Method for detecting bulbus fritillariae cirrhosae medicinal material based on Taqman fluorescent probe double-marking linear correction quantitative PCR (Polymerase Chain Reaction)

Abstract

The invention discloses a primer and probe combination for detecting a bulbus fritillariae cirrhosae medicinal material based on Taqman fluorescent probe double-marking linear correction quantitative PCR (Polymerase Chain Reaction). The invention further discloses a method for detecting the bulbus fritillariae cirrhosae medicinal material based on Taqman fluorescent probe double-marking linear correction quantitative PCR, the bulbus fritillariae cirrhosae specific primers and probes designed based on six bulbus fritillariae cirrhosae-based original plant conserved gene sequences and the 18sRNA universal reference gene primers and probes are adopted, a sample to be tested and a standard reference substance are amplified at the same time in a PCR reaction system, and the specific primers and probes are used for detecting the specific genes of the bulbus fritillariae cirrhosae medicinal material based on Taqman fluorescent probe double-marking linear correction quantitative PCR. The method comprises the following steps: qualitatively confirming that a test sample contains the bulbus fritillariae cirrhosae component according to amplification results of a bulbus fritillariae cirrhosae specific primer and a probe, then taking a standard reference substance as a template to obtain two standard curves of a bulbus fritillariae cirrhosae specific gene and a 18sRNA gene, and finally quantitatively analyzing the bulbus fritillariae cirrhosae content in the test sample by adopting a double-marking linear correction method. The method can be used for efficiently, specifically and accurately detecting the ingredients and content of the bulbus fritillariae cirrhosae through PCR.

Description

Technical Field

[0001] The invention relates to the field of molecular recognition technology and application technology of traditional Chinese medicine components, and in particular to a method for detecting Fritillaria cirrhosa medicinal materials based on Taqman fluorescent probe double-label linear correction quantitative PCR. Background Art

[0002] Fritillaria chuanbei is a perennial herbaceous plant of the genus Fritillaria L. of the Liliaceae family. Its bulb has a long history as a precious Chinese medicinal material. As a commonly used precious medicinal material in traditional Chinese medicine treatment, it is the main raw material of many Chinese medicine prescriptions or Chinese patent medicine preparations for the treatment of acute tracheitis, bronchitis, tuberculosis and other diseases. It is also an important raw material for many health products. It is also often added as a medicinal food material to health-preserving and nourishing soups. Due to its shortage of resources and high prices, the market has seen the phenomenon of inferior products being sold as good ones, fake products being sold as genuine ones, and mixed with each other, which seriously affects the market order of Fritillaria chuanbei and harms the interests of consumers. Fritillaria chuanbei medicinal materials themselves contain multiple origins and are similar in appearance to other closely related Fritillaria medicinal materials, which increases the difficulty of quality control in clinical application and production and circulation. Appearance adulteration is the main adulteration method of Fritillaria chuanbei medicinal materials, and Fritillaria chuanbei is often sold as a powdered finished product. Quantitative analysis technology is urgently needed to accurately quantify the amount of adulteration in Fritillaria chuanbei products (powder).

[0003] PCR fluorescence quantitative molecular detection technology, which targets specific nucleic acid fragments, has important advantages in accurate quantitative analysis and has been proven in the quantitative detection of corn, rice, beef and other products. On the basis of conventional detection technology, the use of PCR fluorescence quantitative molecular detection technology to detect Fritillaria cirrhosa not only shows higher specificity, but also has better sensitivity.

[0004] However, the current PCR quantitative detection method for Fritillaria cirrhosa only designs detection primers and probes for a single Fritillaria cirrhosa origin, which limits its ability to detect all Fritillaria cirrhosa medicinal origins; in addition, this method only uses one standard curve for relative quantitative detection, which cannot effectively eliminate the influence of interfering factors such as different origins and sample volume differences. Summary of the invention

[0005] In view of the above problems, the present invention aims to provide a primer and probe combination for detecting Fritillaria cirrhosa medicinal materials based on Taqman fluorescent probe double-labeled linear correction quantitative PCR.

