Application of PCR (polymerase chain reaction)-based ratio-type fluorescent system in early detection of TMV (tobacco mosaic virus)

Abstract

The invention belongs to the technical field of plant virus detection, and particularly relates to application of a PCR (polymerase chain reaction)-based ratiometric fluorescent system in early detection of TMV (tobacco mosaic virus). According to the invention, a method for rapidly and quantitatively detecting the tobacco mosaic disease in the early stage is established by utilizing a primer marked by a fluorescent dye tetramethyl rhodamine (TAMRA) and a nucleic acid dye SYBR Green I (SG). The SG has excellent photophysical characteristics, is low in cost, simple and easy to use, can specifically recognize double-helix DNA, and becomes a real-time detection fluorescent dye which is most widely used so far. Compared with a traditional single-signal fluorescence method, the ratio-type fluorescence sensor has the characteristics of high sensitivity and high accuracy. Meanwhile, in order to verify the value of the detection method in practical application, potting and field tobacco leaf samples are selected for analysis, and satisfactory effects are achieved. Therefore, the tobacco mosaic disease can be rapidly and accurately detected at an early stage by combining a ratio-type fluorescence method and a PCR amplification technology, and the primer and the kit have a good application prospect in tobacco virus disease detection.

Description

Technical Field

[0001] The invention belongs to the technical field of plant virus detection, relates to TMV detection, and particularly to application of a PCR-based ratiometric fluorescence system in early TMV detection. Background Art

[0002] As one of the important economic crops in my country, tobacco has the characteristics of large planting area, wide distribution and high income. Tobacco virus disease is the most widely distributed, most common and most harmful disease in tobacco production in my country. The incidence of diseased plants in the field is generally 20%-40%, and can reach up to 40%-80%. There are 16 tobacco virus diseases found in my country, among which tobacco common mosaic disease (TMV), cucumber mosaic disease (CMV) and potato virus Y (PVY) have the highest incidence. Once tobacco is infected with the virus, the chlorophyll in the cells is destroyed, the leaves cannot photosynthesize, and the growth is inhibited, which seriously affects the yield and quality of tobacco leaves, resulting in reduced tobacco yield and economic benefits. So far, there is no effective chemical control measure, and effective therapeutic agents for infected tobacco plants are even more scarce. Therefore, it is urgent to establish a tobacco virus disease detection method with strong sensitivity and high accuracy.

[0003] At present, the detection of tobacco virus diseases mainly includes immunological methods and molecular biological methods. Enzyme-linked immunosorbent assay (ELISA) in immunological methods is widely used in tobacco virus detection, but it has disadvantages such as long time consumption, many interference factors, and easy false positives; reverse transcription PCR reaction (RT-PCR) in molecular biological methods is often used in tobacco virus detection due to its high sensitivity; and the fluorescent quantitative PCR (qRT-PCR) method has the advantages of high sensitivity, strong specificity, and real-time rapid quantitative detection of samples, and has been used in the effective detection of a variety of plant viruses. Patent CN201410118999.X discloses a colloidal gold immunochromatographic test paper for detecting tobacco mosaic virus, which has the advantages of high sensitivity, strong specificity, rapidity, and simplicity; Patent CN202310853578.0 discloses a kit for visual detection of tobacco mosaic disease, which has high accuracy and strong potential for field application.

[0004] Fluorescence spectroscopy refers to the use of certain substances to obtain energy in an excited state when irradiated with ultraviolet light, and the excited state molecules undergo collisions and de-excitation processes to re-emit fluorescence with the characteristics of the substance. Fluorescence analysis has the characteristics of high sensitivity, strong selectivity, small sample size, high resolution, and simple operation. Most fluorescence spectroscopy methods output a single fluorescent signal for the detection target, which is easily interfered by temperature, pH, ion concentration, etc., while ratio fluorescence can solve the problems faced by single signal output. Ratio fluorescence spectroscopy calculates the fluorescence intensity ratio of two emission spectra as the output signal to achieve quantitative analysis of the sample to be tested, which can eliminate background signal interference. Patent CN201811527437.5 discloses a fluorescent ratio probe constructed using a labeled nucleic acid aptamer and a nucleic acid quadruplex dye. The nucleic acid aptamer can specifically identify ochratoxin A, reduce the experimental error caused by the traditional method of detecting a fluorescent signal once, and the process is controllable, simple to operate, and highly sensitive. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes an application of a PCR-based ratiometric fluorescence system in the early detection of TMV.

