LMTIA primer group for rhizoma anemarrhenae detection, kit and application

Abstract

The invention discloses an LMTIA primer group for rhizoma anemarrhenae detection, a kit and application, and belongs to the field of biomolecule detection. The LMTIA primer group comprises a primer ZhiMu-1F2, a primer ZhiMu-1B12, a primer ZhiMu-1LB and a probe ZhiMu-1Pr, the primer ZhiMu-1B12, the primer ZhiMu-1LB and the probe ZhiMu-1Pr. The LMTIA primer group can be used for detecting rhizoma anemarrhenae and has good specificity and sensitivity, the absolute sensitivity can reach 10 pg / mu L, and the relative sensitivity can reach 5%; the detection speed is high, and the sample rhizoma anemarrhenae can be detected under a constant-temperature condition. The invention further designs a plant internal standard primer and a probe for verifying the DNA, whether the DNA to be detected is effective or not can be verified before detection, and the effectiveness and the accuracy of a detection result are ensured. In conclusion, the method for detecting the rhizoma anemarrhenae is established on the basis of the specific primer group, and a technical means is provided for authenticity identification of traditional Chinese medicinal materials.

Description

Technical Field

[0001] The present invention relates to the technical field of biomolecular detection, and particularly to an LMTIA primer set, a kit and an application for detecting Anemarrhenae Rhizoma. Background Art

[0002] Anemarrhenae Rhizoma, commonly known as "Maozhimu", is the dried rhizome of the plant Anemarrhema asphodeloides Bge. of the Liliaceae family. It is dug in spring and autumn, the fibrous roots and sediment are removed, and then it is dried in the sun, or the outer bark is removed and then dried in the sun. It grows in dry and sunny places such as slopes, grasslands or roadsides below an altitude of 1450 meters. Anemarrhenae Rhizoma is cold in nature, bitter and sweet in taste. The combination of bitter cold can clear heat and purge fire, and the combination of sweet cold can nourish yin and moisten dryness. Therefore, Anemarrhenae Rhizoma is specialized in clearing and moistening. It can be applied to all cases of dryness-heat injuring yin, whether it is excess syndrome or deficiency syndrome. As a traditional Chinese medicine with a long application history, Anemarrhenae Rhizoma was first recorded in "Shennong's Herbal Classic" and was listed in the middle grade, which states that "it tastes bitter and cold, mainly treats consumptive thirst, heat in the middle, eliminates pathogenic factors, limb edema, promotes diuresis, tonifies deficiency, and benefits qi". It belongs to the lung, stomach and kidney meridians.

[0003] There have been many reports on the related identification studies of Anemarrhenae Rhizoma and its adulterants. The commonly used identification methods are traditional identification methods such as morphological identification, microscopic identification and physicochemical identification. Both morphological identification and microscopic identification need to be completed by experts who have been engaged in traditional Chinese medicine identification for a long time. The professional difficulty is great and it is not easy to master; moreover, the chemical components of traditional Chinese medicine are complex, the physicochemical identification methods have poor specificity and are easily interfered by external factors. With the continuous development of science and technology, molecular biological identification technology has been applied in the field of traditional Chinese medicine identification, such as gene DNA barcoding technology. This method has high accuracy and is especially suitable for seed or origin identification, but it requires relatively complex professional equipment and technology. There are various identification methods for Anemarrhenae Rhizoma. Among them, the gene detection method has the characteristics of short time, strong specificity and high sensitivity, and has great research value. At present, there is no report on the application of LMTIA technology in the detection of Anemarrhenae Rhizoma components. Applying LMTIA technology to the identification of Anemarrhenae Rhizoma and establishing an LMTIA detection method for detecting Anemarrhenae Rhizoma has very important practical significance. Summary of the Invention

[0004] The purpose of the present invention is to provide an LMTIA primer set, a kit and an application for detecting Anemarrhenae Rhizoma to solve the problems existing in the above-mentioned prior art. The primer set designed by the present invention can be used for the detection of Anemarrhenae Rhizoma, has good specificity and sensitivity, and the detection speed is fast, providing a technical means for the authenticity identification of traditional Chinese medicine.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a set of LMTIA primers for the detection of Anemarrhenae Rhizoma, and the primer set comprises the following primers and probes:

[0007] Primer ZhiMu-1F2: 5'-CGACGCACAGCTTTTGAGTGGTGGACGATACGCAC-3';

[0008] Primer ZhiMu-1B12: 5'-GCTGTGCGTCGTTTTTAGGATGCACCGCTTGGG-3';

[0009] Primer ZhiMu-1LB: 5'-CGGTCCTTGCTCTCGG-3';

[0010] Probe ZhiMu-1Pr: 5'-CGGTCCTTGCTCTCA-3'.

[0011] Optionally, a quenching group is connected to the 5'-end of the probe ZhiMu-1Pr, and a fluorescent group is connected to the 3'-end thereof.

[0012] The present invention also provides an LMTIA kit for the detection of Anemarrhenae Rhizoma, which comprises the above-mentioned primer set.

[0013] The present invention also provides the use of the above-mentioned primer set or the above-mentioned kit in the detection of Anemarrhenae Rhizoma.

[0014] The present invention also provides a method for detecting Anemarrhenae Rhizoma, which comprises the following steps:

[0015] Extract the DNA of the sample to be tested, perform LMTIA reaction using the above-mentioned primer set, and judge whether the sample to be tested contains Anemarrhenae Rhizoma according to the presence or absence of an amplification curve.

[0016] Optionally, if an amplification curve appears in the amplification result, it is judged that the sample to be tested contains Anemarrhenae Rhizoma; if no amplification curve appears in the amplification result, it is judged that the sample to be tested does not contain Anemarrhenae Rhizoma.

[0017] Optionally, in the reaction system of the LMTIA reaction, the molar ratio of the primer ZhiMu-1F2, the primer ZhiMu-1B12, the primer ZhiMu-1LB and the probe ZhiMu-1Pr is 8:8:2:5.

[0018] Optionally, the reaction system of the LMTIA reaction is 2 μL of reaction premix, 0.4 μL of DNA polymerase, 0.16 μL of primer ZhiMu-1F2, 0.16 μL of primer ZhiMu-1B12, 0.04 μL of primer ZhiMu-1LB, 0.1 μL of probe ZhiMu-1Pr and 2 μL of DNA template, and DEPC water is added to make up to 10 μL.

[0019] Optionally, the reaction conditions for the LMTIA reaction are isothermal amplification at 61 °C for 20 min.

[0020] Optionally, before performing the LMTIA reaction on the sample DNA, it further includes the step of verifying the effectiveness of the DNA using plant internal standard primers and probes.

[0021] The plant internal standard primers and probes are: primer ZB-6F3: 5'-GTAGGCCACTATCTTTTGCATCATTCAAATTTCTGCCCTAT-3', primer ZB-6B13: 5'-GATAGTGGCCTACTTTTGTCACCCGTCACCACCATG-3', primer ZB-56LF: 5'-TACCATCGAAAGTTG-3' and probe ZB-56Pr: 5'BHQ2-TACCATCGAAAGTTA-JOE.

[0022] The present invention discloses the following technical effects:

[0023] The present invention designs an LMTIA primer set for detecting Anemarrhena asphodeloides, which includes primers and probes. The primer set has good specificity and sensitivity. The absolute sensitivity can reach 10 pg / μL, and the relative sensitivity can reach 5%; the detection speed is fast, and the detection of the sample Anemarrhena asphodeloides can be realized under isothermal conditions.

[0024] The present invention also designs plant internal standard primers and probes for verifying DNA, which can verify whether the DNA to be tested is effective before detection, ensuring the effectiveness and accuracy of the detection results.

[0025] The present invention establishes a method for detecting Anemarrhena asphodeloides based on a specific primer set, providing a technical means for the authenticity identification of Chinese medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0027] Figure 1 It is a comparison diagram of the ITS sequences of Anemarrhena asphodeloides and Anemarrhena tenuifolia;

[0028] Figure 2 It is the melting curve of the target gene of Anemarrhena asphodeloides;

[0029] Figure 3 It is the amplification result diagram of the optimization of the LMTIA amplification temperature of Anemarrhena asphodeloides;

[0030] Figure 4 Amplification result diagram for specific determination of Anemarrhenae asphodeloides LMTIA;

[0031] Figure 5 Amplification result diagram for absolute sensitivity determination of Anemarrhenae asphodeloides LMTIA;

[0032] Figure 6 Amplification result diagram for relative sensitivity determination of Anemarrhenae asphodeloides LMTIA;

[0033] Figure 7 Amplification result diagram for the internal standard of Anemarrhenae asphodeloides plants in the sample;

[0034] Figure 8 Amplification result diagram for authenticity identification of Anemarrhenae asphodeloides from different commercial origins detected by LMTIA. Detailed implementation manners

[0035] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0036] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0038] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0039] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0040] For the sources of the reagents used in the present invention, see Table 1 for details.

