Primer probe combination and kit for detecting Chinese semen cuscutae and / or southern semen cuscutae and application of primer probe combination and kit

Abstract

The invention discloses a primer probe combination and a kit for detecting Chinese dodder seeds and / or southern dodder seeds and application of the primer probe combination and the kit, and belongs to the technical field of molecular detection. The primer and probe combination comprises a TuSi < Z-F >, a TuSi < Z-B >, a TuSi < Z-LF >, a Nan TuSi < Z-F >, a Nan TuSi < Z-B >, a Nan TuSi < Z-LB >, a Nan TuSi < Z-LF >, a TuSi < Z-LF > Pr and a Nan TuSi < Z-LF > Pr, the TuSiZ-LF Pr and the NanTuSiZ-LF Pr are connected with different fluorescent groups, and the fluorescent groups are different from one another. The primer probe combination provided by the invention has good specificity and sensitivity, is high in detection speed, can realize dual detection of Chinese dodder seeds and southern dodder seeds in a sample under a constant temperature condition, and provides technical support for identification of Chinese dodder seeds and southern dodder seeds as well as identification of adulteration and counterfeiting of Chinese medicinal materials.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and particularly relates to a primer-probe combination, a kit and an application for detecting Cuscuta chinensis and / or Cuscuta australis. Background Art

[0002] Cuscuta was first recorded in "Shennong Ben Cao Jing" under the original name of Zihu, and was renamed Cuscuta in "Ben Cao Tu Jing". It is cold in nature, slightly bitter in taste, and belongs to the liver and gallbladder meridians, with the effects of relieving exterior syndrome and expelling fever, soothing the liver and relieving depression, and ascending yang qi. It has a history of application for more than 2,000 years. "Chinese Pharmacopoeia" stipulates that Cuscuta is the dry and mature seeds of the Convolvulaceae plant Cuscuta australis R.Br. or Cuscuta chinensis Lam. When the fruits are mature in autumn, the plants are harvested, dried, the seeds are beaten, and the impurities are removed. According to different characters, they are commonly known as "Cuscuta australis" and "Cuscuta chinensis" respectively. The quality of germplasm has an important impact on the quality of medicinal materials. The wild resources of Cuscuta in China are relatively scarce and mainly rely on cultivation, resulting in a large number and chaos of cultivated germplasms of Cuscuta, and significant differences in the characters of Cuscuta in different cultivation areas. Under the influence of different production areas, different growth patterns, etc., Cuscuta will show differences in quality and medicinal effects, to a certain extent causing confusion in the varieties and uneven quality of Cuscuta medicinal plants. Coupled with the common phenomena of mixing and adulterating Cuscuta from different production areas, it is difficult to guarantee the true quality of Cuscuta, and the safety and effectiveness of clinical use of Cuscuta are greatly threatened. Therefore, establishing a scientific, accurate and stable identification method for Cuscuta chinensis and Cuscuta australis is beneficial to overcoming the technical "shortcomings" in authenticity detection such as screening and identification of traditional Chinese medicines, and has very important practical significance for promoting the high-quality and rapid development of the traditional Chinese medicine industry in China.

[0003] In the market, due to the various tonic effects of Cuscuta and large demand, while its natural output is relatively limited, resulting in a tight market supply, which in turn has led to the behavior of some illegal vendors adulterating for the pursuit of high profits. This adulteration phenomenon has become quite common and serious, posing a potential threat to the interests and health of consumers. Adulterated Cuscuta not only reduces the medicinal effect but may also harm the health of users. Some illegal vendors, in order to make a profit, will adulterate impurities such as thousand-spike millet, small sesame, millet, hyoscyamus, rapeseed or even sand grains. These adulterated substances may be similar to Cuscuta in appearance but actually do not have the medicinal value of Cuscuta.