[0006] The technical solution of the present invention is achieved in this way: On the one hand, the present invention provides a ratiometric fluorescence system based on PCR, including a forward primer F, a reverse primer R-TAMRA, a nucleic acid dye SG working solution, 2×Taq Plus Master Mix and a PBS buffer.

[0007] Preferably, the nucleotide sequence of the forward primer F is as shown in SEQ ID No. 1; the nucleotide sequence of the reverse primer R-TAMRA is as shown in SEQ ID No. 2, and its 5' end is modified with a TAMRA group; the volume ratio of the forward primer F and the reverse primer R-TAMRA is 1:1.

[0008] Preferably, the PBS buffer has a concentration of 10 mmol / L, a pH of 7.4, and a NaCl concentration of 100 mmol / L.

[0009] On the other hand, the application of the PCR-based ratiometric fluorescence system in the early detection of TMV is as follows: (1) Using the cDNA of the leaves of the sample to be tested as a template and the primer pair F and R-TAMRA as primers, PCR is performed to obtain a reaction product; (2) The reactants were mixed with the nucleic acid dye SG working solution, and the resulting reaction solution was diluted with PBS buffer, and then the fluorescence signal intensity was measured and compared. SG / I TAMRAThe change in fluorescence intensity was substituted into the linear regression equation to calculate the cDNA concentration of TMV in the sample to be tested, thereby achieving quantitative detection of TMV.

[0010] Preferably, the volume ratio of the above reactants to the nucleic acid dye SG working solution is 1:3.

[0011] Specifically, the amplification system of the above PCR is 50 μL, wherein the volume of the forward primer F and the reverse primer R-TAMRA are both 2 μL and the concentration is 15 μmol / L, the volume of the sample to be tested is 5 μL, the volume of 2×Taq Plus Master Mix is ​​25 μL, and the volume of ddH2O is 16 μL; the amplification program of the PCR is: 95°C 3 min; 95°C 30 s, 60°C 30 s, 72°C 1 min, 35-45 cycles; 72°C 7 min; and stored at 4°C.

[0012] Specifically, the above linear regression equation is: SG / I TAMRA =0.1743lgc + 0.4928(R 2 =0.9965), and the detection limit of the method was 0.89 amol / L.

[0013] Furthermore, the detection parameter of the above fluorescence signal intensity is the wavelength interval △ λ=25 nm, slit width 5.0 nm, scanning speed 600 nm / min, wavelength scanning range 480 nm-640 nm.

[0014] The present invention has the following beneficial effects: 1. The present invention designs specific primers based on the tobacco mosaic virus coat protein gene reported in Genebank, and uses primers labeled with fluorescent dye tetramethylrhodamine (TAMRA) and nucleic acid dye SG to construct a PCR-based ratiometric fluorescence method for early detection of TMV.

[0015] 2. The application of the PCR-based ratiometric fluorescence system of the present invention in early detection of TMV has the following advantages: ① In this application, cDNA of cucumber mosaic virus, potato virus Y and turnip mosaic virus infected tobacco leaves were extracted and added to the reaction solution for amplification as a control group, and other conditions were kept the same. The system I corresponding to the three samples SG / I TAMRA The results were consistent with those of the blank group, with no significant changes, indicating that this method has extremely high selectivity for TMV-infected tobacco leaves.

[0016] ② This application detects I by extracting cDNA from healthy, slightly diseased and severely diseased tobacco leaves grown in pots SG / ITAMRA The results showed that the SG signal value of healthy tobacco leaves was very low, the SG intensity of severely diseased tobacco leaves was the strongest, and the slightly diseased tobacco leaves were the second; a plot was randomly sampled from the Xuchang Science and Education Park of Henan Agricultural University, and the tobacco leaf samples were measured using this system method and the gold standard method, respectively. The comparison showed that the results of the two tests were consistent. Therefore, this method has the advantage of high accuracy in detecting TMV. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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 work.

[0018] Figure 1 This is a schematic diagram of the principle of using PCR-based ratiometric fluorescence method for early detection of tobacco mosaic disease.

[0019] Figure 2 Gel electrophoresis analysis diagram and fluorescence spectrum diagram under different conditions; (A) is the gel electrophoresis analysis diagram, and (B) is the fluorescence spectrum diagram.

