Real-time fluorescent quantitative PCR (Polymerase Chain Reaction) method for identifying thunberg fritillary bulb and counterfeit Hubei fritillary

Abstract

The invention belongs to the fields of traditional Chinese medicines and molecular biological identification, and provides a real-time fluorescent quantitative PCR (polymerase chain reaction) method for identifying thunberg fritillary bulb and adulterants of the thunberg fritillary bulb, namely fritillaria hupehensis, in the method, the thunberg fritillary bulb is identified by detecting nucleotide difference at the 240th position of a matK gene. The method provided by the invention can be used for detecting the counterfeit Hubei fritillary bulb in the thunberg fritillary bulb with high sensitivity and specificity, and can be used for accurately quantifying the counterfeit Hubei fritillary bulb in the thunberg fritillary bulb. And a powerful tool is provided for quality control of fritillaria medicinal materials.

Description

Technical Field

[0001] The present application belongs to the field of traditional Chinese medicine and molecular biology identification. Specifically, the present application provides a real-time fluorescence quantitative PCR method for identifying Fritillaria thunbergii and its mixed product Fritillaria thunbergii. Background Art

[0002] Fritillaria plants have a long history of medicinal use in my country. First described in Shennong's Classic of Materia Medica, Fritillaria thunbergii Miq. is a medium-grade herb. It is the dried bulb of the lily family, Fritillaria thunbergii, known for its expectorant, antitussive, antibacterial, anti-inflammatory, and lung and mastitis-relieving properties. It is primarily produced in Pan'an, Jinhua, Zhejiang, Haishu, Ningbo, and Nantong and Yancheng, Jiangsu. Fritillaria hupehensis Hsiao et KC Hsia is the dried bulb of the lily family, known for its heat-clearing, phlegm-resolving, cough-relieving, and stagnation-dispersing properties. It is clinically used to treat lung-heat coughs and hot-phlegm coughs. It is primarily produced in Hubei, Chongqing, and Hunan. Although both Fritillaria thunbergii and Fritillaria hupehensis belong to the same genus Fritillaria and are closely related, their active ingredients and pharmacological effects differ. In recent years, due to price pressures, some Fritillaria herbs with similar characteristics to Fritillaria thunbergii have been sold and used as genuine Fritillaria thunbergii in the market, with the Hubei Fritillaria being the most common counterfeit. Currently, there is a significant incidence of Hubei Fritillaria being passed off as or adulterated with Fritillaria thunbergii in the market, especially after being processed into medicinal slices. Because Fritillaria thunbergii and Fritillaria hupehensis are very similar in appearance and chemical composition, traditional methods for their identification primarily rely on their appearance and physicochemical characteristics, employing thin-layer chromatography and high-performance liquid chromatography to distinguish their main chemical components. However, when adulteration is present, traditional identification methods prove challenging.

[0003] Real-time fluorescence quantitative PCR technology collects the accumulated fluorescent signal during the DNA amplification process in real time, offering the advantages of real-time monitoring, high specificity, high sensitivity, and rapid and accurate quantification. It can amplify trace samples and has been widely used in food science testing and identification research, microbiological detection and control analysis, and clinical diagnosis. In recent years, real-time fluorescence quantitative PCR has also begun to be applied to the field of Traditional Chinese Medicine research. Its unique ability to specifically identify specific varieties even when multiple medicinal materials are mixed together, coupled with its dual qualitative and quantitative nature, makes it an effective solution to the widespread problem of adulteration of traditional Chinese medicines and decoction pieces, and holds great promise for future applications. Summary of the Invention

[0004] This study ultimately identified a single-nucleotide polymorphism (SNP) site within the chloroplast's matK gene region that can distinguish conserved and interspecific differences between Fritillaria thunbergii and Fritillaria hupehensis. Based on this SNP, an MGB probe was designed, and primers and probes were designed to distinguish between Fritillaria thunbergii and Fritillaria hupehensis. The MGB probe was also designed. The MGB group binds to the minor groove of the DNA double helix, enhancing the binding stability of the probe to the target and enabling precise discrimination of single nucleotide polymorphisms (SNPs) or differences in closely related sequences. Furthermore, the MGB probe reduces nonspecific binding, significantly reducing the risk of cross-reactions even in the presence of highly homologous sequences and improving the specificity of the reaction.

