Primer probe and application thereof in identifying whether nutgrass galingale rhizome is adulterated with nutgrass galingale rhizome

Abstract

The invention discloses a primer probe and application thereof in identifying whether nutgrass galingale rhizome is adulterated with nutgrass galingale rhizome, and belongs to the field of biomolecule detection. An LMTIA primer and a probe for specifically identifying the rhizoma cyperi and the nutgrass galingale rhizome are designed by taking a nutgrass galingale rhizome rRNA gene spacer sequence as a target sequence, the nutgrass galingale rhizome and the nutgrass galingale rhizome can be simultaneously detected through the probe XiangFd-1Pr, and the probe XiangFd-1Pr can only detect the nutgrass galingale rhizome. Therefore, the nutgrass galingale rhizome and the nutgrass galingale rhizome can be identified and whether the nutgrass galingale rhizome is doped in the nutgrass galingale rhizome or not can be identified through the LMTIA primer and the probe, the method for detecting the nutgrass galingale rhizome and the nutgrass galingale rhizome, which is constructed by utilizing the LMTIA primer and the probe, has the characteristics of high speed, high sensitivity and high specificity, and a foundation is laid for rapidly, efficiently and accurately identifying the nutgrass galingale rhizome and the nutgrass galingale rhizome in a sample. And a technical means is provided for authenticity identification of traditional Chinese medicinal materials.

Description

Technical Field

[0001] The present invention relates to the field of biomolecule detection, and particularly to a primer-probe and its application in identifying whether Cyperus rotundus is adulterated with Cyperus stoloniferus. Background Art

[0002] Cyperus rotundus is the dried rhizome of the plant Cyperus rotundus L. of the Cyperaceae family. It is dug in autumn, the hair and beard are singed off, slightly boiled or steamed through in boiling water and then dried in the sun, or directly dried after singeing. It has the effects of soothing the liver and relieving depression, regulating qi and relieving distension, and regulating menstruation and relieving pain. It is used to treat symptoms such as stagnation of liver qi, chest and hypochondrium distending pain, hernia pain, breast distending pain, stagnation of qi in the spleen and stomach, abdominal distension and fullness pain, irregular menstruation, amenorrhea and dysmenorrhea, etc., and is widely used in Chinese patent medicines and various traditional Chinese medicine prescriptions. With the continuous increase in market demand, the phenomenon of counterfeits and adulteration of Cyperus rotundus is becoming increasingly serious, which not only affects the quality and efficacy of medicinal materials, but also poses a potential threat to the health of patients. In-depth understanding of the current situation of Cyperus rotundus and its counterfeits and adulteration is crucial for ensuring the quality and safety of traditional Chinese medicine. The common counterfeits of Cyperus rotundus are Cyperus stoloniferus, Scirpus juncoides and Rhizoma Cyperi Rotundi, among which Cyperus stoloniferus is the most common.

[0003] There are many reports on the related identification studies of Cyperus rotundus and its adulterated products. The commonly used identification methods are traditional identification methods such as morphological identification, microscopic identification and physicochemical identification. Generally, these traditional methods require a full understanding of the properties and characteristics of traditional Chinese medicinal materials to accurately identify, and have relatively high requirements for the professionalism of researchers. With the continuous development of science and technology, molecular biological identification technology has been applied in the field of identification of traditional Chinese medicinal materials, such as PCR detection. Although the PCR method has achieved certain results in the identification of traditional Chinese medicinal materials, due to the very close genetic relationship between Cyperus rotundus and Cyperus stoloniferus, it is difficult to design specific primers for the dispersed different loci, which also leads to certain limitations of PCR detection in distinguishing Cyperus rotundus from Cyperus stoloniferus. 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 20 - 40 minutes, and has high sensitivity and specificity. At present, there is no report on the application of LMTIA technology in the detection of Cyperus rotundus and Cyperus stoloniferus. Summary of the Invention

[0004] The object of the present invention is to provide a primer-probe and its application in identifying whether Cyperus rotundus is adulterated with Cyperus stoloniferus, so as to solve the problems existing in the above-mentioned prior art. By designing specific LMTIA primers and probes, a rapid detection method for Cyperus rotundus and Cyperus stoloniferus is established, providing technical support for the identification of traditional Chinese medicinal materials, and at the same time providing a new method for solving the problem of adulteration and counterfeiting of traditional Chinese medicinal materials.

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

[0006] The present invention provides a primer probe for identifying Cyperus rotundus L. and Cyperus rotundus L. var. compressus L. The primer probe is as follows:

[0007] XiangF-1F1: 5'-GGTCCCTCGGGCTTTTAATCCCGTGAACCATCGAGT-3';

[0008] XiangF-1B1: 5'-GCCTGGGCGTTAGAATTTTTCGGAGGGCCACTGTC-3';

[0009] XiangF-1LF: 5'-GCAACTTGCGTTCAAAG-3';

[0010] XiangF-1LB: 5'-CATCAACGCTCGGTC-3';

[0011] XiangF-1Pr: 5'-CCCATCAACGCTCGGTT-3';

[0012] XiangFd-1Pr: 5'-CCCATCAACGCTCGGTCA-3'.

[0013] Preferably, the 5' ends of XiangF-1Pr and XiangFd-1Pr are both modified with quenching groups, and the 3' ends are both modified with fluorescent groups.

[0014] The present invention also provides a kit for identifying Cyperus rotundus L. and Cyperus rotundus L. var. compressus L., including the above-mentioned primer probe.

[0015] The present invention also provides the application of the above-mentioned primer probe in any of the following:

[0016] (1) Application in identifying whether Cyperus rotundus L. is adulterated with Cyperus rotundus L. var. compressus L.;

[0017] (2) Application in identifying Cyperus rotundus L. and Cyperus rotundus L. var. compressus L.

[0018] The present invention also provides a method for identifying Cyperus rotundus L. and Cyperus rotundus L. var. compressus L., including the following steps:

[0019] Using the DNA of the sample to be tested as a template, performing a real-time fluorescence PCR amplification reaction with the above-mentioned primer probe, and judging whether Cyperus rotundus L. and / or Cyperus rotundus L. var. compressus L. is contained according to whether an exponential amplification curve appears.

[0020] Preferably, the judgment method is as follows:

[0021] When amplifying with the XiangF-1Pr and four primers, an exponential amplification curve appears. When amplifying with the XiangFd-1Pr and four primers, no exponential amplification curve appears. Then the sample to be tested is Cyperus rotundus L.

[0022] When amplifying with the XiangF-1Pr and four primers, an exponential amplification curve appears. When amplifying with the XiangFd-1Pr and four primers, an exponential amplification curve appears. Then the sample to be tested is Cyperus rotundus L. and Cyperus rotundus L. var. proliferus Ohwi, that is, Cyperus rotundus L. is adulterated with Cyperus rotundus L. var. proliferus Ohwi.

[0023] When amplifying with the XiangF-1Pr and four primers, no exponential amplification curve appears. When amplifying with the XiangFd-1Pr and four primers, no exponential amplification curve appears. Then the sample to be tested is neither Cyperus rotundus L. nor Cyperus rotundus L. var. proliferus Ohwi.

[0024] Preferably, the conditions for the real-time fluorescence PCR amplification reaction are: 65°C, isothermal amplification for 20 minutes.

[0025] Preferably, in the real-time fluorescence PCR amplification reaction system: the molar ratio of XiangF-1F1: XiangF-1B1: XiangF-1LF: XiangF-1LB: XiangF-1Pr is 8:8:2:2:5; or the molar ratio of XiangF-1F1: XiangF-1B1: XiangF-1LF: XiangF-1LB: XiangFd-1Pr is 8:8:2:2:5.

[0026] The present invention discloses the following technical effects:

[0027] The present invention discloses a group of LMTIA primer-probes that can be used to identify Cyperus rotundus L. and Cyperus rotundus L. var. proliferus Ohwi. When using these LMTIA primer-probes to detect Cyperus rotundus L. and Cyperus rotundus L. var. proliferus Ohwi, the specificity is the best at 65°C; the absolute sensitivity of the LMTIA primer-probes can reach 10 pg / μL (FAM probe) or 1 pg / μL (JOE probe). The LMTIA primer-probes provided by the present invention have good specificity and sensitivity, fast detection speed, and can detect Cyperus rotundus L. and Cyperus rotundus L. var. proliferus Ohwi in the sample under isothermal conditions, providing a technical means for quickly, efficiently, and accurately identifying Cyperus rotundus L. and Cyperus rotundus L. var. proliferus Ohwi in the sample, as well as the authenticity identification of traditional Chinese medicinal materials. Description of the Drawings

[0028] 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 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 also be obtained based on these drawings.