[0020] Figure 3 This is a diagram showing the effect of SG dosage on the measurement results.

[0021] Figure 4 This is a graph showing the effect of PCR cycle number on the measurement results.

[0022] Figure 5 For I SG / I TAMRA The relationship diagram between different concentrations of templates; (A) is the synchronous scanning fluorescence spectra of different concentrations of TMV templates, (B) is I SG / I TAMRA The linear relationship between the concentration and the logarithm of the target cDNA.

[0023] Figure 6 This is a specific analysis diagram of the ratiometric fluorescence method.

[0024] Figure 7 Schematic diagram of potted sample detection; (A) is a potted picture of healthy, slightly diseased, and severely diseased tobacco leaves, and (B) is a synchronous scanning fluorescence spectrum of healthy, slightly diseased, and severely diseased tobacco leaves.

[0025] Figure 8 Schematic diagram of field sample testing; (A) is a bird's-eye view of the plot, and (B) is a tobacco leaf sample collected from the field. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0028] The nucleotide sequences of the forward primer F and the reverse primer R-TAMRA in the embodiment of the present invention are shown in Table 1.

[0029] Table 1 DNA sequences used in the experiment The detection principle of the present invention is as follows Figure 1 As shown: First, extract RNA from diseased tobacco leaves, reverse transcribe it into cDNA, and use it as a template for PCR reaction. SG is a nucleic acid dye that binds to the minor groove of double-helix DNA and emits green fluorescence. When there is no TMV target gene in the solution, the TAMRA-modified reverse primer and forward primer are in a single-stranded state, and almost do not react with the free SG, showing a weak fluorescence signal. When the TMV-cDNA template is added, a large amount of target double-stranded DNA is generated, and the nucleic acid dye is inserted into the minor groove region of the double helix. The fluorescence of SG in the solution gradually increases. The more target genes there are, the more double strands there are after the PCR reaction, and the stronger the fluorescence of SG. Therefore, the fluorescence intensity ratio of SG to TAMRA in the test solution (I SG / I TAMRA ) to achieve quantitative detection of TMV.

[0030] Example 1 1. Materials and Methods: Cultivation of potted tobacco leaves: Select Nicotiana benthamiana seeds for cultivation, sterilize the prepared MS culture medium and ultrapure water at high temperature, pour the sterilized culture medium into the culture dish and let it stand for half an hour, put several seeds into the centrifuge tube in the clean bench, add 10% H2O2 and shake vigorously for ten minutes, wash the seeds three times with ultrapure water, gently pick up the seeds with a sterilized toothpick and place them on the culture medium, and then place the culture dish in a constant temperature and humidity incubator. The culture conditions are 25℃, 50% humidity, 16 h of light, and 8 h of darkness. After the seeds germinate, select the tobacco seedlings with better growth and transplant them into small pots for artificial cultivation. After the tobacco leaves have grown for 4 weeks, select healthy potted plants and use tobacco mosaic virus incubation solution for injection infection. The inoculated leaves are larger and lower leaves. Gently push the syringe to evenly inject the incubation solution into the entire tobacco leaf. After 3 weeks of inoculation, take severely diseased tobacco leaves and slightly diseased tobacco leaves for the test.

[0031] Extraction and reverse transcription of total RNA from tobacco leaves: The extraction and reverse transcription of total RNA from tobacco leaves were carried out according to the instructions of RNA extraction kit and RNA reverse transcription kit of Novazonic Biotechnology Co., Ltd.

[0032] 2. Detection of tobacco mosaic virus cDNA The PCR amplification system is shown in Table 2: Table 2 PCR amplification system The PCR program was set as 95°C for 3 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, and 45 cycles; 72°C for 7 min, and stored at 4°C. The amplified products were detected by 6% agarose gel electrophoresis.

[0033] 3. Fluorescence spectrum detection of tobacco mosaic disease: The TAMRA-labeled aptamer was prepared into a 100 µM stock solution with dd H2O, and 2 µL of 15 µmol / L forward and reverse primers and 5 µL of 100 pM target cDNA were added for PCR amplification. After the reaction, 10 µL of PCR solution was added to 30 µL of SG working solution, and diluted to a final volume of 3 mL with PBS buffer solution. After mixing evenly, it was placed in a water bath and incubated at 37°C for 15 min, and then gradually cooled to room temperature. The fluorescence spectrum of the measurement system was synchronously scanned, and the wavelength interval was set. △ λ=25 nm, slit width 5.0 nm, scanning speed 600 nm / min, wavelength scanning range 480 nm - 640 nm.

