LMTIA primer, probe and kit for detecting spreading hedyotis herb components and application of LMTIA primer, probe and kit

Abstract

The invention discloses an LMTIA primer, a probe and a kit for detecting components of oldenlandia diffusa and application, and relates to the technical field of biomolecular detection. The invention develops an LMTIA primer and a probe for detecting the spreading hedyotis herb, and the LMTIA primer and the probe comprise a primer BaiHSSC-LF, a primer BaiHSSC-LB, a primer BaiHSSC-F2 and a primer BaiHSSC-B2, and a probe BaiHSSC-Pr. The LMTIA primer and probe combination provided by the invention has remarkable advantages in the aspects of detection speed, sensitivity, specificity, simplicity and convenience in operation and the like, and an efficient and accurate technical means can be provided for detection of oldenlandia components.

Description

Technical Field

[0001] The present invention relates to the technical field of biomolecule detection, and in particular to an LMTIA primer, probe, kit and application for detecting components of the genus Hedyotis. Background Art

[0002] Hedyotis diffusa (Willd.) R.J.Wang, the dried whole herb of Hedyotis diffusa of the genus Hedyotis in the Rubiaceae family, was first recorded in the Guangxi Traditional Chinese Medicine Annals. Now, a variety of ready-to-use traditional Chinese medicine preparations containing Hedyotis diffusa have been developed for clinical use. Hedyotis diffusa has the effects of clearing heat and detoxifying, anti-inflammatory and analgesic, diuretic and detumescence, and promoting blood circulation to remove stasis. Clinically, it can be used to treat malignant tumors, respiratory tract infections, hepatitis, appendicitis, gynecological diseases, etc., and can also be used externally to treat snake bites. Hedyotis diffusa is a commonly used folk herb with a wide source and is used as a whole herb in medicine. It is very easy to be confused with other species of the genus Hedyotis. Affected by different varieties and origins, Hedyotis diffusa will show differences in quality and efficacy, which to a certain extent causes the variety chaos and uneven quality of medicinal plants of the genus Hedyotis. In addition, the phenomena of mixing and adulterating with other species of the genus Hedyotis are common, and the true quality of Hedyotis diffusa is difficult to guarantee. The safety and effectiveness of clinical use of Hedyotis diffusa are greatly threatened. Therefore, establishing a scientific, accurate and stable identification method for the genus Hedyotis is conducive to overcoming the technical "shortcomings" in authenticity detection such as the screening and identification of genuine Chinese medicinal materials, and promoting the high-quality and rapid development of the traditional Chinese medicine industry, which has very important practical significance.

[0003] There are many kinds of identification methods for *Hedyotis diffusa*. Among them, gene detection methods have shown extremely high research value due to their characteristics of short time, strong specificity, and high sensitivity. For example, patent document CN1661090A discloses a specific primer, an identification kit, and an identification method for identifying the species of *Hedyotis diffusa*; while patent document CN107354217A discloses an ISSR molecular marker method for genetic diversity analysis of *Hedyotis diffusa*. These methods are usually based on real-time fluorescence quantitative PCR technology and require 1-2 hours to complete the detection. The Isothermal Amplification of Nucleic Acids with Ladder-shape Melting Curve (LMTIA) is a newly emerging nucleic acid isothermal amplification technology. This technology can achieve stable amplification of nucleic acids within 30 minutes and has high sensitivity and specificity. However, there is no relevant report on the application of LMTIA technology in the detection of components of the genus *Hedyotis*. The purpose of the present invention is to establish an efficient detection method based on LMTIA technology for the identification of components of the genus *Hedyotis*, so as to achieve rapid and accurate detection of components of the genus *Hedyotis*. Summary of the Invention

[0004] The purpose of the present invention is to provide an LMTIA primer, probe, kit, and application for the detection of components of the genus *Hedyotis* to solve the problems existing in the above-mentioned prior art. The combination of LMTIA primers and probes provided by the present invention has significant advantages in terms of detection speed, sensitivity, specificity, and operational simplicity, and can provide an efficient and accurate technical means for the detection of components of the genus *Hedyotis*.

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

[0006] The present invention provides an LMTIA primer and probe combination for the detection of *Hedyotis diffusa*, including primers BaiHSSC-F2, BaiHSSC-B2, BaiHSSC-LF, and BaiHSSC-LB, and probe BaiHSSC-Pr;

[0007] The nucleotide sequences of the primers BaiHSSC-F2, BaiHSSC-B2, BaiHSSC-LF, and BaiHSSC-LB are shown in SEQ ID NO.3-6 respectively; the nucleotide sequence of the probe BaiHSSC-Pr is shown in SEQ ID NO.7.

[0008] Furthermore, a fluorescence quenching group is connected to the 5' end of the probe BaiHSSC-Pr, and a fluorescence reporting group is connected to the 3' end.

[0009] Furthermore, the fluorescence quenching group is the BHQ1 group; the fluorescence reporting group is the Cy5 group.

[0010] The present invention also provides the use of the above-mentioned LMTIA primer and probe combination in the preparation of a product for detecting components of the genus Hedyotis.

[0011] Furthermore, the product is a kit.

[0012] The present invention also provides a product for detecting components of the genus Hedyotis, comprising the above-mentioned LMTIA primer and probe combination.

[0013] Furthermore, the product is a kit.

[0014] Furthermore, the product further comprises a Fast Universal PCR premix and a Bst enzyme.

[0015] The present invention also provides a method for detecting components of the genus Hedyotis, comprising the following steps:

[0016] Genomic DNA of the sample to be detected is extracted;

[0017] Using the genomic DNA as a template, real-time fluorescence PCR isothermal amplification is carried out with the above-mentioned LMTIA primer and probe combination;

[0018] Judgment is made based on the amplification result: if an exponential curve appears in the amplification result graph, the sample to be detected contains components of the genus Hedyotis; if no amplification curve appears in the amplification result, the sample to be detected does not contain components of the genus Hedyotis.

[0019] Furthermore, the temperature of the real-time fluorescence PCR isothermal amplification is 69°C and the time is 20 min.

[0020] The present invention discloses the following technical effects:

[0021] The present invention has developed an LMTIA primer and probe combination for detecting Hedyotis diffusa, including primers BaiHSSC-LF, BaiHSSC-LB, BaiHSSC-F2 and BaiHSSC-B2, and probe BaiHSSC-Pr. The LMTIA primer and probe combination provided by the present invention has significant advantages in terms of detection speed, sensitivity, specificity and operation simplicity, and can provide an efficient and accurate technical means for detecting components of the genus Hedyotis.

[0022] The LMTIA primer and probe combination provided by the present invention can quickly complete the detection of the components of *Hedyotis diffusa*, significantly improving the detection efficiency. Compared with traditional detection methods, the present invention does not require complex temperature cycle control, simplifies the operation process, and shortens the detection time.

[0023] The LMTIA primer and probe combination of the present invention has extremely high sensitivity to the components of *Hedyotis diffusa*, and the lowest detection limit can reach 10 pg / μL. This characteristic enables the accurate detection of its components even when the content of *Hedyotis diffusa* in the sample is extremely low.

[0024] The LMTIA primer and probe combination of the present invention has high specificity for the components of *Hedyotis diffusa*, especially showing better specificity at 69°C. This ensures the accuracy of the detection results and can effectively distinguish *Hedyotis diffusa* from other similar species or impurities.

[0025] The present invention provides a reliable technical means for the authenticity identification of genuine regional Chinese medicinal materials. By using the LMTIA primer and probe combination of the present invention, it is possible to quickly, efficiently, and accurately identify whether the sample contains the components of *Hedyotis diffusa*, thereby providing support for the quality control and authenticity verification of Chinese medicinal materials.

[0026] Since the detection method of the present invention avoids the dependence on expensive equipment, simplifies the experimental operation, and reduces the detection cost, it is more suitable for wide application in laboratories, pharmaceutical enterprises, and Chinese medicine quality supervision departments. Description of the Drawings

[0027] 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 for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is a ladder sequence curve graph of the nucleic acid target sequence of the *Hedyotis* genus;

[0029] Figure 2 It is an amplification result graph of the LMTIA screening primer of the *Hedyotis* genus;

[0030] Figure 3 It is an amplification result graph of the LMTIA temperature measurement of the *Hedyotis* genus;

[0031] Figure 4 It is an amplification result graph of the LMTIA repeatability and stability measurement of the *Hedyotis* genus;

[0032] Figure 5 It is an amplification result graph of the LMTIA specificity measurement of the *Hedyotis* genus;

[0033] Figure 6 It is the amplification result diagram for the sensitivity determination of LMTIA in the genus Hedyotis. Detailed implementation manners

[0034] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0035] It should be understood that the terms described 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 may be independently included or excluded from the range.

[0036] 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 may 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.

[0037] 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 specification of the present invention, 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.

[0038] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, that is, they are intended to include but not limited to.

[0039] The experimental reagents used in the present invention and their sources are shown in Table 1.

[0040] Table 1 Experimental reagents and sources

[0041]

[0042] Example 1 Optimization of the LMTIA primer set

[0043] (1) Design of LMTIA primers and probes

[0044] Extract the DNA of the Hedyotis diffusa standard product using a plant DNA extraction kit (TIANGEN Biochemical), perform plasmid extraction on the extracted DNA, and through steps such as electrophoresis, gel cutting, gel recovery, transformation of competent cells, plasmid extraction, and enzyme digestion electrophoresis verification, screen the bacterial solutions with conforming bands. Send a part for sequencing and save the other part as glycerol bacteria, which can be stored for a long time at -80 °C. Use BLAST to perform sequence alignment analysis on the sequencing results, with the internal transcribed spacer (ITS) as the target sequence, and select sequences with a ladder-like melting temperature through analysis with Oligo7 software (such as Figure 1 shown), and then use Primer3Plus to design the LMTIA primers and probes for Hedyotis diffusa. After extensive screening, the primers and probes shown in Table 2 are obtained (synthesized by General Biosystems (Anhui) Co., Ltd.).

[0045] Table 2 LMTIA primer set and probe sequence

[0046]

[0047] (2) Extraction of template DNA

[0048] Extract the Hedyotis diffusa DNA using a plant genomic DNA extraction kit purchased from TIANGEN Biochemical Technology (Beijing) Co., Ltd. The specific operation steps refer to the instruction manual of the kit.

[0049] (3) Primer screening for the LMTIA reaction system of the present invention

[0050] The reaction system of LMTIA is shown in Table 3.

[0051] Table 3 Screening primer LMTIA reaction system (25 μL)

[0052]

[0053] Among them, BaiHSSC-LF and BaiHSSC-LB in the LMTIA primers are universal primers for the genus Hedyotis diffusa, and the corresponding BaiHSSC-F and BaiHSSC-B need to be added when screening primers.

[0054] Set up the primer screening experiment as shown in Table 4, use the Gentier 96E fully automatic medical PCR analysis system to set temperature gradients of 67 °C, 68 °C, and 69 °C, amplify for 20 minutes respectively, collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total. Each reaction is repeated 2 times, with 2 parallels each time.

[0055] Table 4 Primer screening experiment grouping

[0056]

[0057]

[0058] Note: In experimental groups A1 - A8, primers BaiHSSC - F1 / BaiHSSC - B1 were used; in experimental groups B1 - B8, primers BaiHSSC - F2 / BaiHSSC - B2 were used.

[0059] Observing the amplification curve of Hedyotis diffusa primer optimization (see Figure 2 ), it can be seen that at three temperatures of 67°C, 68°C, and 69°C for Hedyotis diffusa, there was no non - specific amplification caused by primer dimers with primers BaiHSSC - F2 / BaiHSSC - B2. Considering the repeatability and non - specific amplification results of the two sets of primers at four temperatures comprehensively, BaiHSSC - F2 and BaiHSSC - B2 were selected as the optimal primers for LMTIA, and subsequent determinations were carried out.