[0005] On the one hand, the present application provides a real-time fluorescence quantitative PCR method for identifying Fritillaria thunbergii and its mixed product Fritillaria thunbergii. In the method, the nucleotide difference at the 240th position of the matK gene is detected to identify Fritillaria thunbergii.

[0006] Furthermore, in the method, a primer pair and a probe having nucleotide sequences of SEQ ID NO. 1 and SEQ ID NO. 2 are used to perform real-time fluorescence quantitative PCR reaction to amplify genomic DNA of the sample to be tested.

[0007] Furthermore, the nucleotide sequence of the probe is SEQ ID NO.3.

[0008] Furthermore, the method is used to quantitatively detect the amount of Hubei Fritillaria doped in Zhejiang Fritillaria.

[0009] Furthermore, the method comprises: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA of the sample to be tested extracted in step (1) as a template, perform fluorescence quantitative PCR amplification, collect the fluorescence signal, and calculate the Ct value; (3) The Ct value was used to determine whether the Fritillaria thunbergii sample contained the Fritillaria hupehensis. A Ct value of ≤ 35 determined that the Fritillaria hupehensis DNA sample existed, indicating that the Fritillaria thunbergii sample was adulterated with the Fritillaria hupehensis; a Ct value of > 35 determined that the Fritillaria hupehensis DNA sample did not exist, indicating that the Fritillaria thunbergii sample was not adulterated with the Fritillaria hupehensis.

[0010] Furthermore, the method comprises: (1) Prepare pure Fritillaria thunbergii samples and Fritillaria thunbergii samples mixed with different proportions of Fritillaria hupehensis, and extract genomic DNA from the samples; (2) Using the genomic DNA of each sample extracted in step (1) as a template, perform fluorescence quantitative PCR amplification, collect the fluorescence signal, and calculate the Ct value; (3) Construct a standard curve with the logarithm of the adulterated amount of Hubei Fritillaria in the sample as the horizontal axis and the Ct value as the vertical axis; (4) Extracting genomic DNA from the sample to be tested; (5) Using the genomic DNA of the sample to be tested extracted in step (4) as a template, perform fluorescence quantitative PCR amplification, collect the fluorescence signal, and calculate the Ct value; (6) Substitute the Ct value obtained in step (5) into the standard curve obtained in step (3) to calculate the amount of adulteration of Hubei Fritillaria in the test sample.

[0011] Furthermore, the reaction system for the fluorescent quantitative PCR amplification is a 20 μL reaction system, including 10 μL of Takara Premix Tap (2×), 0.3 μL each of upstream and downstream primers, 0.3 μL of probe, 1.1 μL of genomic DNA, and 8 μL of RNase-free ddH2O; Furthermore, the concentrations of the upstream and downstream primers and the probe are 5-20 μmol / L.

[0012] Furthermore, the concentrations of the upstream and downstream primers and the probe were 10 μmol / L.

[0013] Furthermore, the fluorescence quantitative PCR amplification program is: 95°C for 30s; 95°C for 5s, 66°C for 15s, for 45 cycles.

[0014] On the other hand, the present application provides a primer pair and probe combination for distinguishing Fritillaria thunbergii and its mixed product Fritillaria thunbergii, the nucleotide sequence of the primer pair is SEQ ID NO.1 and SEQ ID NO.2.

[0015] Furthermore, the nucleotide sequence of the probe is SEQ ID NO.3.

[0016] On the other hand, the present application provides the use of the above primer pair and probe combination in preparing a kit for distinguishing Zhejiang Fritillaria and its mixed product Hubei Fritillaria or a kit for quantitatively detecting the amount of Hubei Fritillaria impurities in Zhejiang Fritillaria.

[0017] In the embodiment, FAM and MGB groups are exemplarily connected at both ends of the probe to form the probe FAM-TGATTCGAATCAA-MGB. When the nucleotide sequence of the probe is determined, other fluorescent groups / quenching groups can be routinely selected by those skilled in the art.

[0018] The present invention provides a real-time fluorescence quantitative PCR method for distinguishing Fritillaria thunbergii and its common counterfeit, Fritillaria hupehensis, based on specific SNPs. Existing research results show that there are no suitable variant sites in the barcode sequences of Fritillaria thunbergii and Fritillaria hupehensis (primer sets designed based on these differential sites, such as those disclosed in CN107142319A, can complete qualitative detection, but it is difficult to draw a usable annotation curve for quantitative detection, R2 To address this issue, the researchers screened other gene fragments and ultimately discovered a single-base mutation SNP in the matK gene region that could distinguish conserved and interspecific differences between Fritillaria thunbergii and Fritillaria hupehensis. Based on the SNP, they designed an MGB probe, increased the probe's Tm value, and improved the specificity of the hybridization reaction. They also optimized the reaction system to enhance the sensitivity of the detection method. The fluorescence quantitative amplification reaction established with this method has a sensitivity of 5.8 pg / μL for detecting Fritillaria hupehensis DNA. When testing adulterated samples, even Fritillaria thunbergii samples mixed with 1% Fritillaria hupehensis could be accurately detected. This method has excellent specificity, reproducibility, and sensitivity, enabling the quantitative detection of adulteration between Fritillaria thunbergii and Fritillaria hupehensis, providing excellent technical support for the detection of adulteration in Fritillaria thunbergii. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the sequence alignment result of the matK sequence binding region of Fritillaria thunbergii and Fritillaria hupehensis; Figure 2 This is the amplification curve of different adulteration ratios of Fritillaria thunbergii (Thunbergii), with adulteration ratios of 1%, 5%, 25%, 50%, and 100%. Figure 3 This is a standard curve diagram constructed by adulterating Zhejiang Fritillaria with Hubei Fritillaria in different proportions. DETAILED DESCRIPTION

[0020] Example 1: Design of primers and probes.

[0021] The matK gene sequences of Fritillaria thunbergii and Fritillaria thunbergii were downloaded from GenBank, as shown in Table 1. The sequences were aligned using BioEidt software and compared. Figure 1 According to the sequence alignment results, there is a SNP site at position 240.

[0022] Table 1 Sequence information of Fritillaria thunbergii and Fritillaria thunbergii downloaded from GenBank

[0023] There are 240 SNP sites between Fritillaria thunbergii and Fritillaria hupehensis. Based on the principles of intraspecific conservation and interspecific differences, the Fritillaria hupehensis-specific probe and primer sequences were designed as shown in Table 2.

[0024] Table 2 PCR primers and probe sequences of Fritillaria thunbergii

[0025] Example 2: Optimization of the reaction system and reaction procedure of the fluorescence quantitative PCR method.

[0026] Experimental instruments and materials: The instrument used was a Bio-Rad CFX96 Touch fluorescent quantitative PCR instrument from the United States. The probe enzyme used was purchased from Takara Premix Tap (2×) from Bio-Rad Biotechnology (Beijing) Co., Ltd. The primer pairs and probes were synthesized by Anshengda Biotechnology and were all lyophilized powders. They were then diluted to 100 μmol / L with RNase-free ddH2O as a stock solution for later use.

[0027] Extraction of template DNA: The commercially purchased Fritillaria thunbergii and Fritillaria thunbergii samples were scrubbed with 75% ethanol, rinsed with sterile ultrapure water, and dried. They were then crushed into fine powder in a ball mill. 50 mg of the powder was placed in a 1.5 mL centrifuge tube and the total genomic DNA was extracted using a plant genome extraction kit. 50 mg each of the Fritillaria thunbergii (batch number: 120972-201906) and Fritillaria thunbergii (batch number: 120962-202106) reference medicinal materials from the China National Institute for Food and Drug Control were taken, and the reference medicinal material template DNA solution was prepared in the same way. The concentration and purity of the template DNA were determined using a microspectrophotometer.

[0028] Annealing temperature optimization: Using DNA extracted from Fritillaria thunbergii and Fritillaria thunbergii control materials purchased from the China Food and Drug Inspection Institute as templates, amplification was performed using the primers and probes shown in Table 2, the reaction system shown in Table 3, and the amplification program shown in Table 4.

[0029] Table 3 Fluorescence quantitative PCR total reaction system configuration

[0030] Table 4 Real-time fluorescence quantitative PCR reaction procedure

[0031] An annealing temperature of 60–66°C was selected for amplification under the aforementioned reaction conditions. The results are shown in Table 5 (where HBBM-SD represents the standard sample of Hubei Fritillaria and ZBM-SD represents the standard sample of Zhejiang Fritillaria). Nonspecific amplification of Zhejiang Fritillaria occurred at annealing temperatures of 60 and 61°C, but this disappeared when the annealing temperature was increased to 62°C, demonstrating increased specificity of the method. Simultaneous analysis of the amplified products by fluorescent quantitative PCR showed that the purity of the amplified products increased with increasing annealing temperatures, ultimately selecting 66°C as the optimal annealing temperature.

[0032] Table 5 Annealing temperature optimization results

[0033] The optimized fluorescence quantitative PCR program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, extension at 66°C for 15 s, collection of FAM fluorescence signal, and 45 cycles.

[0034] Example 3: Specificity experiment.

[0035] The established quantitative PCR method was used to test the specificity of 10 samples of known species. Real-time quantitative PCR reactions (repeated three times) were performed using the primers and probes listed in Table 2, the reaction system listed in Table 3, and the amplification program listed in Table 4 to evaluate the specificity of the method. The amplification results for the 10 samples are shown in Table 6 (where HBBM1-4 represents Hubei Fritillaria 1-4, and ZBM1-6 represents Zhejiang Fritillaria 1-6). Significant amplification was observed only in the Hubei Fritillaria samples, with Ct values ​​ranging from 18.77 to 30.71 for the four Hubei Fritillaria samples, which are within the specified Ct range for real-time quantitative PCR (all ≤35). The remaining six Zhejiang Fritillaria samples and the negative control showed no signs of amplification. These results are consistent with previous findings and demonstrate the good specificity of this amplification method.

[0036] Table 6 Results of specificity investigation

[0037] Example 4: Repeatability experiment.

[0038] The reproducibility of this method was evaluated by diluting a reference DNA template from Fritillaria thunbergii to 0.58 ng / μl. Amplification was performed using the primers and probes listed in Table 2, the reaction system listed in Table 3, and the amplification procedure listed in Table 4. The results are shown in Table 7, showing that both the intra-assay and inter-assay coefficients of variation were less than 2%. This indicates good reproducibility and low scatter, demonstrating the method's good reproducibility.

[0039] Table 7 Repeatability test results

[0040] Example 5: Sensitivity experiment.

[0041] Hubei Fritillaria reference medicinal material sample DNA (initial concentration: 5.8×10 3 pg / μL) were diluted in 10-fold gradients, with a concentration range of 5.8×10 3 ~5.8×10 -2 pg / μL, using the primers and probes shown in Table 2, the reaction system shown in Table 3, and the amplification program shown in Table 4, the experimental results are shown in Table 8. The concentration of Hubei Fritillaria DNA is 5.8×10 3 ~5.8×10 -2pg / μL, and the corresponding Ct value range was 14.04~36.84. According to the limit of Ct>35, the minimum detection limit was 5.8pg / μL, indicating that the sensitivity of the fluorescence quantitative amplification reaction established in this experiment for the detection of Hubei Fritillaria DNA can reach 5.8pg / μL.

[0042] Table 8 Sensitivity investigation results

[0043] Example 6: Sample adulteration detection experiment.

[0044] DNA samples from a binary mixture of Fritillaria thunbergii and Fritillaria hupehensis were amplified. DNA extraction and fluorescence quantitative amplification were performed using pure Fritillaria thunbergii samples and mixed samples mixed with 1%, 5%, 25%, 50%, and 100% Fritillaria hupehensis. The results showed that the Ct value increased from the pure Fritillaria thunbergii sample to the mixed sample mixed with 1% Fritillaria hupehensis; the Ct value showed a decreasing trend from the mixed sample mixed with 1% to 5%, 25%, 50%, and 100% Fritillaria hupehensis, with significant differences ( Figure 2 The amplification results are shown in Table 9. The results show that the Ct value increases from the pure Fritillaria thunbergii sample to the mixed sample with 1% of Fritillaria thunbergii; the Ct value shows a decreasing trend from the mixed sample with 1% to 5%, 25%, 50%, and 100% of Fritillaria thunbergii. A standard curve was constructed with the logarithm of the adulteration amount of Fritillaria thunbergii in the Fritillaria thunbergii sample as the horizontal axis and the Ct value as the vertical axis ( Figure 3 ).like Figure 3 As shown, the standard curve y=-2.8187x+19.006, R 2 =0.9979. This indicates that the adulteration amount of Hubei Fritillaria samples in Zhejiang Fritillaria samples has a good linear relationship in the range of 1% to 100%. Taking the Ct of 100% Hubei Fritillaria samples as the reference control, the 1 / 2Δ CT The recoveries of samples with other adulterated ratios were calculated and ranged from 81.40% to 113.85% (Table 9). This indicates that the fluorescence quantitative detection method established in this experiment has good accuracy and can be used for quantitative detection of adulterated Fritillaria thunbergii.