[0004] There are various identification methods for Cuscuta chinensis, among which the gene detection method has the characteristics of short time, strong specificity, high sensitivity, etc., and has great research value. Ladder-shape Melting Temperature Isothermal Amplifiction (LMTIA) is a new type of nucleic acid isothermal amplification technology, which can achieve stable amplification of nucleic acid within 30 minutes, and has high sensitivity and specificity. At present, there is no report on the detection of Cuscuta chinensis and Cuscuta australis components by LMTIA technology. Therefore, applying LMTIA technology to the identification of Cuscuta chinensis and / or Cuscuta australis components and establishing an LMTIA detection method for dual detection of Cuscuta chinensis and / or Cuscuta australis components has very important practical significance. Summary of the Invention

[0005] The purpose of the present invention is to provide a primer-probe combination, a kit and their applications for detecting Cuscuta chinensis and / or Cuscuta australis to solve the problems existing in the above-mentioned prior art. The LMTIA primer set and probe provided by the present invention have good specificity and sensitivity, and the detection speed is fast. The components of Cuscuta chinensis and / or Cuscuta australis in the sample can be detected under constant temperature conditions.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] Technical solution one: An LMTIA primer-probe combination for detecting Cuscuta chinensis and / or Cuscuta australis, including TuSiZ-F shown in SEQ ID NO.1, TuSiZ-B shown in SEQ ID NO.2, TuSiZ-LF shown in SEQ ID NO.3, NanTuSiZ-F shown in SEQ ID NO.4, NanTuSiZ-B shown in SEQ ID NO.5, NanTuSiZ-LB shown in SEQ ID NO.6, NanTuSiZ-LF shown in SEQ ID NO.7, TuSiZ-LF Pr shown in SEQ ID NO.8, and NanTuSiZ-LF Pr shown in SEQ ID NO.9;

[0008] Different fluorescent groups are connected to the TuSiZ-LF Pr and NanTuSiZ-LF Pr.

[0009] Technical solution two: The application of the LMTIA primer-probe combination in the preparation of reagents or kits for detecting Cuscuta chinensis and / or Cuscuta australis.

[0010] Technical solution three: A kit for detecting Cuscuta chinensis based on the LMTIA technology, wherein the kit contains the above-mentioned TuSiZ-F, TuSiZ-B, TuSiZ-LF and TuSiZ-LF Pr.

[0011] Technical solution four: A kit for detecting Cuscuta australis based on the LMTIA technology, wherein the kit contains the above-mentioned NanTuSiZ-F, NanTuSiZ-B, NanTuSiZ-LB, NanTuSiZ-LF and NanTuSiZ-LF Pr.

[0012] Technical solution five: A kit for dual detection of Cuscuta chinensis and Cuscuta australis based on the LMTIA technology, wherein the kit contains the above-mentioned TuSiZ-F, TuSiZ-B, TuSiZ-LF, NanTuSiZ-F, NanTuSiZ-B, NanTuSiZ-LB, NanTuSiZ-LF, TuSiZ-LF Pr and NanTuSiZ-LF Pr.

[0013] Technical solution six: An LMTIA detection method for Cuscuta chinensis and / or Cuscuta australis, comprising the following steps:

[0014] Extract the genomic DNA of the sample to be tested;

[0015] Using the genomic DNA as a template, perform an LMTIA reaction with the above-mentioned kit, and observe whether there is an amplification curve; if there is an amplification curve, it indicates that the sample to be tested contains Cuscuta chinensis and / or Cuscuta australis.

[0016] Further, when performing the LMTIA reaction with the above-mentioned kit, the system of the LMTIA reaction is: 5xMix Buffer 2.00 μL, Bst enzyme 0.40 μL, 100 μM TuSiZ-F 0.16 μL, 100 μM TuSiZ-B 0.16 μL, 100 μM TuSiZ-LF 0.04 μL, 100 μM TuSiZ-Pr 0.30 μL, template 2.00 μL, and make up to 10 μL with DEPC-treated water; the temperature of the LMTIA reaction is 57 °C and the time is 20 min.

[0017] Further, when the described kit is used for the LMTIA reaction, the system of the LMTIA reaction is as follows: 2.00 μL of 5xMix Buffer, 0.40 μL of Bst enzyme, 0.16 μL of 100 μM NanTuSiZ-F, 0.16 μL of 100 μM NanTuSiZ-B, 0.04 μL of 100 μM NanTuSiZ-LB, 0.04 μL of 100 μM NanTuSiZ-LF, 0.10 μL of 100 μM NanTuSiZ-Pr, 2.00 μL of template, and made up to 10 μL with DEPC-treated water; the temperature of the LMTIA reaction is 57 °C and the time is 20 min.