[0060] Example 2 Optimization of LMTIA Primer Set and Probe Temperature

[0061] Optimization of the temperature of the LMTIA reaction system of the present invention

[0062] The reaction system of LMTIA is shown in Table 5.

[0063] Table 5 LMTIA reaction system (10 μL)

[0064]

[0065] Among them, in the LMTIA primers, BaiHSSC - LF and BaiHSSC - LB are universal primers for the genus Hedyotis diffusa, BaiHSSC - F2 and BaiHSSC - B2 are primers after screening and optimization, and the probe BaiHSSC - Pr needs to be added when detecting Hedyotis diffusa.

[0066] Using the LMTIA reaction system shown in Table 5 (template DNA is Hedyotis diffusa DNA), the Gentier 96E fully automatic medical PCR analysis system was used to set temperature gradients of 67°C, 68°C, 69°C, and 70°C respectively, the amplification reaction was carried out for 20 minutes, and fluorescence signals were collected once every 30 seconds, with a total of 40 times of fluorescence signals collected. A negative control group was set for each reaction temperature (i.e., using an equal amount of ddH2O instead of Hedyotis diffusa DNA). Each reaction was repeated 2 times, with 2 parallels each time.

[0067] Observing the amplification curve of Hedyotis diffusa temperature optimization (see Figure 3)It can be known that there is no non-specific amplification caused by primer dimer in Hedyotis diffusa Willd. at 4 temperatures of 67 °C, 68 °C, 69 °C and 70 °C. Considering the amplification efficiency and reproducibility of the amplification curve of Hedyotis diffusa Willd. at different temperatures comprehensively, 69 °C was selected as the optimal temperature for LMTIA, and subsequent determinations were carried out.

[0068] Example 3

[0069] A kit for detecting components of the genus Hedyotis, comprising the following reagents:

[0070] Primers BaiHSSC-F2, BaiHSSC-B2, BaiHSSC-LF and BaiHSSC-LB, and probe BaiHSSC-Pr;

[0071] Fast Universal PCR premix;

[0072] Bst enzyme.

[0073] Method for detecting components of the genus Hedyotis using the above kit:

[0074] Genomic DNA of the sample to be tested was extracted;

[0075] Prepare the LMTIA reaction system shown in Table 5 with the reagents of the above kit, and place the prepared LMTIA reaction system in a real-time fluorescence PCR instrument for isothermal (69 °C) amplification for 20 minutes;

[0076] Judgment was made according to the amplification result: if an exponential curve appears in the amplification result graph, the sample contains components of the genus Hedyotis; if no amplification curve appears in the amplification result, the sample does not contain components of the genus Hedyotis.

[0077] Example 4 Repeatability and stability of the LMTIA primers of the present invention

[0078] Take an eight-tube PCR tube, add the prepared reaction reagents shown in Table 5 (the system does not include template DNA, 8 μL in each single tube) to 16 single tubes respectively, add 2 μL of ddH2O to 8 single tubes as negative controls, and add 2 μL of Hedyotis diffusa Willd. DNA to the other 8 single tubes. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 69 °C, drop paraffin oil into each well, cover the lid and centrifuge, repeat 2 times, with 8 parallels each time, collect fluorescence signals once every 30 seconds, and collect a total of 40 fluorescence signals.

[0079] The results are as Figure 4As shown, when only the DNA of *Hedyotis diffusa* is present, an amplification curve exists in the LMTIA reaction, and the amplification curve has good repeatability and high stability. The LMTIA reaction is positive, and other reactions are negative. Therefore, the LMTIA detection method for detecting the components of the genus *Hedyotis* established in the present invention has good repeatability and high stability.

[0080] Example 5 Specificity of the LMTIA primer of the present invention

[0081] Using the LMTIA reaction system shown in Table 5, the template DNA was respectively selected as the DNA of *Hedyotis diffusa*, *Hedyotis corymbosa*, *Hedyotis tenelliflora* and *Hedyotis pinifolia*. The Gentier 96E fully automatic medical PCR analysis system was used to set the temperature at 69°C, and the reaction was amplified for 20 minutes respectively. Fluorescence signals were collected every 30 seconds for a total of 40 times. At the same time, a negative control group was set (i.e., using an equal amount of ddH2O to replace the template DNA). Each reaction was repeated 2 times, with 2 parallels each time.

