A reagent for detecting DNA barcodes of Asarum species and a fluorescence detection method for Asarum species

Abstract

The present invention provides a reagent for detecting the DNA barcode of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. and a fluorescence detection method for Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. The reagent of the present invention includes a specifically upstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with AuNPs and a specifically downstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with a fluorescent label. The sequences of the specifically upstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. and the specifically downstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with a fluorescent label are respectively: 5’-SH-(CH2)6-ATTGACCACGCGAACTTGTGA-3’; 5’-fluorescent label-ACCGTCAACAAACCTGATTGCT-3’. Detection method: Mix the specifically upstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with AuNPs and the specifically downstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with a fluorescent label with a DNA template solution of the to-be-detected Asarum species for PCR reaction amplification, and detect the fluorescence spectral value of the obtained product to achieve qualitative or quantitative detection of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag.

Description

Technical Field

[0001] The present invention belongs to the technical field of spectral analysis, and in particular relates to a reagent for detecting DNA barcodes of Asarum species and a fluorescence detection method for Asarum species. Background Art

[0002] Asarum heterotropoides F. Schmidt var. mandshuricum (Maxim.) Kitag.) is a perennial herbaceous plant in the Aristolochiaceae family and genus Asarum. The main components of most Asarum species are methyl eugenol, safrole, asarone, sassafras acid, datun asarone, and alkaloids with cardiotonic effects. As early as 2002, the World Health Organization (WHO) listed aristolochic acid as a Class 1 carcinogen. In recent years, people have gradually increased their investment in dietary and health care. Cases of aristolochic acid poisoning from Chinese herbal remedies and pills have occurred frequently. Foods with unknown raw material sources or composition pose significant risks and hidden dangers to food safety. Furthermore, asarum plants are used in Chinese herbal medicines, different parts of the plant are used, making it difficult to display the entire plant when sold on the market. This leads to confusion and adulteration of Chinese herbal medicines, disrupting the normal operation of the market economy. Therefore, accurate and rapid identification of asarum components in food is extremely important.

[0003] Over the past decade, a variety of molecular biology techniques have been applied to the rapid identification of fish, including DNA barcoding, PCR, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and gene chip technology. However, all reported studies have focused solely on the biomorphological identification of Aristolochiaceae species and the mass spectrometry identification of their components. Therefore, the development of rapid detection methods, including spectral analysis methods for DNA barcoding fluorescence detection of Asarum liaoresinum, is crucial to meet the testing and inspection needs of market regulation and food safety supervision in my country. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a reagent for detecting DNA barcodes of Asarum species and a fluorescence detection method for Asarum species.

[0005] The first object of the present invention is to provide a reagent for detecting DNA barcodes of Asarum species, comprising an AuNPs-modified Asarum-specific upstream primer and a fluorescent marker-modified Asarum-specific downstream primer.

[0006] The sequences of the Liao Xixin primer-specific upstream primer dd-AH-F and the Liao Xixin primer-specific downstream primer dd-AH-R modified with a fluorescent marker are: 5'-SH-(CH2)6-ATTGACCACGCGAACTTGTGA-3';

[0007] 5′-Fluorescent marker-ACCGTCAACAAACCTGATTGCT-3′.

[0008] In one embodiment of the present invention, the primers for Asarum liaoensis are obtained by sequencing and screening universal primers of Asarum species, wherein the sequences of the upstream and downstream primers Asa-F and Asa-R of the universal primers of Asarum species are: 5'-TATCTCTTTCTTTTTTTCAAAAGG-3'; 5'-ATGGGCTTTTGCCAATGAGCCAATC-3', respectively.

[0009] In one embodiment of the present invention, the kit further comprises a DNA polymerase, a DNA template, water, a buffer solution and dNTPs.

[0010] In one embodiment of the present invention, the fluorescent marker is selected from Cy5, Cy5.5, Cy3 or ROX.

[0011] In one embodiment of the present invention, the AuNPs-modified Asarum liaoensis-specific upstream primer was prepared by mixing AuNPs with a solution of the Asarum liaoensis upstream primer, dd-AH-F, and then adding a sodium chloride solution for reaction. During the reaction, the AuNPs and the thiol primer bond via an Au-S bond. This covalent bond stabilizes the structure, while the sodium chloride solution can age the reaction.

[0012] In one embodiment of the present invention, the molar ratio of the AuNPs to the Asarum-specific upstream primer dd-AH-F is 1:300-500.

[0013] In one embodiment of the present invention, the Asarum species is selected from Asarum simplicifolium, Asarum variegated, Asarum liao or Asarum pubescens.