[0018] Further, when the described kit is used for the LMTIA reaction, the system of the LMTIA reaction is as follows: 3.00 μL of 5xMix Buffer, 0.40 μL of Bst enzyme, 0.32 μL of 100 μM TuSiZ-F, 0.32 μL of 100 μM TuSiZ-B, 0.08 μL of 100 μM TuSiZ-LF, 0.2 μL of 100 μM TuSiZ-Pr, 016 μL of 100 μM NanTuSiZ-F, 016 μL of 100 μM NanTuSiZ-B, 0.04 μL of 100 μM NanTuSiZ-LB, 0.04 μL of 100 μM NanTuSiZ-LF, 0.10 μL of 100 μM NanTuSiZ-Pr, 2.00 μL of template, and made up to 10 μL with DEPC-treated water; the temperature of the LMTIA reaction is 57 °C and the time is 20 min.

[0019] Technical solution seven: Application of the described kit in the identification of adulteration of Cuscuta chinensis and / or Cuscuta australis.

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

[0021] The LMTIA primer-probe combination provided by the present invention for detecting the components of Cuscuta chinensis and / or Cuscuta australis has good specificity and sensitivity, fast detection speed, and can detect the components of Cuscuta chinensis and Cuscuta australis in the sample under isothermal conditions. The LMTIA primer-probe combination of the present invention has better specificity at 57 °C and the sensitivity can reach 1 pg / μL. The present invention has established a rapid detection method for the components of Cuscuta chinensis and / or Cuscuta australis, which can be used to identify Cuscuta chinensis and Cuscuta australis products on the market, solve the problem of difficult identification of adulteration of traditional Chinese medicines, and provide technical support for the identification of adulteration of traditional Chinese medicines. Description of the Drawings

[0022] 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, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 It is a comparison diagram of the ITS sequences of Cuscuta chinensis and Cuscuta australis;

[0024] Figure 2 It is an amplification result diagram of the optimization of the amplification temperature of LMTIA of Cuscuta chinensis;

[0025] Figure 3 It is an amplification result diagram of the optimization of the amplification temperature of LMTIA of Cuscuta australis;

[0026] Figure 4 It is an amplification result diagram of the specificity determination of LMTIA of Cuscuta chinensis;

[0027] Figure 5 It is an amplification result diagram of the specificity determination of LMTIA of Cuscuta australis;

[0028] Figure 6 It is an amplification result diagram of the absolute sensitivity determination of LMTIA of Cuscuta chinensis;

[0029] Figure 7 It is an amplification result diagram of the absolute sensitivity determination of LMTIA of Cuscuta australis;

[0030] Figure 8 It is an amplification result diagram of the relative sensitivity determination of LMTIA of Cuscuta chinensis;

[0031] Figure 9 It is an amplification result diagram of the relative sensitivity determination of LMTIA of Cuscuta australis;

[0032] Figure 10 It is an amplification result diagram of the dual detection of LMTIA of the components of Cuscuta chinensis and Cuscuta australis;

[0033] Figure 11 It is an amplification result diagram of the specificity of the dual detection of LMTIA of the components of Cuscuta chinensis and Cuscuta australis;

[0034] Figure 12 It is an amplification result diagram of the sensitivity of the dual detection of LMTIA of the components of Cuscuta chinensis and Cuscuta australis;

[0035] Figure 13 It is an amplification result diagram of the authenticity identification of the actual samples of Cuscuta from different sources. Detailed implementation manners

[0036] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0037] It should be understood that the terms used in the present invention are only for describing specific embodiments 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.

[0038] 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 said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0039] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0040] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0041] For the sources of the reagents used in the present invention, see Table 1 for details.

[0042] Table 1 Experimental Reagents and Sources

[0043]

[0044]

[0045] For other reagents or materials involved in the embodiments of the present invention, if not otherwise specified, they can be purchased through conventional channels; for the operation methods or detection methods involved, if not otherwise specified, they are carried out according to the conventional methods in the art.

[0046] Example 1

[0047] 1. Design of LMTIA Primer Sets, Probes, and Temperature Optimization

[0048] (1) Design of LMTIA primer sets and probes: Using the ribosomal RNA gene internal transcribed spacer (ITS) sequences of Cuscuta chinensis and Cuscuta australis as target sequences, BLAST sequence alignment analysis was performed to screen for differential sequences between Cuscuta chinensis and Cuscuta australis (as shown in Figure 1 ), and sequences with a ladder-like melting temperature were selected through analysis with Oligo7 software. Subsequently, primers for LMTIA were designed using Primer3Plus. After extensive screening, primer sets and probes with the following sequences (synthesized by General Biosystems (Anhui) Co., Ltd.) were obtained. The specific sequences are shown in Table 2.

[0049] Table 2 Sequences of LMTIA primer sets and probes

[0050]

[0051] (2) Extraction of template DNA: The DNA of Cuscuta was extracted using a plant genomic DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The specific operating steps are described in the kit's instruction manual.

[0052] (3) Temperature optimization of the LMTIA reaction system: The LMTIA reaction system for Cuscuta chinensis is shown in Table 3; the LMTIA reaction system for Cuscuta australis is shown in Table 4; the LMTIA reaction system for Cuscuta chinensis and Cuscuta australis is shown in Table 5.

[0053] Table 3 LMTIA reaction system for Cuscuta chinensis (10 μL)

[0054]

[0055] Table 4 LMTIA reaction system for Cuscuta australis

[0056]

[0057] Table 5 LMTIA reaction system for Cuscuta chinensis and Cuscuta australis

[0058]

[0059] Take PCR octuplets, and add the reaction reagents prepared in Table 3 (excluding template DNA in the system, 8 μL in each single tube) to 4 single tubes respectively, add 2 μL of DEPC-treated water to 2 single tubes as negative controls, and add 2 μL of *Cuscuta chinensis* to 2 single tubes; take PCR octuplets, and add the reaction reagents prepared in Table 4 (excluding template DNA in the system, 8 μL in each single tube) to 4 single tubes respectively, add 2 μL of DEPC-treated water to 2 single tubes as negative controls, and add 2 μL of *Cuscuta australis* to 2 single tubes. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature gradient at 54 °C, 55 °C, 56 °C, and 57 °C, set to collect fluorescence signals once every 30 seconds, and collect a total of 40 fluorescence signals.

[0060] Observe the temperature-optimized amplification curve of *Cuscuta chinensis* ( Figure 2 ), it can be seen that there is no non-specific amplification caused by primer dimers in *Cuscuta chinensis* at the four temperatures of 54 °C, 55 °C, 56 °C, and 57 °C. Considering the amplification efficiency and reproducibility of the amplification curves of *Cuscuta chinensis* at different temperatures comprehensively, 57 °C is selected as the optimal temperature for LMTIA, and subsequent determinations are carried out.

[0061] Observe the temperature-optimized amplification curve of *Cuscuta australis* ( Figure 3 ), it can be seen that there is no non-specific amplification caused by primer dimers in *Cuscuta australis* at the four temperatures of 57 °C, 58 °C, 59 °C, and 60 °C. Considering the amplification efficiency and reproducibility of the amplification curves of *Cuscuta chinensis* at different temperatures comprehensively, 57 °C is selected as the optimal temperature for LMTIA, and subsequent determinations are carried out.

[0062] Both *Cuscuta chinensis* and *Cuscuta australis* select 57 °C as the optimal temperature for LMTIA, which is beneficial to the dual detection of the components of *Cuscuta chinensis* and *Cuscuta australis*.

[0063] Example 3 Specificity of LMTIA primers

[0064] Take PCR octuplets, add the reaction reagents prepared in Table 3 (excluding template DNA in the system, 8 μL in each single tube) to 26 single tubes respectively, add 2 μL of DEPC-treated water to 2 single tubes as negative controls, and add 2 μL of *Cuscuta chinensis* DNA, *Cuscuta australis*, *Cuscuta japonica* DNA, *Cuscuta europaea* DNA, *Cuscuta lupuliformis* DNA, *Perilla frutescens* DNA, *Amaranthus paniculatus* DNA, *Celosia argentea* DNA, *Setaria italica* DNA, *Brassica rapa* ssp. *rapifera* DNA, *Sinapis alba* DNA, *Hyoscyamus niger* DNA, *Brassica napus* DNA to 24 single tubes respectively, set two parallels for each reaction, use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 57 °C, collect fluorescence signals once every 30 seconds, and collect a total of 40 fluorescence signals.

[0065] Take PCR eight - tube strips, and add the prepared reaction reagents in Table 4 (excluding template DNA in the system, 8 μL in each single tube) into 26 single tubes respectively, add 2 μL of DEPC - treated water into 2 single tubes as negative controls, and add 2 μL of Cuscuta australis DNA, Cuscuta chinensis DNA, Cuscuta japonica DNA, Cuscuta europaea DNA, Cuscuta lupuliformis DNA, Perilla frutescens DNA, Amaranthus paniculatus DNA, Celosia argentea DNA, Setaria italica DNA, Brassica rapa var. rapa DNA, Brassica alba DNA, Hyoscyamus niger DNA, Brassica napus DNA into 24 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 57 °C, collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total.

[0066] The results are as Figure 4 and Figure 5 shown. Only when adding Cuscuta chinensis or Cuscuta australis DNA, there is a good amplification curve in the LMTIA reaction, and its LMTIA reaction is positive; no amplification curves appear for other source DNAs, and the reactions are all negative. Therefore, the single - detection LMTIA detection method for Cuscuta chinensis and Cuscuta australis components established in the present invention has high specificity.

[0067] Example 4 Sensitivity of LMTIA Primers

[0068] (1) Absolute sensitivity

[0069] Perform gradient dilution on Cuscuta chinensis DNA, and dilute it to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL respectively. Take PCR eight - tube strips, and add the prepared reaction reagents in Table 3 (excluding template DNA in the system, 8 μL in each single tube) into 14 single tubes respectively, add 2 μL of DEPC - treated water into 2 single tubes as negative controls, add 2 μL of 10 ng / μL Cuscuta chinensis DNA into 2 single tubes, add 2 μL of 1 ng / μL Cuscuta chinensis DNA into 2 single tubes respectively, add 2 μL of 100 pg / μL Cuscuta chinensis DNA into 2 single tubes respectively, add 2 μL of 10 pg / μL Cuscuta chinensis DNA into 2 single tubes respectively, add 2 μL of 1 pg / μL Cuscuta chinensis DNA into 2 single tubes respectively, and add 2 μL of 100 fg / μL Cuscuta chinensis DNA into 2 single tubes respectively. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 57 °C, collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total.

[0070] The DNA of *Cuscuta australis* was serially diluted to 8 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL. Take an eight-strip PCR tube, and add the reaction reagents prepared in Table 4 (the template DNA is not included in the system, 8 μL in each single tube) to 14 single tubes respectively, add 2 μL of DEPC-treated water to 2 single tubes as negative controls, add 2 μL of *Cuscuta australis* DNA at 8 ng / μL to 2 single tubes, add 2 μL of *Cuscuta australis* DNA at 1 ng / μL to 2 single tubes respectively, add 2 μL of *Cuscuta australis* DNA at 100 pg / μL to 2 single tubes respectively, add 2 μL of *Cuscuta australis* DNA at 10 pg / μL to 2 single tubes respectively, add 2 μL of *Cuscuta australis* DNA at 1 pg / μL to 2 single tubes respectively, and add 2 μL of *Cuscuta australis* DNA at 100 fg / μL to 2 single tubes respectively. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 57 °C, collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total.

[0071] The results are as Figure 8 and Figure 9 shown. When the DNA concentrations of *Cuscuta chinensis* are 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL and the DNA concentrations of *Cuscuta australis* are 8 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL, the LMTIA amplification curves are relatively obvious. However, no amplification curve appears when the DNA concentration is 100 fg / μL because the concentration of 100 fg / μL exceeds the detection limit of this method, making the amplification unstable. Therefore, the absolute sensitivity of this method can reach 1 pg / μL. In a 10 μL reaction system, 1 pg of *Cuscuta chinensis* or *Cuscuta australis* DNA extracted from the sample can be detected.

[0072] (2) Relative sensitivity

[0073] Mix Cuscuta chinensis and Cuscuta australis, with the mass fractions of Cuscuta chinensis being 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% respectively. Extract the DNA of the above mixed samples (using a plant genomic DNA extraction kit, Tiangen, and extract according to the steps in the instruction manual) and reserve it as template DNA. Take a PCR octuplet 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, add 2 μL of DEPC-treated water to 2 single tubes as negative controls, and add 2 μL of DNA with the mass fractions of Cuscuta chinensis being 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% to 18 single tubes respectively. Set two parallels for each reaction. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 57°C, collect fluorescence signals once every 30 seconds, and collect a total of 40 fluorescence signals.

[0074] The results are as Figure 8 shown. When the mixed samples contain 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% of Cuscuta chinensis, there are obvious amplification curves for LMTIA; when the mixed samples contain 0.1% of Cuscuta chinensis, there is no amplification curve for LMTIA. The above results indicate that 0.1% exceeds the detection limit of this method for detecting Cuscuta chinensis. Therefore, the relative sensitivity of this method for detecting Cuscuta chinensis can reach at least 0.5%.