[0029] Figure 1 It is the alignment diagram of the 5.8s and ITS2 sequences of Cyperus rotundus and large Cyperus rotundus;

[0030] Figure 2 It is the melting curve of the target gene of Cyperus rotundus

[0031] Figure 3 It is the amplification result diagram of optimizing the temperature for detecting Cyperus rotundus by LMTIA-FAM;

[0032] Figure 4 It is the amplification result diagram of optimizing the temperature for detecting large Cyperus rotundus by LMTIA-FAM

[0033] Figure 5 It is the amplification result diagram of optimizing the temperature for detecting large Cyperus rotundus by LMTIA-JOE;

[0034] Figure 6 It is the amplification result diagram of specific determination by LMTIA-FAM;

[0035] Figure 7 It is the amplification result diagram of specific determination by LMTIA-JOE;

[0036] Figure 8 It is the amplification result diagram of absolute sensitivity determination by LMTIA-FAM;

[0037] Figure 9 It is the amplification result diagram of absolute sensitivity determination by LMTIA-JOE;

[0038] Figure 10 It is the amplification result diagram of relative sensitivity determination by LMTIA-FAM;

[0039] Figure 11 It is the amplification result diagram of relative sensitivity determination by LMTIA-JOE;

[0040] Figure 12 It is the amplification result diagram of detecting actual samples by LMTIA-FAM;

[0041] Figure 13 It is the amplification result diagram of detecting actual samples by LMTIA-JOE. Specific embodiments

[0042] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed 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.

[0043] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended 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.

[0044] 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.

[0045] 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 specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0046] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0047] Example 1: Design of LMTIA Primer Set, Probe and Temperature Optimization

[0048] 1. Design of LMTIA Primer Set and Probe

[0049] Using the ribosomal RNA gene internal transcribed spacer (ITS) sequence of Cyperus rotundus as the target sequence, BLAST sequence alignment analysis was performed to screen for the differential sequences between Cyperus rotundus and Cyperus rotundus var. rotundus (as shown in Figure 1 ), and sequences with a ladder-like melting temperature were selected through Oligo7 software analysis ( Figure 2 ). Then, Primer3Plus was used to design the primers of LMTIA. After extensive screening, the primer set and probe with the following sequences (synthesized by General Biosystems (Anhui) Co., Ltd.) were obtained. The specific sequences are shown in Table 1.

[0050] Table 1 LMTIA primer sets and probe sequences

[0051]

[0052] 2. Extraction of template DNA

[0053] The DNA of Cyperus rotundus was extracted using a plant genomic DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The specific operation steps are shown in the kit's instruction manual.

[0054] 3. Optimization of the temperature of the LMTIA reaction system

[0055] The reaction system of LMTIA is shown in Table 2.

[0056] Table 2 LMTIA reaction system (10 μL)

[0057]

[0058]

[0059] Take 4 PCR strip tubes and randomly divide them into two groups (negative control group and experimental group), with two single tubes in each group. Add each reagent according to the reaction system shown in Table 2 (excluding template DNA, 8 μL in each single tube). Then, the negative control group is added with 2 μL of DEPC-treated water, and the experimental group is added with 2 μL of Cyperus rotundus or Cyperus rotundus var. xanthorrhiza DNA. 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.

[0060] Observe the temperature-optimized amplification curves of LMTIA-FAM for detecting Cyperus rotundus and Cyperus rotundus var. xanthorrhiza (see Figure 3 、 Figure 4 ) and it can be seen that: there is no non-specific amplification caused by primer dimers in Cyperus rotundus and Cyperus rotundus var. xanthorrhiza at the four temperatures of 59°C, 61°C, 63°C, and 65°C. Considering the amplification efficiency and reproducibility of the amplification curves at different temperatures, 65°C is selected as the optimal temperature for LMTIA and subsequent determinations are carried out. Similarly, observe the temperature-optimized amplification curve of LMTIA-JOE for detecting Cyperus rotundus var. xanthorrhiza (see Figure 5 ) and it can be seen that: there is no non-specific amplification caused by primer dimers in Cyperus rotundus var. xanthorrhiza at the four temperatures of 59°C, 61°C, 63°C, and 65°C. Considering the amplification efficiency and reproducibility of the amplification curves of Cyperus rotundus var. xanthorrhiza at different temperatures, 65°C is selected as the optimal temperature for LMTIA and subsequent determinations are carried out.

[0061] Example 2: Specificity detection

[0062] Take 10 PCR strip tubes and randomly divide them into 5 groups (negative control group, experimental group 1, experimental group 2, experimental group 3, experimental group 4). Set two single tubes in each group and add each reagent according to the reaction system shown in Table 2 (excluding template DNA, 8 μL in each single tube). Then, add 2 μL of DEPC-treated water to the negative control group, and add 2 μL of Cyperus rotundus DNA, Cyperus rotundus var. xanthorrhizus DNA, Scirpus juncoides Roxb. DNA, and Rhizoma Anemones Raddeanae DNA to experimental group 1, experimental group 2, experimental group 3, and experimental group 4 respectively. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 65 °C (FAM) or 65 °C (JOE), collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total.

[0063] The results are as Figure 6 and Figure 7 shown. When detecting with LMTIA-FAM, both Cyperus rotundus and Cyperus rotundus var. xanthorrhizus amplified, while other samples did not amplify. When detecting with LMTIA-JOE, only Cyperus rotundus var. xanthorrhizus amplified, and no amplification curves appeared for other common counterfeit DNA samples. Therefore, the established LMTIA methods for Cyperus rotundus and Cyperus rotundus var. xanthorrhizus have high specificity.

[0064] Example 3: Sensitivity detection

[0065] 1. Absolute sensitivity

[0066] Perform gradient dilution on Cyperus rotundus and Cyperus rotundus var. xanthorrhizus DNA, and dilute them to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL respectively. Take 14 PCR strip tubes and add the reaction reagents prepared in Table 2 (excluding template DNA, 8 μL in each single tube). Add 2 μL of DEPC-treated water to 2 single tubes as negative controls. Then, for the remaining 12 single tubes, divide them into groups of two and add different concentrations of Cyperus rotundus DNA samples of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL respectively. Repeat the above steps for the absolute sensitivity of Cyperus rotundus var. xanthorrhizus. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 65 °C (FAM) or 65 °C (JOE), collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total.

[0067] (1) The results of Cyperus rotundus are as Figure 8As shown, when the DNA concentration of Cyperus rotundus was 5 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, making the amplification unstable. Therefore, the absolute sensitivity of this method can reach 10 pg / μL, that is, in a 10 μL reaction system, Cyperus rotundus extracted from the sample can be detected when it reaches 10 pg / μL.

[0068] (2) Results of large Cyperus rotundus are as Figure 9 As shown, when the DNA concentration of large Cyperus rotundus was 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL, the LMTIA amplification curves were relatively obvious. However, when the DNA concentration was 100 fg / μL, no amplification curve appeared because the concentration of 100 fg / μL exceeded the detection limit of this method, making the amplification unstable. Therefore, the absolute sensitivity of this method can reach 1 pg / μL, that is, in a 10 μL reaction system, Cyperus rotundus extracted from the sample can be detected when it reaches 1 pg / μL.

[0069] 2. Relative sensitivity

[0070] (1) Since there is no cross - reaction between Cyperus rotundus and Scirpus triqueter during specific detection, Scirpus triqueter was used as an adulterated drug and mixed with Cyperus rotundus to test the relative sensitivity of this method to Cyperus rotundus. DNA was extracted after mixing Cyperus rotundus and Scirpus triqueter, and the mass fractions of Cyperus rotundus were 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95%, and 99% respectively. DNA of the above - mentioned mixed samples (extracted using a Plant Genomic DNA Extraction Kit, Tiangen Biochemical Technology (Beijing) Co., Ltd., according to the steps in the instruction manual) was taken as template DNA for standby. Take 26 PCR eight - tube strips, randomly divided into 13 groups (negative control group, positive control group, experimental groups 1 - 10), with two single tubes in each group. Add the reaction reagents prepared in Table 2 (excluding template DNA, 8 μL in each single tube), then add 2 μL of DEPC - treated water to the negative control group, 2 μL of Cyperus rotundus standard product to the positive control group, and 2 μL of DNA with Cyperus rotundus volume fractions of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95%, and 99% to experimental groups 1 - 10 respectively. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 65 °C, collect fluorescence signals once every 30 seconds, and collect a total of 40 fluorescence signals.

[0071] (2) Since there is no intersection between Cyperus rotundus and Cyperus rotundus var. xanthorrhizus, Cyperus rotundus and Cyperus rotundus var. xanthorrhizus were selected and mixed to detect the relative sensitivity of LMTIA to Cyperus rotundus var. xanthorrhizus. Cyperus rotundus var. xanthorrhizus and Cyperus rotundus were mixed, and the volume fractions of Cyperus rotundus var. xanthorrhizus were 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% respectively. DNA of the above mixed samples was taken (extracted using a plant genomic DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. according to the steps in the instruction manual) and used as template DNA for standby. Take 26 PCR octuplets and randomly divide them into 13 groups (negative control group, positive control group, experimental groups 1 - 10). Each group has two single tubes, and the reaction reagents prepared in Table 2 (excluding template DNA, 8 μL in each single tube) are added respectively. Then, 2 μL of DEPC-treated water is added to the negative control group, 2 μL of the Cyperus rotundus var. xanthorrhizus standard product is added to the positive control group, and 2 μL of DNA with the volume fractions of Cyperus rotundus var. xanthorrhizus being 0.1%, 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% are added to experimental groups 1 - 10 respectively. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 65 °C, collect fluorescence signals once every 30 seconds, and collect 40 fluorescence signals in total;

[0072] The results are as Figure 10 shown. When testing the relative sensitivity of LMTIA to Cyperus rotundus, when the mixed samples contain 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% of Cyperus rotundus, there are obvious amplification curves; when the mixed samples contain 0.1% and 0.5% of Cyperus rotundus, there are no amplification curves. Therefore, the relative sensitivity of this method for detecting Cyperus rotundus can reach at least 1%. Similarly, as Figure 11 shown, when testing the relative sensitivity of LMTIA to Cyperus rotundus var. xanthorrhizus, when the mixed samples contain 0.5%, 1%, 5%, 10%, 20%, 50%, 80%, 90%, 95% and 99% of Cyperus rotundus var. xanthorrhizus, there are obvious amplification curves; when the mixed samples contain 0.1% of Cyperus rotundus var. xanthorrhizus, there is no amplification curve. Therefore, the relative sensitivity of this method for detecting Cyperus rotundus var. xanthorrhizus can reach at least 0.5%.

[0073] Example 4 The LMTIA primer set and probe of the present invention were used for the authenticity identification of Cyperus rotundus from different commercial origins.

[0074] Take 20 PCR eight-strip tubes and randomly divide them into 10 groups (negative control group, positive control group, experimental groups 1 - 8). Set two single tubes in each group and add the reaction reagents prepared in Table 2 (excluding template DNA, 8 μL in each single tube). Then, add 2 μL of DEPC-treated water to the negative control group, add 2 μL of cyperus rotundus standard or large cyperus rotundus standard to the positive control group, and add DNA of samples 1 from Huaxian County, Henan Province, DNA of samples 2 from Huaxian County, Henan Province, DNA of samples from Nanzhao County, Nanyang City, Henan Province, DNA of samples from Yuzhou District, Yulin City, Guangxi Zhuang Autonomous Region, DNA of samples from Gangnan District, Guigang City, Guangxi Zhuang Autonomous Region, DNA of samples sold on Taobao, and DNA of samples from Haikou City, Hainan Province to experimental groups 1 - 8 respectively. Use the Gentier 96E fully automatic medical PCR analysis system to set the temperature at 65 °C, collect fluorescence signals once every 60 seconds, and collect a total of 40 fluorescence signals. Detect with FAM and JOE probes respectively once.