[0014] The second purpose of the present invention is to provide a fluorescence detection method for detecting Asarum liaoensis, using the reagent for detection.

[0015] In one embodiment of the present invention, the following steps are included: mixing an AuNPs-modified Asarum-specific upstream primer, a fluorescent marker-modified Asarum-specific downstream primer, and a solution containing a DNA template of the Asarum species to be tested to perform PCR amplification, and detecting the fluorescence spectrum value of the resulting product to achieve qualitative or quantitative detection of Asarum.

[0016] In one embodiment of the present invention, the standard curve for quantitative detection is prepared by the following method: a series of Asarum species DNA templates with different concentrations are amplified by PCR system, and the fluorescence intensity value of the resulting product is detected at an excitation wavelength of 610 nm and an emission wavelength of 670 nm to obtain a standard curve with the logarithm of the Asarum species DNA template concentration as the horizontal axis and F0-F as the vertical axis, wherein F0 is the fluorescence intensity value of the blank and F is the fluorescence intensity value of the resulting product.

[0017] In one embodiment of the present invention, the concentration of the Asarum liaoensis DNA template is 0.1×10 -4 -0.4×10 1 ng / μL.

[0018] In one embodiment of the present invention, the concentration of the Asarum liaoensis DNA template is 0.348×10 -4 -0.348×10 1 ng / μL.

[0019] In one embodiment of the present invention, the Asarum species is selected from Asarum simplicifolium, Asarum variegated, Asarum liao or Asarum pubescens.

[0020] The present invention introduces DNA barcode technology into the detection of Asarum liaoxi. AuNPs and fluorescent dyes are respectively labeled on the upstream and downstream primers of Asarum liaoxi. When the distance between the fluorescent dye and the AuNPs is within 10 nm, a fluorescence resonance energy (FRET) effect is generated between the fluorescent dye and the AuNPs, that is, the fluorescence energy of the fluorescent dye (fluorescence donor) is transferred to the AuNPs (fluorescence acceptor). When targeted DNA is present in the analyte, after successful PCR amplification, the distance between the AuNPs and the fluorescent dye is shortened. As the concentration of the added DNA template changes, the degree of quenching of the fluorescence intensity of the fluorescent dye by the AuNPs is different, thereby establishing a relationship between the fluorescence intensity and the concentration of the Asarum liaoxi DNA template, establishing a standard curve, and realizing high-sensitivity detection of Asarum liaoxi samples. The introduction of fluorescence detection technology into DNA barcodes significantly improves the accuracy and sensitivity of detection.

[0021] The above technical solution of the present invention has the following advantages over the prior art:

[0022] The fluorescence spectrum analysis method for detecting Liao Xixin proposed in the present invention has high sensitivity and specificity. The PCR technology has the characteristics of strong specificity, high sensitivity, simplicity and rapidity, low purity requirements and exponential growth of products. At the same time, the fluorescence of the fluorescent dye is used as the detection signal, which greatly improves the sensitivity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0024] Figure 1 is a TEM image of the gold particles AuNPs prepared in Example 2;

[0025] Figure 2 It is characterized by agarose gel electrophoresis in Example 2;

[0026] Figure 3 Schematic diagram of the DNA barcode principle for detecting Asarum liaoresinum in Example 2;

[0027] Figure 4 Figure 1 shows the specificity validation results of universal primers for Asarum species;

[0028] Figure 5 The fluorescence spectra and standard curves of the fluorescence sensor in Example 2 are obtained when different concentrations of Asarum liaoensis are present. 1-6 in the figure represent concentrations of 0.348×(10,10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 )ng / μL fluorescence spectrum. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0030] Example 1

[0031] 1. Synthesis of AuNPs and modification of upstream primers:

[0032] ① 100 mL of 0.01% HAuCl4 solution was heated to boiling while stirring. 4 mL of 1% trisodium citrate solution was quickly added to the HAuCl4 solution. The solution was stirred and heated until it turned orange. The entire reaction time was 10-15 min. The AuNPs were stored at 4°C for later use. The TEM of the obtained AuNPs was as follows: Figure 1 shown.

[0033] ② Centrifuge 1mL of the prepared 15±2nm AuNPs at 8500g for 10min, discard the supernatant, resuspend the precipitate in 100μL of ultrapure water, store at 4℃ and set aside. Take 50μL of the prepared AuNPs and 50μL of 10μM dd-AH-F primer solution and react at room temperature for 12h. Add 2M sodium chloride solution to make the final concentration 50mM and react overnight. Then centrifuge the reaction solution at 8500g for 10min, discard the supernatant, disperse the precipitate with 50μL of ultrapure water, store at 4℃ and set aside.

[0034] 2. Liao Xixin DNA template using Yisheng Extract Plant DNA with a Plant DNA Kit. The specific steps are as follows:

[0035] 1. Sample preprocessing:

[0036] 1). Add 400 μL of lysis buffer LB and 4 μL of RNase A (10 mg / mL) to a 1.5 mL centrifuge tube, mix well, and store at room temperature.

[0037] 2) Take 20 mg dry weight of Asarum sieboldii tissue, grind the tissue into powder in liquid nitrogen, transfer it to the above 1.5 mL centrifuge tube, vortex to mix, and collect the liquid on the tube wall by centrifugation.

[0038] 3) Place in a 65°C warm bath for 10 minutes, inverting and mixing the sample twice every 2-3 minutes.

[0039] 4). Add 130 μL of binding solution BD, mix thoroughly, and place on ice for 5 minutes.

[0040] 5) Centrifuge at 12000 rpm for 10 min. Transfer the supernatant to a new 1.5 mL centrifuge tube.

[0041] 6). Calculate the volume of the supernatant, add 1.5 times the volume of protein removal solution PL*, and mix immediately by pipetting.

[0042] 2. DNA Extraction

[0043] 1). Place DNA adsorption column P1 into a 2 mL collection tube and set aside.

[0044] 2) Add the above sample pretreatment solution (including precipitate) to DNA adsorption column P1, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0045] 3) Place the DNA adsorption column P1 back into the collection tube, add 600 μL of rinse solution W*, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0046] 4). Repeat step 3.

[0047] 5) Place the DNA adsorption column P1 back into the collection tube and centrifuge the empty column at 12000 rpm at room temperature for 2 minutes to remove the residual rinse solution W*.

[0048] 6. Place DNA adsorption column P1 in a new 1.5 mL centrifuge tube. Add 50-100 μL of elution buffer to the center of DNA adsorption column P1 and incubate at room temperature for 5 minutes. Then, centrifuge at 12,000 rpm for 1 minute. Collect the filtrate, which is the DNA solution, and store the DNA at -20°C. The concentration of the extracted DNA was measured using a Thermo Fisher NanoDrop One ultra-micro-volume UV spectrophotometer and was 8.7 ng / μL.

[0049] 3. Take 10 μL of Asarum liaoensis DNA template and dilute it 10-fold to 0.348×(10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 )ng / μL (respectively expressed as 10, 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 ,), the above-mentioned DNA with different concentrations was used as template for PCR amplification, and positive control (ordinary PCR) and negative control (template was 0) were performed at the same time.

[0050] Table 1 Universal primers (SEQ ID NO. 1-2) and specific primers (SEQ ID NO. 3-4) for specific identification of Asarum species

[0051]

[0052] PCR reaction system and amplification procedure:

[0053]

[0054]

[0055] Amplification procedure: pre-denaturation at 94°C for 3 min;

[0056] Denature at 98°C for 30 seconds;

[0057] Anneal at 58°C for 30 seconds;

[0058] Extension at 68°C for 10 s, 35 cycles;

[0059] Extend at 68°C for 3 min and store at 4°C to obtain the PCR product.

[0060] Example 2

[0061] Agarose gel electrophoresis characterization: 2% agarose gel was prepared with TAE buffer and the above PCR products (positive control, 0.348×0, 0.348×10 -4 , 0.348×10 -3 , 0.348×10 -2 , 0.348×10 -1 , 0.348×10 0 , 0.348×10 1 ) ng / μL was subjected to agarose gel electrophoresis: the electrophoresis voltage was 100 V, the electrophoresis time was 30 min, and the obtained agarose gel electrophoresis was as shown in FIG. Figure 2 As shown, the conventional PCR (positive control) was consistent with the expected result of 117 bp, and the DNA template was 0.348×10 -2 , 0.348×10 -1 , 0.348×10 0 , 0.348×10 1 ) can be amplified successfully, while 0.348×10 -4 , 0.348×10 -3 No obvious bands.