[0075] Mix Cuscuta australis and Cuscuta chinensis, with the mass fractions of Cuscuta australis being 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% (, extract the DNA of the above mixed samples (using a plant genomic DNA extraction kit, Tiangen, and extract according to the steps in the instruction manual) and reserve it as template DNA. Take a PCR octuplet tube, add the reaction reagents prepared in Table 4 (the system does not include template DNA, 8 μL in each single tube) to 26 single tubes respectively, add 2 μL of DEPC-treated water to 2 single tubes as negative controls, and add 2 μL of DNA with the mass fractions of Cuscuta australis being 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% to 18 single tubes respectively. Set two parallels for each reaction. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 57°C, collect fluorescence signals once every 30 seconds, and collect a total of 40 fluorescence signals.

[0076] The results are as Figure 9As shown, when the mixed samples contained 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% of *Cuscuta australis*, there were obvious amplification curves for LMTIA; when the mixed samples contained 0.05% of *Cuscuta australis*, there was no amplification curve for LMTIA. The above results indicate that 0.05% exceeded the detection limit of this method for detecting *Cuscuta australis*. Therefore, the relative sensitivity of this method for detecting *Cuscuta australis* can reach at least 0.1%.

[0077] Example 5: Use of the LMTIA primer set and probe for the dual detection of the components of *Cuscuta chinensis* and *Cuscuta australis*

[0078] Take a PCR octuplet tube, add the reaction reagents prepared in Table 5 (the system does not include template DNA, 8 μL in each single tube) to 4 single tubes respectively, add 2 μL of DEPC-treated water to 2 single tubes as negative controls, and add 2 μL of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* (DNA extracted after mixing *Cuscuta chinensis* and *Cuscuta australis* according to a mass ratio of 1:1) to 2 single tubes. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 57°C, collect fluorescence signals once every 30 seconds, and collect a total of 40 fluorescence signals.

[0079] The results showed that good amplification curves ( Figure 10 ) appeared when the LMTIA primer set and probe of the present invention were used for the simultaneous detection of the mixed product of *Cuscuta chinensis* and *Cuscuta australis*. The components of *Cuscuta chinensis* and *Cuscuta australis* contained therein showed positive LMTIA reactions respectively, and had high specificity. Therefore, the LMTIA primer set and probe of the present invention can be used for the dual detection of the components of *Cuscuta chinensis* and *Cuscuta australis*, that is, for the simultaneous identification of the components of *Cuscuta chinensis* and *Cuscuta australis*.

[0080] Example 6: Specificity detection of the use of the LMTIA primer set and probe for the dual detection of the components of *Cuscuta chinensis* and *Cuscuta australis*

[0081] Take a PCR octuplet tube, add the reaction reagents prepared in Table 5 (the system does not include template DNA, 8 μL in each single tube) to 26 single tubes respectively, add 2 μL of DEPC-treated water to 2 single tubes as negative controls, and add 2 μL of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis*, the DNA of *Cuscuta japonica*, the DNA of *Cuscuta europaea*, the DNA of *Cuscuta lupuliformis*, the DNA of *Perilla frutescens*, the DNA of *Amaranthus paniculatus*, the DNA of *Celosia argentea*, the DNA of *Setaria italica*, the DNA of *Brassica rapa subsp. rapifera*, the DNA of *Sinapis alba*, the DNA of *Hyoscyamus niger*, and the DNA of *Brassica napus* to 24 single tubes respectively, and set 2 parallels for each reaction. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 57°C, collect fluorescence signals once every 30 seconds, and collect a total of 40 fluorescence signals.

[0082] The results are as Figure 11 shown. The results show that only when the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* is added, there is a good amplification curve in the LMTIA reaction, and the LMTIA reaction is positive; no amplification curve appears for the DNA of other provenances, and the reactions are all negative. Therefore, the dual detection of the LMTIA detection method for the detection of the components of *Cuscuta chinensis* and *Cuscuta australis* established in the present invention has high specificity.