[0045] Table 9 Amplification results of adulterated Fritillaria thunbergii and Hubei Fritillaria thunbergii

Claims

1. A real-time fluorescence quantitative PCR method for identifying Fritillaria thunbergii and its counterfeit Fritillaria thunbergii, characterized in that: The method uses a primer pair with nucleotide sequences of SEQ ID NO.1 and SEQ ID NO.2 and a probe to perform real-time fluorescence quantitative PCR reaction to amplify genomic DNA of the sample to be tested, so as to detect the nucleotide difference at the 240th position of the matK gene to identify the Fritillaria thunbergii.

2. The method according to claim 1, characterized in that The nucleotide sequence of the probe is SEQ ID NO.

3.

3. The method according to claim 1 or 2, characterized in that The method is used for quantitatively detecting the amount of contamination of Fritillaria thunbergii in Fritillaria thunbergii.

4. The method according to claim 3, characterized in that The method comprises: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA of the sample to be tested extracted in step (1) as a template, perform fluorescence quantitative PCR amplification, collect the fluorescence signal, and calculate the Ct value; (3) The Ct value was used to determine whether the Fritillaria thunbergii sample contained the Fritillaria hupehensis. A Ct value of ≤ 35 determined that the Fritillaria hupehensis DNA sample existed, indicating that the Fritillaria thunbergii sample was adulterated with the Fritillaria hupehensis; a Ct value of > 35 determined that the Fritillaria hupehensis DNA sample did not exist, indicating that the Fritillaria thunbergii sample was not adulterated with the Fritillaria hupehensis.

5. The method according to claim 3, characterized in that The method comprises: (1) Prepare pure Fritillaria thunbergii samples and Fritillaria thunbergii samples mixed with different proportions of Fritillaria hupehensis, and extract genomic DNA from the samples; (2) Using the genomic DNA of each sample extracted in step (1) as a template, perform fluorescence quantitative PCR amplification, collect the fluorescence signal, and calculate the Ct value; (3) Construct a standard curve with the logarithm of the adulterated amount of Hubei Fritillaria in the sample as the horizontal axis and the Ct value as the vertical axis; (4) Extracting genomic DNA from the sample to be tested; (5) Using the genomic DNA of the sample to be tested extracted in step (4) as a template, perform fluorescence quantitative PCR amplification, collect the fluorescence signal, and calculate the Ct value; (6) Substitute the Ct value obtained in step (5) into the standard curve obtained in step (3) to calculate the amount of adulteration of Hubei Fritillaria in the test sample.

6. The method according to claim 4 or 5, characterized in that The fluorescent quantitative PCR amplification reaction system is a 20 μL reaction system, including Takara Premix Tap 2×10 μL, upstream and downstream primers 0.3 μL each, probe 0.3 μL, genomic DNA 1.1 μL and RNase-free ddH2O 8 μL; the concentrations of upstream and downstream primers and probe are 5-20 μmol / L.

7. The method according to claim 6, characterized in that The fluorescence quantitative PCR amplification program was as follows: 95° C. for 30 s; 95° C. for 5 s, and 66° C. for 15 s, for 45 cycles.

8. A primer pair and probe combination for distinguishing Fritillaria thunbergii from its counterfeit Fritillaria thunbergii, characterized in that: The nucleotide sequences of the primer pair are SEQ ID NO.1 and SEQ ID NO.

2.

9. The primer pair and probe combination according to claim 8, characterized in that The nucleotide sequence of the probe is SEQ ID NO.

3.

10. Use of the primer pair and probe combination according to claim 8 or 9 in preparing a kit for distinguishing Fritillaria thunbergii from its mixed product Fritillaria hupehensis or a kit for quantitatively detecting the amount of Fritillaria hupehensis doped in Fritillaria thunbergii.

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