[0006] The technical solution of the present invention is as follows:

[0007] The primer and probe combination includes Fritillaria cirrhosa-specific primers and probes designed based on the conserved gene sequences of six Fritillaria cirrhosa-derived plants in the 2020 edition of the Chinese Pharmacopoeia, and primers and probes designed based on the 18sRNA universal internal reference gene sequence; wherein the Fritillaria cirrhosa-specific primers and probes are:

[0008] Upstream primer sequence CBM-YF: 5'-CAGACTGAGACATGTCGAACAAGA-3'

[0009] Downstream primer sequence CBM-YR: 5'-CCTCTCGGGTTCGCAACA-3'

[0010] Taqman fluorescent probe BMH-TP:

[0011] FAM-CTCAKATTRTTGCTTCMGAATTTRCATTATGTC-TAMRA;

[0012] The six Chuanbei-based plants include Chuanbei, dark purple Fritillaria, Gansu Fritillaria, Suosa Fritillaria, Wabu Fritillaria, and Taibai Fritillaria of the Liliaceae family;

[0013] The 18sRNA universal internal reference gene primers and probes are:

[0014] Upstream primer sequence 18sRNA-F: 5'-TCTGCCCTATCAACTTTCGATGGTA-3'

[0015] Downstream primer sequence 18sRNA-R: 5'-AATTTGCGCGCCTGCTGCCTTCCTT-3'

[0016] Taqman fluorescent probe 18sRNA-P:

[0017] FAM-CCGTTTCTCAGGCTCCCTCTCCGGAATCGAACC-TAMRA.

[0018] On the other hand, a method for detecting Fritillaria cirrhosa medicinal materials by Taqman fluorescent probe double-label linear correction quantitative PCR is also provided, using the Fritillaria cirrhosa specific primers and probes described in claim 1, and 18sRNA universal internal reference gene primers and probes, and amplifying the test sample and the standard reference substance simultaneously in a PCR reaction system, firstly qualitatively confirming that the test sample contains Fritillaria cirrhosa components based on the amplification results of the Fritillaria cirrhosa specific primers and probes, and then using the standard reference substance as a template to obtain two standard curves of the Fritillaria cirrhosa specific gene and the 18sRNA gene, and finally using the double-label linear correction method to quantitatively analyze the Fritillaria cirrhosa content in the test sample.

[0019] Preferably, the process of qualitatively confirming whether the test sample contains Fritillaria cirrhosa components based on the amplification results of Fritillaria cirrhosa-specific primers and probes is as follows: determine whether the positive control and the test sample have amplification signals when the Ct value is ≤35, and the blank control does not show an amplification signal; if so, it is qualitatively determined that the test sample contains Fritillaria cirrhosa components; when the positive control has an amplification signal at a Ct value ≤35, and the blank control and the sample to be tested do not show an amplification signal, or the sample to be tested has an amplification Ct value >35, it is qualitatively determined that the test sample does not contain Fritillaria cirrhosa components.

[0020] Preferably, the method of quantitatively detecting and analyzing the Fritillaria cirrhosa content in the test sample using the double-label linear correction method is: Fritillaria cirrhosa component content = (Fritillaria cirrhosa specific gene content in the test sample / 18sRNA gene content in the test sample) ÷ (Fritillaria cirrhosa specific gene content in the standard reference substance / 18sRNA gene content in the standard reference substance) × 100%.

[0021] Preferably, the concentrations of the Fritillaria cirrhosa-specific primers and probes, and the 18sRNA universal internal reference gene primers and probes are all 10 μmol / l.

[0022] Preferably, in the PCR reaction system, the DNA template solution of the test sample is diluted to obtain a DNA dilution solution with a concentration of 40 to 60 ng / μl.

[0023] Preferably, the PCR reaction system comprises the following components:

[0024]

[0025]

[0026] Preferably, in the PCR reaction system, the standard control substance is diluted 10 times in a gradient manner, and the concentrations of the gradient concentration templates are, from high to low, 50 ng / μl, 5 ng / μl, 0.5 ng / μl, 0.05 ng / μl, 0.005 ng / μl and 0.0005 ng / μl.

