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 method for detecting a bulbus fritillariae cirrhosae medicinal material based on Taqman fluorescent probe double-marking linear correction quantitative PCR (Polymerase Chain Reaction), which comprises the following steps: amplifying a test sample and a standard reference substance in a PCR reaction system based on bulbus fritillariae cirrhosae specific primers and probes designed on the basis of six bulbus fritillariae cirrhosae-based original plant conserved gene sequences, and 18sRNA universal reference gene primers and probes; the method comprises the following steps: qualitatively determining whether a test sample contains the bulbus fritillariae cirrhosae component or not according to amplification results of a bulbus fritillariae cirrhosae specific primer and a probe, 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 percentage content of the bulbus fritillariae cirrhosae component of the test sample by adopting a double-marking linear correction method. According to the method, the bulbus fritillariae cirrhosae components can be accurately and reliably quantified, bulbus fritillariae cirrhosae and non-bulbus fritillariae cirrhosae medicinal materials are effectively identified, the detection universality of all certified bulbus fritillariae cirrhosae bases specified by pharmacopeia is ensured, and missing detection or false detection is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular recognition technology and application of traditional Chinese medicine ingredients, and particularly relates to a method for detecting Fritillaria cirrhosa Bulbus based on Taqman fluorescent probe double-standard linear correction quantitative PCR. BACKGROUND

[0002] Fritillaria cirrhosa Bulbus is a perennial herb of the genus Fritillaria L. in the Liliaceae family. Its bulb has a long history as a precious traditional Chinese medicine. It is a precious medicine commonly used in traditional Chinese medicine treatment and is the main raw material of many traditional Chinese medicine prescriptions or Chinese patent medicine preparations for treating acute bronchitis, bronchitis, tuberculosis and other diseases. It is also an important raw material for many health products and is often added to health tonifying soup as a medicinal food material. Due to its resource shortage and high price, there are phenomena of using inferior goods as good ones, confusing true and false, and mixing in the market, which seriously affects the market order of Fritillaria cirrhosa Bulbus and damages the interests of consumers. Fritillaria cirrhosa Bulbus itself contains multiple origins, and its appearance is similar to other related Fritillaria Bulbus, which increases the difficulty of quality control in clinical application and production circulation. Counterfeiting is the main counterfeiting method of Fritillaria cirrhosa Bulbus, and Fritillaria cirrhosa Bulbus is often sold as a powder product, and there is an urgent need for quantitative analysis technology to accurately quantify the amount of adulteration and mixing in Fritillaria cirrhosa Bulbus products (powder).

[0003] PCR fluorescent quantitative molecular detection technology with specific nucleic acid fragments as targets has important advantages in precise quantitative analysis and has been applied to the quantitative detection of corn, rice, beef and other products. Based on conventional detection technology, PCR fluorescent quantitative molecular detection technology is used to detect Fritillaria cirrhosa Bulbus, which not only shows higher specificity, but also has better sensitivity.

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

[0005] The prior art disclosed in the prior art with the publication number CN108707686A discloses a PCR detection method based on Fritillaria cirrhosa specific endogenous genes. The method uses the cloned Fritillaria cirrhosa species-specific endogenous gene sequence as the basis to design specific primer pairs BMH-YF and BMH-YR, and establishes a high-specificity PCR detection technology system for Fritillaria cirrhosa identification. However, this technical solution still has significant defects: first, this method can only realize qualitative detection, and cannot quantitatively analyze the Fritillaria cirrhosa components, making it difficult to meet the market demand for precise control of Fritillaria cirrhosa content; second, this method relies on electrophoresis detection of PCR products, which is complicated and susceptible to non-specific amplification products, resulting in reduced accuracy of result interpretation; in addition, it cannot eliminate the influence of factors such as differences in sample DNA extraction efficiency on the detection results, and the detection stability and reliability are insufficient.

[0006] Therefore, the applicant has designed a method for detecting Fritillaria cirrhosa medicinal materials based on Taqman fluorescent probe double-standard linear correction quantitative PCR (patent publication number CN119464537A, hereinafter referred to as the "prior scheme"). The prior scheme is based on the principle of TaqMan fluorescent probe quantitative PCR technology, and designs Fritillaria cirrhosa specific primers and TaqMan probes, as well as 18sRNA internal standard primers and probes. The test sample and standard control are amplified simultaneously in the PCR reaction system, and the Fritillaria cirrhosa content is qualitatively and quantitatively calculated based on the amplification results, in an attempt to solve the above problems. However, upon inspection, the prior scheme cannot achieve the technical effects claimed, and cannot solve the above problems. The main reason is that the probe position does not overlap with the primer amplification region, and the fluorescence signal cannot be generated by Taq enzyme cutting.