[0062] Example 3

[0063] Construction of an analytical method for the detection of Asarum liaoensis based on DNA barcode fluorescence:

[0064] After gradient dilution of Asarum liaoensis DNA template, 0.348×(10 -4 , 10 -3 , 10 -2 , 10 -1 , 10 0 , 10) ng / μL was added to the above PCR system for amplification. The resulting product was diluted to 300 μL with ultrapure water and then subjected to fluorescence spectrum detection with an excitation wavelength of 610 nm and an emission wavelength of 670 nm. The luminescence spectrum of each solution group was tested by fluorescence spectrometer to obtain a standard curve with the logarithm of the concentration of Asarum liaoensis DNA as the horizontal axis and the fluorescence intensity F0-F of the detection probe Cy5 as the vertical axis, where F0 is the fluorescence intensity value of the blank and F is the fluorescence intensity value of each concentration of the resulting product. The emission spectrum of each solution group was tested by fluorescence spectrometer, and each solution group was measured three times. F0-F=159.57Log[AH]+635.06R 2 =0.98.

[0065] Test Case

[0066] 1. Study on the specificity of the detection method:

[0067] The obtained plant samples, such as single-leaf Asarum, variegated tail flower Asarum, long-haired Asarum, Dabie Mountain Aristolochia, Tibetan Aristolochia, yellow-haired Aristolochia, giant flower Aristolochia, beautiful Aristolochia, ear-leaf Aristolochia, Aristolochia, large-leafed Aristolochia, Liao Asarum, Cynanchum indica, and Baiwei, were used with Yisheng DNA was extracted using the Plant DNA Kit, and the extracted DNA concentration was then measured using NanoDrop One. An appropriate amount of DNA template was used for PCR amplification according to the above procedure, and the PCR amplification results were characterized by agarose gel electrophoresis. The results were as follows: Figure 4 As shown, under standard PCR reaction system and reaction conditions, the Asarum species Asarum unifolium, Asarum variegated, and Asarum pubescens produced positive reactions, generating specific target bands, while the other species easily confused with Asarum (Aristolochia, Aristolochia spp., and Cynanchum indica) all showed negative reactions. Therefore, the fluorescence detection method of the present invention has high specificity.

[0068] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A reagent for detecting DNA barcodes of Asarum species, characterized in that: It includes AuNPs-modified Liao Xixin-specific upstream primer dd-AH-F and fluorescent marker-modified Liao Xixin-specific downstream primer dd-AH-R. The AuNPs-modified Liao Xixin specific upstream primer dd-AH-F and the fluorescent marker-modified Liao Xixin specific downstream primer dd-AH-R sequences are: 5'-SH-(CH2)6-ATTGACCACGCGAACTTGTGA-3'; 5'-fluorescent marker-ACCGTCAACAAACCTGATTGCT-3'; The Asarum species is selected from Asarum simplicifolium, Asarum variegated, Asarum liao or Asarum pubescens.

2. The reagent according to claim 1, characterized in that It also includes DNA polymerase, DNA template, water, buffer solution and dNTP.

3. The reagent according to claim 1, characterized in that The fluorescent marker is selected from Cy5, Cy5.5, Cy3 or ROX.

4. The reagent according to claim 1, characterized in that The AuNPs-modified Asarum liaoxilin specific upstream primer is prepared by the following method: AuNPs are mixed with an upstream primer dd-AH-F solution of Asarum liaoxilin for reaction, and then a chloride solution is added and reacted to obtain the AuNPs-modified Asarum liaoxilin specific upstream primer.

5. The reagent according to claim 4, characterized in that The molar ratio of the AuNPs to the upstream primer dd-AH-F of Asarum liaoxi is 1:300-500.

6. A fluorescence detection method for detecting Asarum species, characterized in that: The detection is performed using the reagent described in any one of claims 1 to 5.

7. The fluorescence detection method according to claim 6, characterized in that: The following steps are involved: The AuNPs-modified Asarum-specific upstream primer and the fluorescent marker-modified Asarum-specific downstream primer are mixed with a DNA template solution containing the Asarum species to be detected for PCR reaction amplification, and the fluorescence spectrum value of the obtained product is detected to achieve qualitative or quantitative detection of the Asarum species.

8. The fluorescence detection method according to claim 7, characterized in that: The standard curve for quantitative detection is prepared by the following method: a series of Asarum species DNA templates with different concentrations are amplified by PCR system, and the fluorescence intensity value of the obtained product is detected at an excitation wavelength of 610 nm and an emission wavelength of 670 nm, and the logarithm of the concentration of the Asarum species DNA template is used as the horizontal axis and the logarithm of the concentration of the Asarum species DNA template is used as the horizontal axis. F 0 -F is the standard curve of the ordinate, where F 0 is the fluorescence intensity value of the blank, F is the fluorescence intensity value of the obtained product.

9. The fluorescence detection method according to claim 8, characterized in that: The concentration of the Asarum species DNA template was 0.1×10 -4 -0.4×10 1 ng / μL.

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