[0075] The results are as Figure 12 , Figure 13 and shown in Table 3. When detecting actual samples with LMTIA-FAM, 7 samples were amplified. When detecting actual samples with LMTIA-JOE, 6 samples were amplified. By comparison, it was found that the samples from Nanyang, Henan Province were cyperus rotundus; the samples 2 from Huaxian County, Henan Province, the samples from Yulin City, Guangxi Zhuang Autonomous Region, the samples from Guigang City, Guangxi Zhuang Autonomous Region, the samples sold on Taobao, and the samples from Haikou City, Hainan Province were counterfeits containing large cyperus rotundus; the sample 1 from Huaxian County, Henan Province was neither cyperus rotundus nor large cyperus rotundus. Therefore, the LMTIA primer set and probe of the present invention can be used simultaneously for the authenticity identification of cyperus rotundus from different origins on the market.

[0076] Table 3 Amplification situations of cyperus rotundus from different origins

[0077]

[0078]

[0079] 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 should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer-probe for differentiating Cyperus rotundus L. and Cyperus rotundus L. var. compressus L., characterized in that, The primer-probes are as follows: XiangF-1F1: 5'-GGTCCCTCGGGCTTTTAATCCCGTGAACCATCGAGT-3'; XiangF-1B1: 5'-GCCTGGGCGTTAGAATTTTTCGGAGGGCCACTGTC-3'; XiangF-1LF: 5'-GCAACTTGCGTTCAAAG-3'; XiangF-1LB: 5'-CATCAACGCTCGGTC-3'; XiangF-1Pr: 5'-CCCATCAACGCTCGGTT-3'; XiangFd-1Pr: 5'-CCCATCAACGCTCGGTCA-3'.

2. The primer-probe according to claim 1, wherein, The 5'-ends of the said XiangF-1Pr and XiangFd-1Pr are both modified with quenching groups, and the 3'-ends are both modified with fluorescent groups.

3. A kit for identifying Cyperi Rhizoma and Cyperi Rhizoma Rotundus, characterized in that, Comprising the primer-probes described in claim 1 or 2.

4. Use of the primer-probes described in claim 1 or 2 in any of the following: (1) Use in identifying whether Cyperus rotundus is adulterated with Cyperus stoloniferus; (2) Use in identifying Cyperus rotundus and Cyperus stoloniferus.

5. A method for identifying Cyperi Rhizoma and Cyperi Rhizoma Rotundus, characterized in that, Comprising the following steps: Using the DNA of the sample to be tested as a template, performing a real-time fluorescence PCR amplification reaction with the primer-probes described in claim 1 or 2, and judging whether Cyperus rotundus and / or Cyperus stoloniferus is / are contained according to whether an exponential amplification curve appears.

6. The method according to claim 5, characterized in that, The method of the said judgment is: When amplifying with the said XiangF-1Pr and the four primers, if an exponential amplification curve appears, and at the same time when amplifying with the said XiangFd-1Pr and the four primers, no exponential amplification curve appears, then the sample to be tested is Cyperus rotundus; When amplifying with the said XiangF-1Pr and the four primers, if an exponential amplification curve appears, and at the same time when amplifying with the said XiangFd-1Pr and the four primers, an exponential amplification curve appears, then the sample to be tested is Cyperus rotundus and Cyperus stoloniferus, that is, Cyperus rotundus is adulterated with Cyperus stoloniferus; When amplifying with the said XiangF-1Pr and the four primers, if no exponential amplification curve appears, and at the same time when amplifying with the said XiangFd-1Pr and the four primers, no exponential amplification curve appears, then the sample to be tested is neither Cyperus rotundus nor Cyperus stoloniferus.

7. The method according to claim 5, characterized in that The conditions of the said real-time fluorescence PCR amplification reaction are: 65°C, isothermal amplification for 20 minutes.

8. The method according to claim 5, wherein In the said real-time fluorescence PCR amplification reaction system: the molar ratio of XiangF-1F1: XiangF-1B1: XiangF-1LF: XiangF-1LB: XiangF-1Pr is 8:8:2:2:5 or the molar ratio of XiangF-1F1: XiangF-1B1: XiangF-1LF: XiangF-1LB: XiangFd-1Pr is 8:8:2:2:5.

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