[0082] The results are as Figure 5 shown. When only the DNA of *Hedyotis diffusa* is present, a good amplification curve exists in the LMTIA reaction, and the LMTIA reaction is positive; no amplification curve appears for other source DNAs, and the reactions are all negative. Therefore, the LMTIA detection method for detecting the components of the genus *Hedyotis* established in the present invention has high specificity.

[0083] Example 6 Sensitivity of the LMTIA primer of the present invention

[0084] The extracted DNA of the genus *Hedyotis* was serially diluted to 1 ng / μL, 500 pg / μL, 100 pg / μL, 10 pg / μL and 1 pg / μL. The serially diluted DNA of the genus *Hedyotis* was respectively selected as the DNA template, and the LMTIA reaction system shown in Table 5 was prepared. The Gentier 96E fully automatic medical PCR analysis system was used to set the temperature at 69°C, and the reaction was amplified for 20 minutes respectively. Fluorescence signals were collected every 30 seconds for a total of 40 times. At the same time, a negative control group was set (i.e., using an equal amount of ddH2O to replace the template DNA). Each reaction was repeated 2 times, with 2 parallels each time.

[0085] The results are as Figure 6 shown. When the DNA concentration of *Hedyotis diffusa* is 1 ng / μL, 500 pg / μL, 100 pg / μL and 10 pg / μL, the LMTIA amplification curve is relatively obvious and linear. When the DNA concentration of *Hedyotis diffusa* is 1 pg / μL and the negative control, no amplification curve appears. Therefore, the sensitivity of this method can reach 10 pg / μL. In a 10 μL reaction system, the DNA of *Hedyotis diffusa* extracted from the sample can be detected as long as it reaches 10 pg.

[0086] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not 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 solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer and probe combination for detecting Hedyotis diffusa, characterized in that, Comprising primers BaiHSSC-F2, BaiHSSC-B2, BaiHSSC-LF and BaiHSSC-LB, and probe BaiHSSC-Pr; The nucleotide sequences of the primers BaiHSSC-F2, BaiHSSC-B2, BaiHSSC-LF and BaiHSSC-LB are shown in SEQ ID NO.3-6 respectively; the nucleotide sequence of the probe BaiHSSC-Pr is shown in SEQ ID NO.

7.

2. The LMTIA primer and probe combination according to claim 1, wherein The 5' end of the probe BaiHSSC-Pr is linked to a fluorescence quenching group, and the 3' end is linked to a fluorescence reporting group.

3. The LMTIA primer and probe combination according to claim 1, wherein The fluorescence quenching group is the BHQ1 group; the fluorescence reporting group is the Cy5 group.

4. Use of an LMTIA primer and probe combination according to any one of claims 1-3 in the preparation of a product for detecting components of the genus Hedyotis.

5. The application according to claim 4, characterized in that, The product is a kit.

6. A product for detecting components of the genus *Hedyotis*, characterized in that, Comprising the LMTIA primer and probe combination according to any one of claims 1-3.

7. The product according to claim 6, characterized in that, The product is a kit.

8. The product according to claim 6, wherein, The product further comprises a Fast Universal PCR premix and Bst enzyme.

9. A method for detecting components of the genus Hedyotis, characterized in that, Comprising the following steps: Genomic DNA of the sample to be detected is extracted; Using the genomic DNA as a template, real-time fluorescence PCR isothermal amplification is carried out with the LMTIA primer and probe combination according to any one of claims 1-3; Judgment is made according to the amplification result: if an exponential curve appears in the amplification result graph, the sample to be detected contains components of the genus Hedyotis; If no amplification curve appears in the amplification result, the sample to be detected does not contain components of the genus Hedyotis.

10. The method according to claim 9, characterized in that, The temperature of the real-time fluorescence PCR isothermal amplification is 69°C and the time is 20 min.

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