[0041] Table 1 Experimental Reagents and Sources

[0042]

[0043] Example 1 Design of LMTIA Primer Set, Probe and Temperature Optimization

[0044] 1. Design of LMTIA Primer Set and Probe

[0045] Using the ribosomal RNA gene internal transcribed spacer (ITS) of Anemarrhena asphodeloides as the target sequence, BLAST sequence alignment analysis was used to screen the differential sequences between Anemarrhena asphodeloides and Anemarrhena gracilis (as shown in Figure 1 ), and sequences with a trapezoidal melting temperature were selected through analysis with Oligo7 software (as shown in Figure 2 ). Furthermore, Primer3Plus was used to design the primers for LMTIA. After extensive screening, a primer set and probe with the following sequences (synthesized by General Biosystems (Anhui) Co., Ltd.) were obtained. The specific sequences are shown in Table 2. Among them, the quenching group (BHQ2) is connected to the 5' end of the probe ZhiMu-1Pr, and the fluorescent group (6-FAM) is connected to the 3' end.

[0046] Table 2 LMTIA Primer Set and Probe Sequences

[0047]

[0048]

[0049] 2. Extraction of Template DNA

[0050] The DNA of Anemarrhena asphodeloides and Anemarrhena gracilis samples was extracted using a plant genomic DNA extraction kit. The specific operation steps are shown in the kit's instruction manual.

[0051] 3. Temperature Optimization of the LMTIA Reaction System of the Present Invention

[0052] The reaction system of LMTIA is shown in Table 3.

[0053] Table 3 LMTIA Reaction System (10 μL)

[0054]

[0055] Take PCR octuplets, and add the reaction reagents prepared in Table 3 (excluding template DNA in the system, 8 μL in each single tube) into 4 single tubes respectively. Add 2 μL of DEPC-treated water into 2 of the single tubes as negative controls, and add 2 μL of Anemarrhena asphodeloides DNA into the other 2 single tubes. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature gradient at 59 °C, 61 °C, 63 °C, and 65 °C, and set to collect fluorescence signals once every 30 seconds, for a total of 40 times (a total of 20 min).

[0056] Observe the amplification curve of Anemarrhena asphodeloides temperature optimization ( Figure 3 ) and it can be seen that there is no non-specific amplification caused by primer dimers at the 4 temperatures of 59 °C, 61 °C, 63 °C, and 65 °C for Anemarrhena asphodeloides. Considering the amplification efficiency and reproducibility of the Anemarrhena asphodeloides amplification curve at different temperatures, select 61 °C as the optimal temperature for LMTIA and conduct subsequent measurements.

[0057] Example 2 Specificity of the LMTIA primer of the present invention

[0058] Take PCR octuplets, and add the reaction reagents prepared in Table 3 (excluding template DNA in the system, 8 μL in each single tube) into 12 single tubes respectively. Use DEPC-treated water as the negative control, and detect Anemarrhena asphodeloides DNA, Anemarrhena japonica Miq. DNA, Cornus officinalis Sieb. et Zucc. DNA, Cyperus rotundus L. DNA, and Saposhnikovia divaricata (Turcz.) Schischk. DNA respectively. Set two parallels for each reaction, use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 61 °C, and collect fluorescence signals once every 30 seconds, for a total of 40 times.

[0059] The results are as Figure 4 shown. Only when Anemarrhena asphodeloides DNA is added, there is a good amplification curve for the LMTIA reaction, and the LMTIA reaction is positive; no amplification curves appear for other source DNAs, and the reactions are all negative. Therefore, the established LMTIA detection method for Anemarrhena asphodeloides detection has high specificity.

[0060] Example 3 Sensitivity of the LMTIA primer of the present invention

[0061] 1. Absolute sensitivity

[0062] The extracted Anemarrhena asphodeloides DNA was serially diluted to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL. Take PCR strip tubes, and add the reaction reagents prepared in Table 3 (excluding template DNA in the system, 8 μL in each single tube) to 12 single tubes respectively. Use DEPC-treated water as the negative control, and detect 10 ng / μL Anemarrhena asphodeloides DNA, 1 ng / μL Anemarrhena asphodeloides DNA, 100 pg / μL Anemarrhena asphodeloides DNA, 10 pg / μL Anemarrhena asphodeloides DNA, and 1 pg / μL Anemarrhena asphodeloides DNA respectively. Set up two parallels for each reaction. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 61 °C, collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total.