[0083] Example 7 Detection of the sensitivity of the LMTIA primer set and probe for the dual detection of the components of *Cuscuta chinensis* and *Cuscuta australis*

[0084] The extracted mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* was serially diluted to 8 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL. Take an eight-strip PCR tube, and add the prepared reaction reagents (the template DNA is not included in the system, 8 μL in each single tube) in 14 single tubes respectively, add 2 μL of DEPC-treated water in 2 single tubes as negative controls, add 2 μL of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* at 8 ng / μL in 2 single tubes respectively, add 2 μL of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* at 1 ng / μL in 2 single tubes respectively, add 2 μL of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* at 100 pg / μL in 2 single tubes respectively, add 2 μL of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* at 10 pg / μL in 2 single tubes respectively, add 2 μL of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* at 1 pg / μL in 2 single tubes respectively, and add 2 μL of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* at 100 fg / μL in 2 single tubes respectively. Use the Gentier96E fully automatic medical PCR analysis system to set the temperature at 57 °C, collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total.

[0085] The results are as Figure 12 shown. When the concentration of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* is 8 ng / μL, 1 ng / μL, 100 pg / μL, and 10 pg / μL, the LMTIA amplification curves are relatively obvious, but no amplification curve appears when the DNA concentration is 1 pg / μL. This is because the concentration of 1 pg / μL exceeds the detection limit of this method, making the amplification unstable. Therefore, the absolute sensitivity of this method can reach 10 pg / μL. In a 10 μL reaction system, 10 pg of the mixed DNA of *Cuscuta chinensis* and *Cuscuta australis* extracted from the sample can be detected.

[0086] Example 8 Identification of the authenticity of commercially available *Cuscuta chinensis* and / or *Cuscuta australis* using the LMTIA primer set and probe

[0087] Take PCR octuplets, and add the reaction reagents prepared in Table 5 (excluding template DNA in the system, 8 μL in each single tube) to 26 single tubes, add 2 μL of DEPC-treated water to 2 single tubes as negative controls, add 2 μL of Cuscuta chinensis DNA to 2 single tubes as positive controls for Cuscuta chinensis, add 2 μL of Cuscuta australis DNA to 2 single tubes as positive controls for Cuscuta australis, add 2 μL of commercially available Cuscuta chinensis DNA from Inner Mongolia 1 to 2 single tubes, add 2 μL of commercially available Cuscuta chinensis DNA from Inner Mongolia 2 to 2 single tubes, add 2 μL of commercially available Cuscuta chinensis DNA from Inner Mongolia 3 to 2 single tubes, add 2 μL of commercially available Cuscuta chinensis DNA from Ningxia 1 to 2 single tubes, add 2 μL of commercially available Cuscuta chinensis DNA from Ningxia 2 to 2 single tubes, add 2 μL of commercially available Cuscuta chinensis DNA from Ningxia 3 to 2 single tubes, add 2 μL of commercially available Cuscuta chinensis DNA from Gansu Province to 2 single tubes, add 2 μL of commercially available Cuscuta chinensis DNA from Northeast China to 2 single tubes, add 2 μL of commercially available Cuscuta chinensis DNA from Yuzhou to 2 single tubes, and add 2 μL of commercially available Cuscuta chinensis DNA from Henan Province to 2 single tubes. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 57 °C, collect fluorescence signals once every 30 seconds, and collect a total of 40 fluorescence signals.

[0088] The results are as Figure 13 shown. The LMTIA primer set and probe of the present invention can simultaneously detect positive controls for Cuscuta chinensis and Cuscuta australis, and good amplification curves appear. When detecting Cuscuta chinensis from other production areas, it is found that the commercially available Cuscuta chinensis products from Henan Province contain both Cuscuta chinensis components and Cuscuta australis components; the commercially available Cuscuta australis from Gansu Province, Northeast China, Yuzhou, Inner Mongolia 1, 2, 3, and Ningxia 1, 2, 3 only contain Cuscuta australis components and do not contain Cuscuta chinensis components. Therefore, the LMTIA primer set and probe of the present invention can be simultaneously used for the authenticity identification of commercially available Cuscuta chinensis and / or Cuscuta australis from different production areas.