[0007] Therefore, it is necessary to adjust the prior scheme in order to achieve the corresponding purpose and solve the above technical problems. SUMMARY

[0008] In view of the above problems, the present application aims to provide a method for detecting Fritillaria cirrhosa medicinal materials based on Taqman fluorescent probe double-standard linear correction quantitative PCR and corresponding primer and probe combinations. This method can realize high-specificity identification, precise quantitative analysis and universal detection of all genuine base materials of Fritillaria cirrhosa medicinal materials, avoiding missed detection or false detection, and improving the accuracy and reliability of the detection.

[0009] The technical solution of the present application is as follows:

[0010] The application provides a primer and probe combination for detecting Fritillaria cirrhosa Bulbus based on Taqman fluorescent probe double-mark linear correction quantitative PCR, which comprises Fritillaria cirrhosa Bulbus specific primers and probes designed based on the conservative gene sequence (NCBI Genbank No. MN151370.1) of six Fritillaria cirrhosa Bulbus original plants (Fritillaria cirrhosa Bulbus, Fritillaria unibracteata Bulbus, Fritillaria przewalskii Bulbus, Fritillaria megalophylla Bulbus, Fritillaria walujewii Bulbus and Fritillaria taipaiensis Bulbus) stipulated in the 2020 edition of Chinese Pharmacopoeia, and primers and probes designed based on the 18sRNA universal internal reference gene sequence; wherein the Fritillaria cirrhosa Bulbus specific primers and probes are:

[0011] the upstream primer sequence CBM-YF is 5'-CAGACTGAGACATGTCGAACAAGA-3'

[0012] the downstream primer sequence CBM-YR is 5'-CCTCTCGGGTTCGCAACA-3'

[0013] the Taqman fluorescent probe CBM-YP is FAM-ACCAACCGTGGGAGATTACCATCACAAA-TAMRA.

[0014] The upstream primer corresponds to the 142-165th position of the target sequence, the downstream primer corresponds to the 196-213th position, and the probe corresponds to the 170-197th position. The position of the probe used is in the amplification region of the upstream and downstream primers, and real-time fluorescent quantitative PCR can be realized.

[0015] The six Fritillaria cirrhosa Bulbus original plants include Fritillaria cirrhosa Bulbus, Fritillaria unibracteata Bulbus, Fritillaria przewalskii Bulbus, Fritillaria megalophylla Bulbus, Fritillaria walujewii Bulbus and Fritillaria taipaiensis Bulbus;

[0016] The 18sRNA universal internal reference gene primers and probes are:

[0017] the upstream primer sequence 18s RNA-F is 5'-TCTGCCCTATCAACTTTCGATGGTA-3'

[0018] the downstream primer sequence 18s RNA-R is 5'-AATTTGCGCGCCTGCTGCCTTCCTT-3'

[0019] the Taqman fluorescent probe 18s RNA-P is:

[0020] FAM-CCGTTTCTCAGGCTCCCTCTCCGGAATCGAACC-TAMRA.

[0021] In another aspect, the application provides a method for detecting Fritillaria cirrhosa by Taqman fluorescent probe double-standard linear correction quantitative PCR, which adopts the above-mentioned Fritillaria cirrhosa specific primers and probes, and 18sRNA universal internal reference gene primers and probes, and simultaneously amplifies the test sample and the standard control in the PCR reaction system. First, it is determined whether the test sample contains Fritillaria cirrhosa components according to the amplification results of the Fritillaria cirrhosa specific primers and probes, and then two standard curves of the Fritillaria cirrhosa specific gene and the 18sRNA gene are obtained by taking the standard control as a template, and the double-standard linear correction method is used to quantitatively analyze the content of Fritillaria cirrhosa in the test sample.

[0022] Preferably, the process of determining whether the test sample contains Fritillaria cirrhosa components according to the amplification results of the Fritillaria cirrhosa specific primers and probes is as follows: it is determined whether the positive control and the test sample have amplification signals when the Ct value is less than or equal to 35, and the blank control does not have amplification signals; if yes, it is determined that the test sample contains Fritillaria cirrhosa components; if the positive control has amplification signals when the Ct value is less than or equal to 35, and the blank control and the test sample do not have amplification signals, or the test sample has amplification signals when the Ct value is greater than 35, it is determined that the test sample does not contain Fritillaria cirrhosa components.