[0063] The results are as Figure 5 shown. When the concentration of Anemarrhena asphodeloides DNA was 10 ng / μL, 1 ng / μL, 100 pg / μL, and 10 pg / μL, the LMTIA amplification curves were relatively obvious. However, when the DNA concentration was 1 pg / μL, no amplification curve appeared because the concentration of 1 pg / μL exceeded the detection limit of this method, resulting in unstable amplification. Therefore, the absolute sensitivity of this method can reach 10 pg / μL. In a 10 μL reaction system, 10 pg of Anemarrhena asphodeloides DNA extracted from the sample can be detected.

[0064] 2. Relative sensitivity

[0065] Mix Anemarrhena asphodeloides with Anemarrhena gracilis, with the mass fractions of Anemarrhena asphodeloides being 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95%, and 99% respectively. Take the DNA of the above mixed samples (extracted using a plant genomic DNA extraction kit according to the steps in the instruction manual) and use it as the template DNA for standby. Take PCR strip tubes, and add the reaction reagents prepared in Table 3 (excluding template DNA in the system, 8 μL in each single tube) to 20 single tubes respectively. Add 2 μL of DEPC-treated water to 2 single tubes as the negative control, and add 2 μL of Anemarrhena asphodeloides DNA with mass fractions of 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95%, and 99% to the other 18 single tubes respectively. Set up two parallels for each reaction. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 61 °C, collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total.

[0066] The results are as Figure 6As shown in the figure, when the mixed samples contained 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% Anemarrhenae Rhizoma, there were obvious amplification curves for LMTIA; when the mixed samples contained 1% Anemarrhenae Rhizoma, there was no amplification curve for LMTIA. The above results indicate that 1% exceeded the detection limit of this method for detecting Anemarrhenae Rhizoma. Therefore, the relative sensitivity of this method for detecting Anemarrhenae Rhizoma can reach at least 5%.

[0067] Example 4 Identification of the authenticity of Anemarrhenae Rhizoma from different origins

[0068] Before the actual sample detection, in order to ensure the effectiveness of the extracted DNA, the extracted DNA was verified by the plant internal standard method first. Take an eight-strip PCR tube, configure the reaction reagents according to Table 5 (the system does not include template DNA, 8 μL in each single tube), where DEPC water (enzyme-free water) was used as the negative control, and the DNA of Anemarrhenae Rhizoma from Inner Mongolia sold in Zhang Zhongjing Pharmacy, Anemarrhenae Rhizoma from Hebei 1 sold in the market, Anemarrhenae Rhizoma from Hebei 2 sold in the market, Anemarrhenae Rhizoma from Anhui 1 sold in the market, Anemarrhenae Rhizoma from Henan sold in the market, Anemarrhenae Rhizoma from Bozhou, Anhui sold in the market, Anemarrhenae Rhizoma from Hebei 3 sold in the market, Anemarrhenae Rhizoma from Anhui 2 sold in the market, Anemarrhenae Rhizoma from Anhui 3 sold in the market, and Anemarrhenae Rhizoma from Hebei 4 sold in the market were used as the test samples, and were added to the eight-strip tube in turn. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 60 °C, collect the optical signal once every 30 seconds, and collect a total of 40 optical signals.

[0069] The results are as Figure 7 shown. Good amplification curves appeared for the DNA of the actual samples in this detection, indicating that the DNA of the samples used in this experiment was all effective.

[0070] Table 4 Sequences of LMTIA plant internal standard primers and probes

[0071]

[0072] Table 5 Plant internal standard reaction system

[0073]

[0074] Then, the actual sample detection was carried out: take an eight-strip PCR tube, add the reaction reagents prepared in Table 3 (the system does not include template DNA, 8 μL in each single tube) to 24 single tubes respectively, use DEPC-treated water as the negative control, and use the DNA of Anemarrhenae Rhizoma standard as the positive control, and detect the DNA of Anemarrhenae Rhizoma from Inner Mongolia sold in Zhang Zhongjing Pharmacy, Anemarrhenae Rhizoma from Hebei 1, Anemarrhenae Rhizoma from Hebei 2, Anemarrhenae Rhizoma from Hebei 3, Anemarrhenae Rhizoma from Hebei 4, Anemarrhenae Rhizoma from Anhui 1, Anemarrhenae Rhizoma from Anhui 2, Anemarrhenae Rhizoma from Anhui 3, Anemarrhenae Rhizoma from Henan and Anemarrhenae Rhizoma from Bozhou, Anhui respectively. Each reaction was set with two parallels. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 61 °C, collect the fluorescence signal once every 30 seconds, and collect a total of 40 fluorescence signals.