[0034] 4. Feasibility analysis In order to verify the feasibility of this method, agarose gel electrophoresis and fluorescence spectroscopy were used to detect the Figure 2 (A) As shown in the electrophoresis diagram, the marker is used to mark the size of the DNA molecular weight (band M). When there is no TMV-cDNA in the reaction solution, there is no characteristic band of the PCR product (band a). After adding the target gene, a band is generated at 105 bp (band b). By comparison, it can be seen that the position of the characteristic band of the template double strand is consistent (band c). Figure 2 (B) As shown in the fluorescence spectrum, in the absence of target cDNA, the TAMRA-labeled aptamer is in a single-stranded state and does not react with the aqueous solution of the nucleic acid dye SG, and the fluorescence signal intensity is low (curve Blank). After adding 1aM of target cDNA, SG enters the minor groove region of the double-stranded DNA and binds to it, and the SG fluorescence is enhanced (curve 1 aM). The further the template concentration is increased, the stronger the SG fluorescence signal is (curve 100 pM). The fluorescence intensity of the dye TAMRA does not change due to different concentrations of the target gene.

[0035] Example 2 The experimental conditions for the application of the PCR-based ratiometric fluorescence system in the early detection of TMV in this application are optimized: 1. SG dosage The fluorescence intensity was investigated under the condition of buffer solution pH = 7.4, reaction temperature 37℃, and light protection for 15 min. The template concentration added to the system was kept consistent and uniformly selected as 100 pmol / L. As the amount of SG increased, I SG / I TAMRA The value gradually increases until the volume of SG working solution reaches 30 µL, and then the amount of SG is increased. SG / I TAMRA There is no obvious change and the line is almost flat, so 30 µLSG is finally selected for subsequent experiments ( Figure 3 ).

[0036] 2. PCR cycle number Different PCR cycle numbers were investigated under the conditions of reaction temperature 37°C, reaction time 15 min, and template concentration 100 pmol / L. The results are as follows Figure 4 As shown, when the cycle is between 5 and 35, I SG / I TAMRA The ratio increases rapidly with the increase of the number of turns. When the number of turns is greater than 35, I SG / I TAMRA The ratio remains basically unchanged and the line is almost flat. This is because in the PCR reaction solution, increasing the number of cycles can generate more DNA double strands, but when the number of cycles is too large, the amount of SG fluorescence is limited and cannot be inserted into the redundant double helix structure. Therefore, the number of PCRs selected in this experiment is 40.

[0037] 3. Working curve Under known optimized conditions, I SG / I TAMRA The relationship between different template concentrations, Figure 5 (A) is the synchronous scanning fluorescence spectra of TMV templates with different concentrations. Figure 5 (B) is a linear relationship between ISG / ITAMRA and the logarithm of target cDNA concentration. As can be seen from the figure, in the range of 100 pmol / L to 1 amol / L, I SG / I TAMRA It has a good linear relationship with the TMV template concentration, and the fitted regression equation is I SG / I TAMRA =0.1743lgc + 0.4928(R 2 =0.9965), and the method detection limit was 0.89 amol / L (3σ / slope). Therefore, the ratiometric fluorescence dual-signal detection method has extremely high sensitivity.

[0038] 4. Selectivity of the method In order to improve the selectivity of the detection method, the cDNA of cucumber mosaic virus (CMV), potato virus Y (PVY) and turnip mosaic virus (TuMV) infected tobacco leaves were extracted as three templates and added to the reaction solution for amplification as a control group, while keeping other conditions the same. SG / I TAMRA The results are as follows Figure 6 As shown, the system I corresponding to the three samples SG / I TAMRA The results were consistent with those of the blank group, with no significant changes, indicating that this method has extremely high selectivity for TMV-infected tobacco leaves.

[0039] Application Examples Actual sample testing 1. Potted sample testing: In order to verify the accuracy of the method, this experiment used a light incubator to artificially cultivate tobacco leaves, from which Figure 7 The most representative healthy, slightly diseased and severely diseased tobacco leaves shown in (A) were tested. