonkinensis DNA; 2: Green cloak powder; 3: Throat disease Ling tablet; 4: Double throat wind powder; 5: Toothache Ning drop pill; 6: Guilin watermelon cream tablet; 7: Throat disease Ling capsule; 8: Heat acne pill; 9: DEPC water. DETAILED DESCRIPTION

[0029] The various illustrative embodiments of the present application will now be described in detail below. The detailed description is not intended to be limiting of the present application, but rather is to provide a more detailed description of certain aspects, features and embodiments of the present application.

[0030] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, solvent amounts, and the like, there can be multiple minimum and maximum values for the range. Each minimum and maximum value is specifically and individually disclosed. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the present application and does not pose a limitation on the scope of the application unless otherwise claimed. No language is intended to indicate that the present application will necessarily encompass any particular embodiment.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, and publications mentioned herein are incorporated by reference for the disclosure and description thereof to the extent that such incorporation does not conflict with the explicit teachings of this specification. Although the foregoing application has been described in some detail by way of illustration and example for purposes of clarity and understanding, it is readily apparent to those of ordinary skill in the art in this field that various changes and modifications can be contrary to or depart from the scope and spirit of this application, without departing from the scope of the application. The description is illustrative and not restrictive, and the scope of the application is, therefore, indicated by the appended claims, rather than by the foregoing description.

[0032] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the description and examples provided herein. The description and examples are illustrative and not restrictive.

[0033] With respect to the use of "comprising", "including", "containing", "having" and "ensing" and the like, these terms are used in the sense of "open ended" and are intended to mean including but not limited to.

[0034] Example 1LMTIA primer set design and temperature optimization

[0035] 1. Primer design

[0036] The ITS sequence of Sophora flavescens was searched from the NCBI database (http: / / www.ncbi.nlm.nih.gov / ). After the target sequence was determined according to the searched Sophora flavescens sequence and the comparison of similarity, the Oligo 7 software was used to select the target sequence with a ladder-shaped melting temperature curve, and the selected sequence was subjected to bioinformatics analysis. Then, the LMTIA primer was designed using the online software Primer3Plus, and the designed primer was synthesized by General Biotech (Anhui) Co., Ltd. The sequences of the 3 groups of primers designed and synthesized according to the target sequence by using the Primer 3Plus software are shown in Table 1.

[0037] Table 1 LMTIA primer sequences of Sophora flavescens

[0038]

[0039] Set the speed of the low speed centrifuge to 12000 rpm, centrifuge the primers for 1 minute. Then add TE buffer to the primers as required, and put them into the adjustable mixer for mixing.

[0040] 2. Extraction of DNA

[0041] Use the powerful electric grinder to grind the radix sophorae tonkinensis raw materials (purchased from Henan Yuzhou, produced in Guangxi) and the purchased Chinese patent medicines containing radix sophorae tonkinensis into powder. Take 100 mg of each and place them in 1.5 mL centrifuge tubes. Use the DNA extraction kit to extract the DNA of radix sophorae tonkinensis and each Chinese patent medicine.

[0042] Use the Qubit 4 fluorometer to detect the concentration of the radix sophorae tonkinensis and each Chinese medicine gene, and rely on the Nano Drop One ultramicro nucleic acid protein detector for purity detection. Store the extracted radix sophorae tonkinensis DNA in a -20°C refrigerator for future use. The purchased Chinese patent medicine names and manufacturers are shown in Table 2.

[0043] Table 2: Purchased Chinese patent medicines containing radix sophorae tonkinensis

[0044]

[0045] 3. Screening of LMTIA primers

[0046] Use a single 10 μL reaction system for LMTIA-dye detection, and the system composition is shown in Table 3.

[0047] Table 3: LMTIA-dye reaction system composition

[0048]

[0049] Radix sophorae tonkinensis gene and DEPC water were used as positive and negative controls, respectively. Use the fluorescence quantitative PCR instrument to set the temperature gradient: 52°C, 54°C, 56°C, and 58°C. The fluorescence reagent is SYBR Green, and 1 minute and 30 seconds is the time required for one cycle. A total of 40 cycles are set, and the instrument collects fluorescence signals during the period. According to the amplification of the radix sophorae tonkinensis sample, determine the most suitable set of primers.

[0050] Screen the designed 3 groups of radix sophorae tonkinensis primers. Use the real-time fluorescence PCR instrument, set the reaction temperature to 52°C, 54°C, 56°C, and 58°C; set 40 cycles. The obtained radix sophorae tonkinensis PCR amplification chart is shown in Figures 1-3 .

[0051] Since the second and third sets of primers for detecting S. glabra designed in the application all appeared false positive in the later cycles, the first set of primers (SdouG-1F1, SdouG-1B1, SdouG-1LF and SdouG-1LB) was selected for subsequent experiments.

[0052] 4. LMTIA temperature optimization experiment

[0053] The improved S. glabra reaction system was used for determination. After the S. glabra system was prepared according to Table 3, DEPC water and S. glabra gene were used as negative and positive controls, respectively. The Archimed fluorescence quantitative PCR instrument of Kunpeng Gene (Beijing) was used, and 1 minute and 30 seconds were required for one cycle, and a total of 40 cycles were set. The optimal temperature for amplification of S. glabra primers was selected according to the amplification curve.

[0054] The first set of primers was selected, and on the basis of the pre-experiment, temperatures of 57°C, 58°C and 59°C were selected for S. glabra LMTIA temperature optimization. After 40 times of fluorescence signal collection, the S. glabra DNA amplification was obtained, as shown in Figure 4 .

[0055] It can be seen from the observation Figure 4 that only the positive control S. glabra gene was amplified, and DEPC water did not appear to be amplified. The S. glabra gene at 58°C had strong fluorescence signal, early amplification and good repeatability. Therefore, the amplification temperature of 58°C was selected for subsequent experiments.

[0056] Example 2 Sensitivity experiment of LMTIA

[0057] In order to determine the sensitivity of the established LMTIA-dye method, the S. glabra gene was gradually diluted to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL and 10 fg / μL using TE buffer, and was stored in the refrigerator.

[0058] After the S. glabra system was prepared according to Table 3, the optimized LMTIA-dye method reaction system was used for detection using the fluorescence quantitative PCR instrument. After the experiment was completed, the sensitivity of the S. glabra primer system was determined according to the amplification of DNA of different concentrations.

[0059] Under the condition of 58°C, 1 ng / μL of S. glabra gene was used as a positive control, and DEPC water was used as a negative control. After 40 times of fluorescence signal collection in the real-time fluorescence PCR instrument, the amplification graph of each S. glabra concentration was obtained, as shown in Figure 5 .

[0060] According to the amplification graph, the sensitivity of the S. glabra primer system was determined. Figure 5It can be seen that the sample of Sophora flavescens appeared amplification at the concentration of 1 ng / μL, 100 pg / μL, 10 pg / μL and 1 pg / μL, while the sample of Sophora flavescens at the concentration of 100 fg / μL, 10 fg / μL and the negative control DEPC water did not appear amplification, so the sensitivity of Sophora flavescens system was 1 pg / μL.

[0061] Example 3 Limit of detection experiment of LMTIA

[0062] The single 10 μL reaction system was prepared according to Table 3, and DEPC water and Sophora flavescens gene were set as negative control and positive control respectively. The Sophora flavescens system was used for detection, and after the experiment was completed, the minimum detection limit of Sophora flavescens simulation sample was analyzed and judged.

[0063] The mixed DNA system of Sophora flavescens content of 100%, 20%, 10%, 5%, 1%, 0.1% and 0.01% was prepared using Sophora flavescens DNA gene and Pueraria DNA gene. The Sophora flavescens gene with 100% content was used as positive control, and water was used as negative control. After 40 times of fluorescence signal collection in real-time fluorescence PCR instrument, the amplification graph of each Sophora flavescens content was obtained, as shown in Figure 6 .

[0064] According to Figure 6 It can be seen that the Sophora flavescens content of 100%, 20%, 10%, 5%, 1% and 0.1% in the mixed system appeared amplification, and 0.01% did not appear amplification. Among them, the amplification curve of the system with 100% Sophora flavescens DNA content was the highest, and it began to amplify at a lower cycle number, while the system with 0.1% Sophora flavescens DNA content still could detect Sophora flavescens although it amplified late. It showed that the method could detect the sample with Sophora flavescens content of 0.1% and above.

[0065] Example 4 Specific detection of LMTIA

[0066] The single 10 μL reaction system was prepared according to Table 3 for testing. The improved Sophora flavescens system was used for detection by using fluorescence quantitative PCR instrument. After the experiment was completed, whether the optimized Sophora flavescens system had specificity was judged according to the amplification curve.

[0067] The DNA of Sophora flavescens, Chinese yam, Pueraria, Saposhnikovia divaricata and Ophiopogon japonicus was selected for specific detection of Sophora flavescens system. After 40 times of fluorescence signal collection in fluorescence quantitative PCR instrument, the amplification graph of genomic DNA of Sophora flavescens, Chinese yam, Pueraria, Saposhnikovia divaricata and Ophiopogon japonicus was obtained, and the amplification of genomic DNA of the five medicinal materials was as shown in Figure 7 .

[0068] From Figure 7As shown, only the Sophora flavescens genomic DNA was amplified in the Sophora flavescens system, while no amplification was observed in the four Chinese medicinal herbs, namely, Dioscorea opposita, Pueraria root, Saposhnikovia divaricata, and Radix Ophiopogonis, as well as the negative control water. This demonstrates the specificity of the Sophora flavescens primer system and the high specificity of the developed method, with no cross-reactions with other species. It can specifically distinguish Sophora flavescens genes from genes of other species and is suitable for detecting Sophora flavescens genes in traditional Chinese medicine products.

[0069] Example 5 LMTIA real sample detection

[0070] After preparing a single 10 μL reaction system according to Table 3, using 1 ng / μL Sophora flavescens gene as a positive control and DEPC water as a negative control, the LMTIA-dye detection method established in the present invention was used to detect Sophora flavescens-derived components in seven commercially available Chinese patent medicines containing Sophora flavescens in their ingredients. A fluorescent quantitative PCR instrument was used with the cycle number set to 40 to detect the Sophora flavescens system.

[0071] After DNA gene extraction, the seven drugs were tested using the Sophora flavescens system LMTIA-dye method to see if they contained Sophora flavescens. The test results are as follows: Figure 8 shown.

[0072] Depend on Figure 8 The amplification of DNA from Sophora flavescens and seven Chinese patent medicines was observed, indicating that all seven medicines contain Sophora flavescens. The LMTIA system achieved a 100% detection rate for the seven Chinese patent medicines containing Sophora flavescens in their ingredient lists, demonstrating high sensitivity and specificity.

[0073] For the Sophora flavescens system, the experimental results show that the amplification effect is best when the first set of primers is selected in the system and the temperature is 58°C. Under this primer system and temperature, the detection concentration of Sophora flavescens can reach as low as 1pg / μL. For a mixed system containing gene DNA of other samples, the minimum detection limit of Sophora flavescens is 0.1%.

[0074] The LMTIA detection system used in this experiment demonstrated excellent cyclic amplification results in sensitivity, detection limit, and specificity testing for Sophora flavescens. This assay not only specifically distinguishes Sophora flavescens products from counterfeit products but also effectively detects Sophora flavescens components in pharmaceutical matrices, demonstrating high sensitivity. The assay is quick and easy to perform. Based on the test results for actual samples, the LMTIA assay is suitable for Sophora flavescens differentiation studies.

[0075] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A primer set for identifying Sophora flavescens components using LMTIA technology, characterized in that: The following primers are included: SdouG-1F1: 5'-ATACACGTGTCGCAACTTTTCCCATCGGCCCGGAGA-3'; SdouG-1B1: 5'-GTATCCAAAAGACTCTTTTTTTCGCTACGTTCTTCATCGA-3'; SdouG-1LF:5'-ACCACGCAAGCACCG-3'; SdouG-1LB: 5'-CGGATATCTCGGCTC-3'.

2. A kit for identifying Sophora flavescens components using LMTIA technology, characterized in that: Comprising the primer set according to claim 1.

3. Use of the primer set according to claim 1 or the kit according to claim 2 in identifying components of Sophora flavescens in traditional Chinese medicine or health products.

4. A detection method for identifying components of Sophora flavescens based on LMTIA technology, characterized in that: The following steps are involved: Obtain genomic DNA of the sample to be tested, use the genomic DNA as a template, and perform LMTIA reaction using the primer set according to claim 1, and determine whether the sample to be tested contains Sophora flavescens components based on the presence or absence of an amplification curve.

5. The detection method according to claim 4, wherein The LMTIA reaction system includes: 0.39 μL of primers SdouG-1F1, SdouG-1B1, SdouG-1LF and SdouG-1LB, 4.4 μL of universal LMTIA reaction premix, 3.21 μL of DEPC water and 2 μL of DNA template.

6. The detection method according to claim 5, wherein In the LMTIA reaction system, the molar ratio of SdouG-1F1, SdouG-1B1, SdouG-1LF and SdouG-1LB is 2:2:1:

1.

7. The detection method according to claim 4, wherein The procedure of the LMTIA reaction is: isothermal amplification at 58° C., 1 minute 30 seconds as one cycle, and 40 cycles in total.

8. The detection method according to claim 4, wherein The judgment method is: if an amplification curve appears in the amplification result, the sample to be tested contains the Sophora flavescens component; if no amplification curve appears in the amplification result, the sample to be tested does not contain the Sophora flavescens component.

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