[0027] Preferably, the reaction steps of the PCR reaction system are as follows: S1, pre-denaturation treatment: heating the PCR reaction system to 95°C for 10 minutes; S2, denaturation treatment: heating the PCR reaction system to 95°C again for 15 seconds; S3, annealing stage: lowering the temperature of the PCR reaction system to between 56 and 58°C for 40 to 60 seconds, and repeating this process for 40 to 42 cycles.

[0028] The beneficial effects of the present invention are:

[0029] (1) The fluorescent quantitative PCR technology developed by the present invention using the specific gene of Fritillaria cirrhosa has strong specificity, high amplification efficiency, high accuracy and good repeatability, and can distinguish Fritillaria cirrhosa and its confused products and counterfeits, and detect products containing Fritillaria cirrhosa ingredients. The technology is based on the detection primers and probes designed based on the conserved genes of the six original plants of Fritillaria cirrhosa medicinal materials, and can effectively distinguish Fritillaria cirrhosa from non-Fritillaria cirrhosa medicinal materials, and is suitable for detecting any original component of Fritillaria cirrhosa.

[0030] (2) The present invention utilizes 18sRNA gene-specific primers and Taqman probes, combined with a double-label linear correction method, to accurately quantify the components of Fritillaria cirrhosa in a sample and achieve quantitative evaluation of the content. The 18sRNA gene is used as a housekeeping gene to compare the expression differences of the target gene in different tissue samples to ensure the stability and reliability of the results. As an endogenous control, the 18sRNA gene compensates for the variation in sample processing and ensures the accuracy of the expression of the target gene. The double-label linear correction method eliminates the interference of sample source and volume differences, and achieves accurate quantification of the Fritillaria cirrhosa content.

[0031] (3) The present invention compares the content of the Fritillaria cirrhosa component in the test sample with the standard reference substance, thereby calculating the actual content of the Fritillaria cirrhosa component in the test sample. Using the standard reference substance as a reference can reduce experimental errors, improve the accuracy of quantitative analysis, and can be used for comparison between different samples to ensure the consistency and comparability of the results.

[0032] (4) The technology of the present invention can directly perform PCR reaction on genomic DNA as a template. It is simple to operate, has good stability, strong applicability, and can be easily promoted to ordinary molecular detection institutions for use.

[0033] (5) The technology of the present invention is a powerful supplement to the technical system of authenticity identification and quantitative determination of Fritillaria cirrhosa, and will make important contributions to combating illegal counterfeiting and selling of counterfeit goods, clearing up the market order, protecting the rights and interests of consumers, and ensuring the safety of Fritillaria cirrhosa for edible and medicinal use. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0035] Figure 1 It is the specific detection profile of Taqman fluorescent probe PCR of different Fritillaria;

[0036] Figure 2 It is the amplification curve of 18sRNA universal internal reference gene in the standard control;

[0037] Figure 3 This is the amplification curve of the specific endogenous gene of Fritillaria cirrhosa in the standard reference substance;

[0038] Figure 4 It is a double-label curve diagram of the Fritillaria cirrhosa-specific endogenous gene and the 18sRNA universal internal reference gene. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and examples, but it is not intended to limit the scope of the present invention, and is only illustrative. It should be noted that, in the absence of conflict, the embodiments in this application and the technical features in the embodiments can be combined with each other. It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meanings as those of ordinary skill in the art to which this application belongs. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0040] In a specific embodiment, when detecting Fritillaria cirrhosae by quantitative PCR based on Taqman fluorescent probe double-label linear correction, the specific steps are as follows:

[0041] (1) Preparation of detection primers and Taqman fluorescent probes

[0042] Fritillaria cirrhosa-specific PCR accurate detection primers:

[0043] Upstream primer sequence CBM-YF: 5'-CAGACTGAGACATGTCGAACAAGA-3'

[0044] Downstream primer sequence CBM-YR: 5'-CCTCTCGGGTTCGCAACA-3'

[0045] Fritillaria cirrhosa-specific Taqman fluorescent probe:

[0046] BMH-TP: FAM-CTCAKATTRTTGCTTCMGAATTTRCATTATGTC-TAMRA

[0047] 18sRNA universal internal reference gene quantitative detection primers:

[0048] Upstream primer sequence 18s RNA-F: 5'-TCTGCCCTATCAACTTTCGATGGTA-3'

[0049] Downstream primer sequence 18s RNA-R: 5'-AATTTGCGCGCCTGCTGCCTTCCTT-3'

[0050] 18sRNA universal internal reference gene Taqman fluorescent probe:

[0051] 18s RNA-P: FAM-CCGTTTCTCAGGCTCCCTCTCCGGAATCGAACC-TAMRA

[0052] In this example, the concentrations of the Fritillaria cirrhosa-specific primers and probe, and the 18sRNA universal internal reference gene primers and probe were all 10 μmol / l.

[0053] The above primer pair CBM-YF / CBM-YR and Taqman fluorescent probe BMH-TP are designed based on the nucleotide sequence of the specific gene fragment in Fritillaria cirrhosa, and can achieve specific detection of Fritillaria cirrhosa and products containing Fritillaria cirrhosa. The primer pair 18s RNA-F / 18s RNA-R is designed based on the universal internal reference gene sequence of 18sRNA, and is intended to detect eukaryotic organisms. The above Taqman fluorescent probes and corresponding primer pairs can be used in combination.

[0054] (2) Preparation of DNA template solution

[0055] DNA extracted from samples of the Liliaceae plants Chuan Fritillaria (also known as Fritillaria cirrhosa), Suosa Fritillaria, Dark Purple Fritillaria, Wabu Fritillaria, Gansu Fritillaria, Taibai Fritillaria, Zhejiang Fritillaria, Ping Fritillaria, Hubei Fritillaria, Iberian Fritillaria, Tu Fritillaria, and Guangcigu was diluted to obtain a DNA dilution solution with a concentration of 50 ng / μl, which was used as the test sample. The DNA solution extracted from the standard reference substance (Dark Purple Fritillaria) (purchased from the China Food and Drug Inspection Institute) was used as a positive control, and the extraction blank and sterilized pure water (H2O) were used as blank controls.

[0056] (3) Preparation of gradient concentration template of standard reference substance (dark purple Fritillaria)

[0057] The standard curve working solution containing 5 different concentration points was prepared by diluting the standard control DNA solution by 10 times. These concentration points were arranged in descending order: 50ng / μl, 5ng / μl, 0.5ng / μl, 0.05ng / μl, 0.005ng / μl and 0.0005ng / μl, and each concentration gradient was set up with 3 parallel samples.

[0058] (4) Preparation of PCR reaction system

[0059] Add the prepared DNA dilution into the reaction system.

[0060] The Fritillaria cirrhosa specific detection reaction system includes the following components:

[0061]

[0062] The 18sRNA gene detection reaction system includes the following components:

[0063]

[0064] In this example, two qPCR Master Mix reagents were used: 2× Taq Prob qPCR Master Mix (ABI, Canada) and 2× qPCR Master Mix (Toyobo Co., Ltd., Osaka, Japan). Any one of the reagents can meet the experimental requirements. The choice of reagents should be based on the specific requirements of the experimental instrument. At the same time, the PCR reaction system also needs to be appropriately adjusted according to the type of reagents selected.

[0065] (5) PCR amplification

[0066] The PCR reaction system was heated to 95°C for 10 minutes; then the PCR reaction system was heated to 95°C again for 15 seconds; finally, the temperature of the PCR reaction system was lowered to 57°C for 60 seconds, and this process was repeated 40 cycles.

[0067] In this example, a fluorescence quantitative PCR instrument (ABI applied Biosystems 7500 instrument) produced by ABI was used.

[0068] (6) Implementation results

[0069] Determination of the qualitative results of Fritillaria cirrhosa: The Fritillaria cirrhosa-specific primer set and probe are used to simultaneously amplify the test sample and the standard control (dark purple Fritillaria). If the positive control and the sample to be tested have an amplification signal when the Ct value is ≤35, and the blank control has no amplification signal, it can be determined that the tested sample contains Fritillaria cirrhosa components; on the contrary, if the positive control has an amplification signal when the Ct value is ≤35, and the blank control and the sample to be tested have no amplification signal, or the amplification Ct value of the sample to be tested is >35, it can be determined that the tested sample does not contain Fritillaria cirrhosa components.

[0070] Determination of the quantitative results of Fritillaria cirrhosa: The Fritillaria cirrhosa specific primer set and probe and the 18sRNA primer set and probe were used to amplify the test sample and the standard reference substance (dark purple Fritillaria cirrhosa) simultaneously. Using the standard reference substance as a template, two standard curves of the Fritillaria cirrhosa specific gene and the 18sRNA gene were obtained. The slope of the standard curve obtained by the Fritillaria cirrhosa specific primer set and probe was between -3.134 and -3.095, and the correlation coefficient R 2 The slope of the standard curve obtained by the 18sRNA gene amplification primer set and probe is between -3.260 and -3.104, and the correlation coefficient R 2The amplification efficiency is greater than 0.98, and the amplification efficiency is within the range of 90% to 110%. The content of Fritillaria cirrhosa in the test sample was quantitatively analyzed by double-standard linear calibration. The Fritillaria cirrhosa components were quantitatively estimated for the test sample and the standard reference substance (dark purple Fritillaria cirrhosa) according to the standard curves of the 18sRNA gene and the Fritillaria cirrhosa-specific gene. The content of Fritillaria cirrhosa components in the test sample was calculated according to the following formula:

[0071] Fritillaria cirrhosa component content = (Fritillaria cirrhosa specific gene content in the test sample / 18sRNA gene content in the test sample) ÷ (Fritillaria cirrhosa specific gene content in the standard reference substance / 18sRNA gene content in the standard reference substance) × 100%.

[0072] In this embodiment, the Fritillaria cirrhosa specific primer set and Taqman probe in the present invention have no amplification signal in the blank control, and there is a strong amplification fluorescence signal in the original species of Fritillaria cirrhosa such as dark purple Fritillaria, Gansu Fritillaria, Wabu Fritillaria, curled leaf Fritillaria, Taibai Fritillaria, and Suosha Fritillaria, with strong specificity, Ct average value of 20 to 26, and threshold limit of 0.01704, which is judged as positive. However, in other closely related Fritillaria such as Zhejiang Fritillaria, Ping Fritillaria, Hubei Fritillaria, Yi Fritillaria, Tu Fritillaria, Guang Cigu and non-Fritillaria DNA, there is an extremely weak amplification signal (Ct value>35) or no amplification signal. Among them, the amplification Ct value in Yi Fritillaria is more than 35.23, and the amplification Ct value in the samples of Zhejiang Fritillaria, Ping Fritillaria, and Hubei Fritillaria is greater than 38, which is a non-specific target amplification (see Table 1), and there is no amplification signal in the samples of Tu Fritillaria and Guang Cigu, and they are all judged as negative. The specific detection results of Taqman fluorescent probe PCR for dark purple Fritillaria, Gansu Fritillaria, Wabu Fritillaria, curled leaf Fritillaria, Taibai Fritillaria, Sophora flavescens, Zhejiang Fritillaria, Ping Fritillaria, Hubei Fritillaria, Iberia Fritillaria, Tu Fritillaria, and Guangcigu are shown as follows Figure 1 According to the Ct value, the Fritillaria cirrhosae medicinal materials and non-Fritillaria cirrhosae medicinal materials can be clearly distinguished, and the Fritillaria cirrhosae-specific primer and probe combination has good specificity.

[0073] Table 1 Specificity of primers and Taqman fluorescent probe PCR detection system of Fritillaria cirrhosa

[0074]

[0075] Furthermore, the double-label linear calibration method was used to quantitatively analyze the content of Fritillaria cirrhosa components in the samples. The 53 ng / μl Fritillaria cirrhosa standard reference DNA template was diluted 10 times with sterile double distilled water, and 2 μl was taken for fluorescence PCR detection. The template content of each concentration gradient in the reaction system was from high to low: 106, 21.2, 4.24, 0.85, 0.17, and 0.03 ng, respectively, and 3 parallel experiments were performed for each gradient. The standard reference (dark purple Fritillaria) was amplified simultaneously using the Fritillaria cirrhosa-specific primer set and probe as well as the 18sRNA primer set and probe. Figure 2The figure shows the amplification curve of the 18sRNA universal internal reference gene in the standard control; Figure 3 The graph shows the amplification curve of the specific endogenous gene of Fritillaria cirrhosa in the standard reference substance. The Ct value of the obtained 18sRNA gene amplification was linearly fitted, as shown in FIG. Figure 4 The double-label curve of the specific endogenous gene of Fritillaria cirrhosa and the universal internal reference gene of 18sRNA is shown. The standard curve shows an obvious linear growth and a good linear relationship: y = -3.260x + 23.924, R 2 =0.999. The relative standard deviation RSD was <0.7%, with good repeatability and an amplification efficiency of 102.642%. Similarly, the amplification efficiency of the specific endogenous gene of Fritillaria cirrhosa was 110.00. The linear fit was high, with linear growth and good linear relationship: y=-3.095x+29.872, R 2 =0.998. The standard reference substance (dark purple Fritillaria) was quantitatively estimated based on the standard curves of the 18sRNA gene and the Fritillaria cirrhosa specific gene. The content of the Fritillaria cirrhosa component in the test sample was calculated according to the following formula:

[0076] Fritillaria cirrhosa component content = (Fritillaria cirrhosa specific gene content in the test sample / 18sRNA gene content in the test sample) ÷ (Fritillaria cirrhosa specific gene content in the standard reference substance / 18sRNA gene content in the standard reference substance) × 100%.

[0077] Fritillaria cirrhosa and other Fritillaria bulb powders were mixed in a 1:1 ratio to prepare a test sample (Fritillaria cirrhosa content 50%), numbered Test1, and quantitatively detected by the method described in the present invention. The results are shown in Table 2. The 18sRNA gene and the Fritillaria cirrhosa-specific endogenous gene BMH of the Test1 sample were amplified normally, and the Ct average values ​​were 24.11 and 28.64, respectively. In this test, the standard curves of the 18sRNA gene and the Fritillaria cirrhosa-specific gene were quantitatively estimated to be 216.520ng and 141.921ng for the positive control (dark purple Fritillaria), respectively, and correspondingly, the values ​​for the test samples were 73.19ng and 22.15ng, respectively. According to the above formula, the Fritillaria cirrhosa content in the Test1 sample was 46.17%, which was 3.83 percentage points different from the theoretical value, and the relative deviation was 7.66%. The deviation rate of the test result is much less than the internationally recognized value of 25%, and the reliability is good.

[0078] Table 2 Taqman fluorescent probe double-label linear calibration quantitative PCR test results

[0079]

[0080] In summary, the present invention achieves high specificity and accurate quantitative analysis of Fritillaria cirrhosa by designing specific primers and probes and introducing double-label linear correction technology. It is simple, easy to operate and easy to promote, effectively promotes market supervision and consumer protection, and makes significant contributions to the standardization of Fritillaria cirrhosa detection technology and industry progress.

[0081] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A primer and probe combination for detecting Fritillaria cirrhosae by Taqman fluorescent probe double-label linear correction quantitative PCR, characterized in that: Including Fritillaria cirrhosa-specific primers and probes designed based on the conserved gene sequences of six Fritillaria cirrhosa-based plants in the 2020 edition of the Chinese Pharmacopoeia, and primers and probes designed based on the 18sRNA universal internal reference gene sequence; wherein the Fritillaria cirrhosa-specific primers and probes are: Upstream primer sequence CBM-YF: 5'-CAGACTGAGACATGTCGAACAAGA-3' Downstream primer sequence CBM-YR: 5'-CCTCTCGGGTTCGCAACA-3' Taqman fluorescent probe BMH-TP: FAM-CTCAKATTRTTGCTTCMGAATTTRCATTATGTC-TAMRA; The six Chuanbei-based plants include Chuanbei, dark purple Fritillaria, Gansu Fritillaria, Suosa Fritillaria, Wabu Fritillaria, and Taibai Fritillaria of the Liliaceae family; The 18sRNA universal internal reference gene primers and probes are: Upstream primer sequence 18s RNA-F: 5'-TCTGCCCTATCAACTTTCGATGGTA-3' Downstream primer sequence 18s RNA-R: 5'-AATTTGCGCGCCTGCTGCCTTCCTT-3' Taqman fluorescent probe 18s RNA-P: FAM-CCGTTTCTCAGGCTCCCTCTCCGGAATCGAACC-TAMRA.