[0075] The results are as Figure 8 shown. For the positive control of Anemarrhena asphodeloides detected by the LMTIA primer set and probe of the present invention, good amplification curves all appeared. When detecting Anemarrhena asphodeloides from other producing areas, it was found that commercially available Anemarrhena asphodeloides from Inner Mongolia, Hebei 1, Hebei 2, Hebei 3, Hebei 4, Bozhou of Anhui, Anhui 1, Anhui 2, Anhui 3 and Henan all contained the components of Anemarrhena asphodeloides and were genuine products. Therefore, the LMTIA primer set and probe of the present invention can be used simultaneously for the authenticity identification of commercially available Anemarrhena asphodeloides from different producing areas.

[0076] The embodiments described above are only used to describe the preferred mode of the present invention, rather than to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A LMTIA primer set for detecting Rhizoma Anemarrhenae, characterized in that: The primer set comprises the following primers and probes: Primer ZhiMu-1F2: 5'-CGACGCACAGCTTTTGAGTGGTGGACGATACGCAC-3'; Primer ZhiMu-1B12: 5'-GCTGTGCGTCGTTTTTTAGGATGCACCGCTTGGG-3'; Primer ZhiMu-1LB: 5′-CGGTCCTTGCTCTCGG-3′; Probe ZhiMu-1Pr: 5′-CGGTCCTTGCTCTCA-3′.

2. The primer set according to claim 1, characterized in that The 5' end of the probe ZhiMu-1Pr is connected to a quenching group, and the 3' end is connected to a fluorescent group.

3. A LMTIA kit for detecting Rhizoma Anemarrhenae, characterized in that: Comprising the primer set according to claim 1 or 2.

4. Use of the primer set according to claim 1 or 2 or the kit according to claim 3 in detecting Rhizoma Anemarrhenae.

5. A method for detecting Rhizoma Anemarrhenae, characterized in that: The following steps are involved: Extract the DNA of the sample to be tested, perform LMTIA reaction using the primer set described in claim 1 or 2, and judge whether the sample to be tested contains Anemarrhena asphodeloides based on the presence or absence of an amplification curve.

6. The method according to claim 5, characterized in that If an amplification curve appears in the amplification result graph, it is judged that the sample to be tested contains Anemarrhena asphodeloides; if no amplification curve appears in the amplification result graph, it is judged that the sample to be tested does not contain Anemarrhena asphodeloides.

7. The method according to claim 5, characterized in that In the reaction system of the LMTIA reaction, the molar ratio of the primer ZhiMu-1F2, the primer ZhiMu-1B12, the primer ZhiMu-1LB and the probe ZhiMu-1Pr is 8:8:2:

5.

8. The method according to claim 5, characterized in that The reaction system of the LMTIA reaction is 2 μL of reaction premix, 0.4 μL of DNA polymerase, 0.16 μL of primer ZhiMu-1F2, 0.16 μL of primer ZhiMu-1B12, 0.04 μL of primer ZhiMu-1LB, 0.1 μL of probe ZhiMu-1Pr and 2 μL of DNA template, and DEPC water is added to 10 μL.

9. The method according to claim 5, characterized in that The reaction conditions of the LMTIA reaction are isothermal amplification at 61° C. for 20 min.

10. The method according to claim 5, characterized in that Before performing LMTIA reaction on the sample DNA, the method further includes the step of using plant internal standard primers and probes to verify whether the DNA is effective; The plant internal standard primers and probes are: primer ZB-6F3: 5'-GTAGGCCACTATCTTTTGCATCATTCAAATTTCTGCCCTAT-3', primer ZB-6B13: 5'-GATAGTGGCCTACTTTTGTCACCCGTCACCACCATG-3', primer ZB-56LF: 5'-TACCATCGAAAGTTG-3' and probe ZB-56Pr: 5'BHQ2-TACCATCGAAAGTTA-JOE.

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