[0089] The above-described embodiments 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 LMTIA primer-probe combination for detecting Cuscuta chinensis and / or Cuscuta australis, characterized in that, Including TuSiZ-F shown in SEQ ID NO.1, TuSiZ-B shown in SEQ ID NO.2, TuSiZ-LF shown in SEQ ID NO.3, NanTuSiZ-F shown in SEQ ID NO.4, NanTuSiZ-B shown in SEQ ID NO.5, NanTuSiZ-LB shown in SEQ ID NO.6, NanTuSiZ-LF shown in SEQ ID NO.7, TuSiZ-LF Pr shown in SEQ ID NO.8, and NanTuSiZ-LF Pr shown in SEQ ID NO.9; The TuSiZ-LF Pr and NanTuSiZ-LF Pr are linked with different fluorescent groups.

2. Use of the LMTIA primer-probe combination according to claim 1 in the preparation of a reagent or kit for detecting Cuscuta chinensis and / or Cuscuta australis.

3. A kit for detecting Cuscuta chinensis based on the LMTIA technology, characterized in that, The kit contains TuSiZ-F, TuSiZ-B, TuSiZ-LF, and TuSiZ-LF Pr as described in claim 1.

4. A kit for detecting Cuscuta australis based on the LMTIA technology, characterized in that, The kit contains NanTuSiZ-F, NanTuSiZ-B, NanTuSiZ-LB, NanTuSiZ-LF, and NanTuSiZ-LF Pr as described in claim 1.

5. A kit for dual detection of Cuscuta chinensis and Cuscuta australis based on the LMTIA technology, characterized in that, The kit contains TuSiZ-F, TuSiZ-B, TuSiZ-LF, NanTuSiZ-F, NanTuSiZ-B, NanTuSiZ-LB, NanTuSiZ-LF, TuSiZ-LF Pr, and NanTuSiZ-LF Pr as described in claim 1.

6. A method for detecting LMTIA of Cuscuta chinensis and / or Cuscuta australis, characterized in that, Including the following steps: Extracting genomic DNA of the sample to be tested; Using the genomic DNA as a template, performing an LMTIA reaction with the kit according to any one of claims 3-5, and observing whether there is an amplification curve; If there is an amplification curve, it indicates that the sample to be tested contains Cuscuta chinensis and / or Cuscuta australis.

7. The LMTIA detection method according to claim 6, wherein When performing the LMTIA reaction with the kit according to claim 3, the system of the LMTIA reaction is: 2.00 μL of 5x Mix Buffer, 0.40 μL of Bst enzyme, 0.16 μL of 100 μM TuSiZ-F, 0.16 μL of 100 μM TuSiZ-B, 0.04 μL of 100 μM TuSiZ-LF, 0.30 μL of 100 μM TuSiZ-Pr, 2.00 μL of template, and made up to 10 μL with DEPC-treated water; the temperature of the LMTIA reaction is 57 °C and the time is 20 min.

8. The LMTIA detection method according to claim 7, wherein When performing the LMTIA reaction using the kit described in claim 4, the system for the LMTIA reaction is as follows: 2.00 μL of 5x Mix Buffer, 0.40 μL of Bst enzyme, 0.16 μL of 100 μM NanTuSiZ-F, 0.16 μL of 100 μM NanTuSiZ-B, 0.04 μL of 100 μM NanTuSiZ-LB, 0.04 μL of 100 μM NanTuSiZ-LF, 0.10 μL of 100 μM NanTuSiZ-Pr, 2.00 μL of template, and made up to 10 μL with DEPC-treated water; the temperature of the LMTIA reaction is 57 °C and the time is 20 min.

9. The LMTIA detection method according to claim 7, characterized in that, When performing the LMTIA reaction using the kit described in claim 5, the system for the LMTIA reaction is as follows: 3.00 μL of 5x Mix Buffer, 0.40 μL of Bst enzyme, 0.32 μL of 100 μM TuSiZ-F, 0.32 μL of 100 μM TuSiZ-B, 0.08 μL of 100 μM TuSiZ-LF, 0.2 μL of 100 μM TuSiZ-Pr, 0.16 μL of 100 μM NanTuSiZ-F, 0.16 μL of 100 μM NanTuSiZ-B, 0.04 μL of 100 μM NanTuSiZ-LB, 0.04 μL of 100 μM NanTuSiZ-LF, 0.10 μL of 100 μM NanTuSiZ-Pr, 2.00 μL of template, and made up to 10 μL with DEPC-treated water; the temperature of the LMTIA reaction is 57 °C and the time is 20 min.

10. Use of the kit according to any one of claims 3 - 5 in the identification of adulteration and fraud of Cuscuta chinensis and / or Cuscuta australis.

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