2. A method for detecting Fritillaria cirrhosae Bulbs based on Taqman fluorescent probe double-label linear correction quantitative PCR, characterized in that: The Fritillaria cirrhosa-specific primers and probes as described in claim 1, and the 18sRNA universal internal reference gene primers and probes are used to simultaneously amplify the test sample and the standard reference substance in a PCR reaction system. First, it is qualitatively confirmed that the test sample contains Fritillaria cirrhosa components based on the amplification results of the Fritillaria cirrhosa-specific primers and probes. Then, two standard curves of the Fritillaria cirrhosa-specific gene and the 18sRNA gene are obtained using the standard reference substance as a template. Finally, a double-label linear correction method is used to quantitatively analyze the Fritillaria cirrhosa content in the test sample.

3. The method for detecting Fritillaria cirrhosae by Taqman fluorescent probe double-label linear correction quantitative PCR according to claim 2, characterized in that: The process of qualitatively confirming whether the test sample contains Fritillaria cirrhosa components based on the amplification results of Fritillaria cirrhosa-specific primers and probes is as follows: determine whether the positive control and the test sample have amplification signals when the Ct value is ≤35, and the blank control has no amplification signal. If so, it is qualitatively determined that the test sample contains Fritillaria cirrhosa components; when the positive control has an amplification signal at a Ct value ≤35, and the blank control and the sample to be tested have no amplification signal, or the sample to be tested has an amplification Ct value >35, it is qualitatively determined that the test sample does not contain Fritillaria cirrhosa components.

4. The method for detecting Fritillaria cirrhosae by Taqman fluorescent probe double-label linear correction quantitative PCR according to claim 2, characterized in that: The method for quantitatively analyzing the content of Fritillaria cirrhosa in the test samples using the double-standard linear correction method is: Fritillaria cirrhosa component content = (Fritillaria cirrhosa specific gene content in the test sample / 18sRNA gene content in the test sample) ÷ (Fritillaria cirrhosa specific gene content in the standard reference substance / 18sRNA gene content in the standard reference substance) × 100%.

5. The method for detecting Fritillaria cirrhosae Bulbs by Taqman fluorescent probe double-label linear correction quantitative PCR according to claim 2, characterized in that: The concentrations of the Fritillaria cirrhosa-specific primers and probes, and the 18sRNA universal internal reference gene primers and probes are all 10 μmol / l.

6. The method for detecting Fritillaria cirrhosae by Taqman fluorescent probe double-label linear correction quantitative PCR according to claim 2, characterized in that: In the PCR reaction system, the DNA template solution of the test sample is diluted to obtain a DNA dilution solution with a concentration of 40 to 60 ng / μl.

7. The method for detecting Fritillaria cirrhosae by Taqman fluorescent probe double-label linear correction quantitative PCR according to claim 6, characterized in that: The PCR reaction system includes the following components:

8. The method for detecting Fritillaria cirrhosae Bulbs by Taqman fluorescent probe double-label linear correction quantitative PCR according to claim 2, characterized in that: In the PCR reaction system, the standard control substance was diluted 10 times in a gradient manner, and the concentrations of the gradient concentration templates were, from high to low, 50 ng / μl, 5 ng / μl, 0.5 ng / μl, 0.05 ng / μl, 0.005 ng / μl and 0.0005 ng / μl.

9. The method for detecting Fritillaria cirrhosae Bulbs based on Taqman fluorescent probe double-label linear correction quantitative PCR according to claim 2, characterized in that: The reaction steps of the PCR reaction system are as follows: S1, pre-denaturation treatment: heating the PCR reaction system to 95°C for 10 minutes; S2, denaturation treatment: heating the PCR reaction system to 95°C again for 15 seconds; S3, annealing stage: lowering the temperature of the PCR reaction system to between 56 and 58°C for 40 to 60 seconds, and repeating this process for 40 to 42 cycles.

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