[0023] Preferably, the method for quantitatively analyzing the content of Fritillaria cirrhosa in the test sample by the double-standard linear correction method is as follows: the content of Fritillaria cirrhosa components = (the content of the Fritillaria cirrhosa specific gene in the test sample / the content of the 18sRNA gene in the test sample) ÷ (the content of the Fritillaria cirrhosa specific gene in the standard control / the content of the 18sRNA gene in the standard control) × 100%; the double-standard linear correction method includes: taking the standard control as a template, respectively amplifying the Fritillaria cirrhosa specific gene and the 18sRNA gene, and drawing two standard curves; according to the amplification results of the two genes in the test sample, the respective contents are calculated through the standard curves, and the relative content is calculated according to the above formula. The percentage content of the Fritillaria cirrhosa components in the test sample = the Fritillaria cirrhosa characteristic value of the test sample ÷ (the content of the Fritillaria cirrhosa specific gene in the standard control / the content of the 18sRNA gene in the standard control) × 100%; the Fritillaria cirrhosa characteristic value of the test sample = (the content of the Fritillaria cirrhosa specific gene in the test sample / the content of the 18sRNA gene in the test sample).

[0024] Preferably, the concentration of the Fritillaria cirrhosa specific primers and probes and the 18sRNA universal internal reference gene primers and probes is 10 μmol / L.

[0025] Preferably, in the PCR reaction system, the DNA template solution of the test sample is diluted to obtain a DNA diluent with a concentration of 40-60 ng / μl.

[0026] Preferably, the PCR reaction system comprises the following components:

[0027]

[0028] As preferred, the reaction steps of the PCR reaction system are as follows: S1, pre-denaturation treatment: heating the PCR reaction system to 95℃ for 10 minutes; S2, denaturation treatment: heating the PCR reaction system to 95℃ again for 15 seconds; S3, annealing stage: reducing the temperature of the PCR reaction system to between 56 and 58℃ for 40 to 60 seconds, and repeating the denaturation and annealing process for 40 to 42 cycles.

[0029] The beneficial effects of the present application are:

[0030] (1) The method of the present application uses specific primers and Taqman probes of endogenous genes and 18sRNA genes of Fritillaria cirrhosa, combined with double-standard linear correction method, to accurately quantify the Fritillaria cirrhosa components in the sample, and realize the quantitative evaluation of the content. The method for detecting Fritillaria cirrhosa medicinal materials of the present application determines the percentage content of Fritillaria cirrhosa components based on the double-standard linear correction method (target gene / internal reference gene ratio), that is, (Fritillaria cirrhosa specific gene content of the test sample / 18sRNA gene content of the test sample) ÷ (Fritillaria cirrhosa specific gene content of the standard control / 18sRNA gene content of the standard control) × 100%, which further eliminates the errors from three sources of "original diversity", "matrix inhibition" and "extraction efficiency difference", significantly improves the quantitative accuracy and reliability of the evaluation results, and is suitable for precise detection of complex samples (such as powders and traditional Chinese medicines).

[0031] The method of the present application uses standard controls as references, which can reduce experimental errors, improve the accuracy of quantitative analysis, and can be used for comparison between different samples, ensuring the consistency and comparability of the results.

[0032] (2) The method of the present application is a fluorescence quantitative PCR technology developed by using Fritillaria cirrhosa specific genes, which has strong specificity, high amplification efficiency, high accuracy and good repeatability, can distinguish Fritillaria cirrhosa and its confused and fake products, and can detect products containing Fritillaria cirrhosa components. The technology is based on detection primers and probes designed based on the conserved genes of six original plants of Fritillaria cirrhosa medicinal materials, which can effectively identify Fritillaria cirrhosa and non-Fritillaria cirrhosa medicinal materials, and is suitable for detecting any Fritillaria cirrhosa original component, ensuring the detection universality of all genuine Fritillaria cirrhosa originals specified in the pharmacopoeia, and avoiding missed detection or false detection.

[0033] (3) The method can directly perform PCR reaction on genomic DNA as a template, and has simple operation, good stability, strong applicability, and is easy to popularize to ordinary molecular detection institutions. The application realizes the balance of amplification efficiency and specificity by limiting the concentrations of primers and probes, the concentration of DNA template, and the annealing temperature. The application realizes the qualitative and quantitative integrated detection of Fritillaria walujewii, shortens the detection cycle to 2 hours, and does not need electrophoresis steps, and can be directly used for quality control of complex matrix samples such as Chinese patent medicines and medicinal meals, and provides a standardized technical means for cracking the adulteration behavior of Fritillaria walujewii.

[0034] The method is a powerful supplement to the identification and quantitative determination technology system of Fritillaria walujewii, and will make important contributions to cracking the illegal behaviors of making and selling fake products, clearing the market order, protecting the rights and interests of consumers, and ensuring the edible and medicinal safety of Fritillaria walujewii. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0036] Figure 1 Taqman fluorescent probe PCR specific detection map for different Fritillaria;

[0037] Figure 2 Fritillaria walujewii specific endogenous gene amplification curve map in the standard control;

[0038] Figure 3 Fritillaria walujewii specific endogenous gene amplification curve map in the standard control;

[0039] Figure 4 Fritillaria walujewii specific endogenous gene amplification curve map in the standard control; DETAILED DESCRIPTION

[0040] The present application will be further described below in combination with the drawings and examples, but is not limited to the scope of the present application, and is only illustrative. It should be noted that the embodiments in the present application and the technical features in the embodiments can be combined with each other without conflict. It should be noted that all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs, unless otherwise specified. The experimental methods used in the following examples are conventional methods, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0041] In one specific embodiment, when detecting Fritillaria cirrhosa by Taqman fluorescent probe double-label linear correction quantitative PCR, the specific steps are as follows:

[0042] (1) Preparation of detection primers and Taqman fluorescent probe

[0043] Fritillaria cirrhosa specific PCR precise detection primer:

[0044] Upstream primer sequence CBM-YF: 5'-CAGACTGAGACATGTCGAACAAGA-3'

[0045] Downstream primer sequence CBM-YR: 5'-CCTCTCGGGTTCGCAACA-3'

[0046] Fritillaria cirrhosa specific Taqman fluorescent probe:

[0047] CBM-YP: FAM-ACCAACCGTGGGAGATTACCATCACAAA-TAMRA.

[0048] 18sRNA universal internal reference gene quantitative detection primer:

[0049] Upstream primer sequence 18s RNA-F: 5'-TCTGCCCTATCAACTTTCGATGGTA-3'

[0050] Downstream primer sequence 18s RNA-R: 5'-AATTTGCGCGCCTGCTGCCTTCCTT-3'

[0051] 18sRNA universal internal reference gene Taqman fluorescent probe:

[0052] 18s RNA-P: FAM-CCGTTTCTCAGGCTCCCTCTCCGGAATCGAACC-TAMRA

[0053] The concentrations of the Fritillaria cirrhosa specific primers and probes, and the 18sRNA universal internal reference gene primers and probes in this embodiment are all 10 μmol / L. The 18sRNA universal internal reference gene is suitable for plant-derived samples.

[0054] The primer pair CBM-YF / CBM-YR and the Taqman fluorescent probe CBM-YP 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 components. The primer pair 18s RNA-F / 18s RNA-R is designed based on the sequence of the universal internal reference gene 18sRNA, and is intended for detection of eukaryotes. The above Taqman fluorescent probes can be used in combination with the corresponding primer pairs, respectively.

[0055] (2) Preparation of DNA template solution

[0056] DNA extracted from samples of lily family plants Fritillaria cirrhosa D. Don (also known as Fritillaria delavayi Franch), Fritillaria meusii, Fritillaria unibracteata, Fritillaria walujewii, Fritillaria przewalskii, Fritillaria taipaiensis, Fritillaria thunbergii Miq, Fritillaria pallidiflora, Fritillaria hupehensis, Fritillaria iuliana, Fritillaria davidii, and Fritillaria unibracteata var. wabuensis were diluted to obtain a DNA dilution solution with a concentration of 50 ng / μl, which was used as the test sample. DNA extracted from a standard control (Fritillaria atropurpurea, purchased from the China Institute for Food and Drug Control) was used as the positive control, and blank extraction and sterilized pure water (H2O) were used as the blank controls.

[0057] (3) Preparation of standard control (Fritillaria atropurpurea) gradient concentration templates

[0058] In some embodiments, after the standard control DNA solution was gradient diluted in the PCR reaction system, the Fritillaria cirrhosa specific primers and probes, and the 18sRNA primers and probes were used to perform PCR amplification reactions simultaneously. In this embodiment, the standard control DNA solution was gradient diluted by 5 times to prepare a standard curve working solution containing 6 different concentration points. Three parallel samples were prepared for each concentration gradient.

[0059] (4) Preparation of PCR reaction system

[0060] The prepared DNA dilution solution was added to the reaction system.

[0061] The Fritillaria cirrhosa specific detection reaction system included the following components:

[0062]

[0063] The 18sRNA gene detection reaction system included the following components:

[0064]

[0065] In this embodiment, two qPCR Master Mix reagents were used: 2x TaqProb qPCR Master Mix (ABI, Canada) and 2x qPCR Master Mix (Toyobo Co., Ltd., Osaka, Japan). Either reagent can optionally be used to meet the experimental requirements. The selection of reagents should be based on the specific requirements of the experimental instrument, and the PCR reaction system also needs to be adjusted according to the category of the selected reagent. The qPCR Master Mix (2x) should include: HotStart DNA polymerase ≥ 0.05 U / μl, dNTPs ≥ 0.4 mM, MgCl2≥ 3 mM, stabilizer and optimized buffer. It is recommended to use ABI TaqProb qPCR Master Mix or equivalent reagents. When using non-specified reagents, the Mg 2+ concentration and annealing temperature and the amplification efficiency is verified by experiment to be 90%~110%.

[0066] (5) PCR amplification

[0067] 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 reduced to 57°C for 60 seconds, and the process was repeated for 40 cycles.

[0068] In this embodiment, the fluorescent quantitative PCR instrument produced by ABI Company (ABI applied Biosystems 7500 instrument) was used.

[0069] (6) Implementation results

[0070] Crown Fritillary qualitative result judgment: The test sample and standard control (dark purple Fritillary) were amplified at the same time using the crown Fritillary specific primer group and probe. If there is an amplification signal in the positive control and the sample to be detected at Ct value ≤ 35, while the blank control does not appear amplification signal, it can be determined that the sample contains crown Fritillary component; on the contrary, the positive control has amplification signal at Ct value ≤ 35, while the blank control and the sample to be detected do not appear amplification signal, or the amplification Ct value of the sample to be detected is > 35, it can be determined that the sample does not contain crown Fritillary component.

[0071] Crown Fritillary quantitative result judgment: The test sample and standard control (dark purple Fritillary) were amplified at the same time using the crown Fritillary specific primer group and probe and the 18sRNA primer group and probe. The standard control was used as a template to obtain two standard curves of the crown Fritillary specific gene and the 18sRNA gene. The standard curve slope of the crown Fritillary specific primer group and probe is between -3.134 and -3.095, the correlation coefficient R 2greater than 0.98, and the amplification efficiency is in the range of 90% to 110%. The standard curve slope of the 18sRNA gene amplification primer group and the probe is between -3.260 and -3.104, and the correlation coefficient R is greater than 0.99. 2 greater than 0.98, and the amplification efficiency is in the range of 90% to 110%. The double-standard linear correction method is used for quantitative analysis, the Ct value of the gradient dilution solution of the standard control product is used as the ordinate (Y axis), the logarithmic value (log [template amount (ng)]) of the DNA template amount of the Fritillaria cirrhosa specific gene (or 18sRNA gene) added to the reaction system is used as the abscissa (X axis), the least square method is used for linear regression fitting, and the standard curve equation Y=aX+b is obtained.

[0072] Further, the content of the Fritillaria cirrhosa component in the test sample is calculated according to the following formula: 10 [template amount (ng)] as the abscissa (X axis), the least square method is used for linear regression fitting, and the standard curve equation Y=aX+b is obtained.

[0073] Further, the content of the Fritillaria cirrhosa component in the test sample is calculated according to the following formula:

[0074] The content of the Fritillaria cirrhosa component = (the content of the Fritillaria cirrhosa specific gene in the test sample / the content of the 18sRNA gene in the test sample) ÷ (the content of the Fritillaria cirrhosa specific gene in the standard control product / the content of the 18sRNA gene in the standard control product) x 100%.

[0075] In order to more clearly illustrate the quantitative analysis process, the present application divides the quantitative calculation of the Fritillaria cirrhosa component in the test sample into two steps: first, the 'Fritillaria cirrhosa characteristic value' is defined as the normalized ratio of the expression amount of the Fritillaria cirrhosa specific gene and the internal reference gene in the test sample, and the calculation formula is: the Fritillaria cirrhosa characteristic value = the content of the Fritillaria cirrhosa specific gene in the test sample / the content of the 18sRNA gene in the test sample;

[0076] Secondly, the characteristic value of the test sample is corrected based on the normalized ratio of the standard control product, and the percentage content of the Fritillaria cirrhosa component in the test sample is obtained, and the calculation formula is: the percentage content of the Fritillaria cirrhosa component in the test sample = (the Fritillaria cirrhosa characteristic value of the test sample) ÷ (the content of the Fritillaria cirrhosa specific gene in the standard control product / the content of the 18sRNA gene in the standard control product) x 100%.

[0077] The two-step calculation is completely equivalent to the original formula 'Bulbus Fritillariae Cirrhosae component content = (test sample Bulbus Fritillariae Cirrhosae specific gene content / test sample 18sRNA gene content) ÷ (standard control Bulbus Fritillariae Cirrhosae specific gene content / standard control 18sRNA gene content) x 100%', which is only a mathematical form of splitting, aiming to more intuitively reflect the double-standard linear correction logic of 'eliminating sample differences by internal reference gene normalization → eliminating systematic errors by standard control correction'. The gene content refers to the DNA concentration (ng / μl) calculated by the standard curve, wherein the standard curve takes the gradient concentration of the standard control as the abscissa and the Ct value as the ordinate, and the least square method is used for fitting.

[0078] In this embodiment, the Bulbus Fritillariae Cirrhosae specific primer set and Taqman probe in the present application have no amplification signal in the blank control, have strong amplification fluorescence signal in Bulbus Fritillariae Cirrhosae base stock such as F. unibracteata, F. przewalskii, F. walujewii, F. delavayi, F. taipaiensis and F. megalophylla, have strong specificity, the average Ct value is 20-26, the threshold limit is 0.01704, and it is determined to be positive. In other near-source Fritillariae and non-Fritillariae DNA such as F. thunbergii, F. delavayi, F. hupehensis, F. iulianae, F. ussuriensis and F. decipiens, there is very weak amplification signal (Ct value > 35) or no amplification signal. The amplification Ct value in F. iulianae is more than 35.23, the amplification Ct value in F. thunbergii, F. delavayi and F. hupehensis is more than 38, which is non-specific target amplification (see Table 1), there is no amplification signal in F. ussuriensis and F. decipiens, and they are all determined to be negative. The Taqman fluorescence probe PCR specificity detection map results of F. unibracteata, F. przewalskii, F. walujewii, F. delavayi, F. taipaiensis, F. megalophylla, F. thunbergii, F. delavayi, F. hupehensis, F. iulianae, F. ussuriensis and F. decipiens are shown in Table 1. Figure 1 According to the Ct value, the Bulbus Fritillariae Cirrhosae medicinal material can be obviously distinguished from the non-Bulbus Fritillariae Cirrhosae medicinal material, and the Bulbus Fritillariae Cirrhosae specific primer and probe combination has good specificity. ' / ' in the table indicates that the amplification signal (Ct value > 35) is not detected.

[0079] Table 1 Specificity of Bulbus Fritillariae Cirrhosae primer and Taqman fluorescence probe PCR detection system

[0080]

[0081]

[0082] Furthermore, the content of Fritillaria cirrhosa components in the samples was quantitatively analyzed using a double-standard linear correction method. The 53 ng / μl Fritillaria cirrhosa standard DNA template was serially diluted 5-fold with sterile double-distilled water, and 2 μl of each diluted sample was used for fluorescent PCR detection. The template concentrations in the reaction system, from highest to lowest, were: 10⁶, 21.2, 4.24, 0.85, 0.17, and 0.03 ng, with each gradient performed in triplicate. Fritillaria cirrhosa-specific primers and probes, as well as 18S rRNA primers and probes, were used to simultaneously amplify the standard control (Dark Purple Fritillaria cirrhosa). Figure 2 The image shows the amplification curve of the 18SRNA universal internal reference gene in the standard control; as shown. Figure 3 The figure shows the amplification curve of the specific endogenous gene of Fritillaria cirrhosa in the standard control. The Ct values ​​of the obtained 18SRNA gene amplification were linearly fitted, as shown below. Figure 4 The figure shows a double-labeled curve of the Fritillaria cirrhosa-specific endogenous gene and the 18SRNA universal internal reference gene. The standard curve shows a clear linear increase with a good linear relationship: y = -3.260x + 23.924, R 2 =0.999. The relative standard deviation (RSD) was <0.7%, indicating good repeatability, with an amplification efficiency of 102.642%. Similarly, the amplification of the specific endogenous gene of Fritillaria cirrhosa was highly efficient, with an amplification efficiency of 110.00%. The linear fit was high, showing linear growth and a good linear relationship: y = -3.095x + 29.872, R... 2 =0.998. The standard reference (Dark Purple Fritillaria) was quantitatively estimated based on the standard curves of the 18sRNA gene and the Fritillaria cirrhosa-specific gene, respectively. The content of Fritillaria cirrhosa components in the test sample was calculated using the following formula:

[0083] 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 / 18sRNA gene content in the standard reference) × 100%.

[0084] Bulbus fritillariae cirrhosae and other fritillaria materials were mixed at a ratio of 1 : 1 to prepare a test sample (containing 50% of Bulbus fritillariae cirrhosae) numbered Test 1, which was subjected to quantitative detection by the method described in the present application. The results are shown in Table 2. The 18sRNA gene and the Bulbus fritillariae cirrhosae specific endogenous gene CBM in the Test 1 sample were normally amplified, with average Ct values of 24.11 and 28.64, respectively. In this test, the standard curves of the 18sRNA gene and the Bulbus fritillariae cirrhosae specific gene were used to quantitatively evaluate the positive control (dark purple fritillaria) at 216.520 ng and 141.921 ng, respectively, and the test sample at 73.19 ng and 22.15 ng, respectively. The Bulbus fritillariae cirrhosae content in the Test 1 sample was calculated to be 46.17% according to the above formula, which was 3.83 percentage points different from the theoretical value. The average relative deviation of three parallel experiments was 7.66%, and the RSD value was 2.3%. The deviation rate of the detection results was much smaller than the internationally recognized value of 25%, and the reliability was good.

[0085] Table 2 Test results of Taqman fluorescent probe double standard linear correction quantitative PCR method

[0086]

[0087]

[0088] The conventional double standard linear analysis method is as follows: based on the two standard curves of the target gene (Bulbus fritillariae cirrhosae specific gene) and the internal reference gene (18sRNA gene), the absolute initial copy number of the target gene (Bulbus fritillariae cirrhosae specific gene) and the internal reference gene (18sRNA gene) in the test sample is determined. In order to eliminate the operation error between different samples, the absolute amount of the target gene after correction is obtained by dividing the copy number of the target gene by the copy number of the internal reference gene. That is, the characteristic value (absolute copy number after normalization) = Q_target gene (sample) / Q_internal reference gene (sample). In order to make the results easier to understand and compare between different laboratories, this characteristic value can be further converted into a more general unit. For example, if the total RNA amount of the sample is recorded: characteristic value (copy number / ng RNA) = [Q_target gene (sample) / Q_internal reference gene (sample)] / (total RNA amount * loading volume proportion).

[0089] In order to eliminate the operation error (such as extraction efficiency difference) between different batches of samples of Fritillaria cirrhosa, the ratio of the target gene (Fritillaria cirrhosa specific gene) and the internal reference gene (18sRNA gene) is introduced to characterize the Fritillaria cirrhosa characteristic value of the test sample according to the double standard linear analysis method; in order to eliminate the systematic error between different varieties of Fritillaria cirrhosa (such as genetic diversity, matrix inhibition), the Q_target gene (standard control) / Q_internal reference gene (standard control) value is introduced to correct the Fritillaria cirrhosa characteristic value. Further test results prove that, while introducing the 18sRNA internal reference gene, the "ratio of ratios" is used, that is,

[0090] [Q_target gene (sample) / Q_internal reference gene (sample)] / [Q_target gene (standard control) / Q_internal reference gene (standard control)]x100%, that is,

[0091] (the content of Fritillaria cirrhosa specific gene in the test sample / the content of 18sRNA gene in the test sample)÷(the content of Fritillaria cirrhosa specific gene in the standard control / the content of 18sRNA gene in the standard control)x100%

[0092] The content characteristic of Fritillaria cirrhosa in the test sample can further eliminate the errors from three sources of "genetic diversity", "matrix inhibition" and "extraction efficiency difference", and improve the detection accuracy and reliability. According to the embodiment of the present application, the detection result of the present application method for the adulterated sample with a theoretical content of 50% is 46.17%, the average relative deviation of 3 parallel experiments is 7.66%, and the RSD value is 2.3%, which is far lower than the internationally recognized deviation threshold of 25%.

[0093] Those skilled in the art can directly deduce that, in the quantitative detection of Fritillaria cirrhosa medicinal materials with multiple origins, due to the differences in genomic DNA extraction efficiency of different original plants and the possible presence of PCR inhibitors in the sample matrix, when only a single target gene standard curve is used for quantification, it is easy to be affected by non-specific interference in the sample processing process, resulting in large deviation in the quantitative result. Based on the "Fritillaria cirrhosa specific gene + 18sRNA internal reference gene" double standard linear correction system described in the present application, by introducing 18sRNA as a housekeeping gene (its expression is stable in different samples and is not affected by the matrix), the influence of sample DNA extraction efficiency difference and PCR inhibitors can be effectively compensated, and combined with the correction formula of "test sample ratio / standard control ratio", the systematic error of single target quantification can be significantly reduced. This technical effect can be directly understood and verified by those skilled in the art based on the primer probe design, reaction system optimization and example data described in the application document, without relying on additional experimental evidence.

[0094] In summary, the present application realizes high specificity and accurate quantitative analysis of Fritillaria cirrhosa by designing specific primers and probes and introducing double-mark linear correction technology, is simple and easy to promote, effectively promotes market supervision and consumer protection, and makes a significant contribution to the standardization of Fritillaria cirrhosa detection technology and the progress of the industry.

[0095] The above is only a preferred embodiment of the present application, not any form of limitation on the present application, although the present application has been disclosed as above with a preferred embodiment, however, not to limit the present application, any person skilled in the art, without departing from the technical solution range of the present application, can make some changes or modifications to the above disclosed technical content for equivalent embodiments with equivalent changes, but as long as it does not deviate from the technical solution of the present application, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application, still belongs to the range of the technical solution of the present application.

Claims

1. A primer and probe combination for detecting Fritillaria cirrhosa by Taqman fluorescent probe double standard line linear correction quantitative PCR, characterized in that, The specific primer and probe of the Fritillariae cirrhosae Bulbus are designed based on the conservative gene sequences of six original plants of Fritillariae cirrhosae Bulbus in the 2020 edition of Chinese Pharmacopoeia, and the primer and probe of the 18sRNA universal internal reference gene are designed based on the sequence of the 18sRNA universal internal reference gene. The upstream primer sequence CBM-YF is 5'-CAGACTGAGACATGTCGAACAAGA-3' The downstream primer sequence CBM-YR is 5'-CCTCTCGGGTTCGCAACA-3' The Taqman fluorescent probe CBM-YP is FAM-ACCAACCGTGGGAGATTACCATCACAAA-TAMRA. The six original plants of Fritillariae cirrhosae Bulbus include Fritillaria cirrhosa D.Don, Fritillaria unibracteata Hsiao et K.C.Hsia, Fritillaria przewalskii Maxim., Fritillaria walujewii Prokh., Fritillaria delavayi Franch. and Fritillaria taipaiensis S.C.Chen. The primer and probe of the 18sRNA universal internal reference gene are as follows: The upstream primer sequence 18s RNA-F is 5'-TCTGCCCTATCAACTTTCGATGGTA-3' The downstream primer sequence 18s RNA-R is 5'-AATTTGCGCGCCTGCTGCCTTCCTT-3' The Taqman fluorescent probe 18s RNA-P is FAM-CCGTTTCTCAGGCTCCCTCTCCGGAATCGAACC-TAMRA. The specific primer and probe of the Fritillariae cirrhosae Bulbus and the primer and probe of the 18sRNA universal internal reference gene are used to amplify the test sample and the standard control sample in a PCR reaction system, the presence of the Fritillariae cirrhosae Bulbus in the test sample is qualitatively confirmed according to the amplification results of the specific primer and probe of the Fritillariae cirrhosae Bulbus, then two standard curves of the specific gene and the 18sRNA gene of the Fritillariae cirrhosae Bulbus are obtained by taking the standard control sample as a template, and finally the content of the Fritillariae cirrhosae Bulbus in the test sample is quantitatively analyzed by using a double-standard linear correction method.

2. A method for detecting Fritillaria cirrhosa bulbs based on Taqman fluorescent probe double mark line linear correction quantitative PCR, characterized in that, The method for quantitatively analyzing the content of the Fritillariae cirrhosae Bulbus in the test sample by using the double-standard linear correction method is as follows: the content of the Fritillariae cirrhosae Bulbus = (the content of the specific gene of the Fritillariae cirrhosae Bulbus in the test sample / the content of the 18sRNA gene in the test sample) ÷ (the content of the specific gene of the Fritillariae cirrhosae Bulbus in the standard control sample / the content of the 18sRNA gene in the standard control sample) × 100%. The process for qualitatively confirming the presence of the Fritillariae cirrhosae Bulbus in the test sample according to the amplification results of the specific primer and probe of the Fritillariae cirrhosae Bulbus is as follows: whether the positive control and the test sample have amplification signals when the Ct value is less than or equal to 35, and whether the blank control has no amplification signal, are determined, if yes, it is determined that the test sample contains the Fritillariae cirrhosae Bulbus; if not, it is determined that the test sample does not contain the Fritillariae cirrhosae Bulbus.

3. The method according to claim 2, wherein the method is characterized in that, The concentrations of the specific primer and probe of the Fritillariae cirrhosae Bulbus and the primer and probe of the 18sRNA universal internal reference gene are both 10 μmol / L.

4. The method according to claim 2, wherein the method is characterized in that, ​ 5. The method according to claim 2, wherein the method is characterized in that, In the PCR reaction system, the DNA template solution of the test sample is diluted to obtain a DNA diluent with a concentration of 40-60 ng / μl.

6. The method according to claim 5, wherein the method is characterized in that, The PCR reaction system comprises the following components:

7. The method according to claim 2, wherein the method is characterized by, The reaction steps of the PCR reaction system are as follows: S1, pre-denaturation treatment: heating the PCR reaction system to 95℃ for 10 minutes; S2, denaturation treatment: heating the PCR reaction system to 95℃ again for 15 seconds; S3, annealing stage: reducing the temperature of the PCR reaction system to between 56 and 58℃ for 40-60 seconds, and repeating the denaturation and annealing process for 40-42 cycles.

Images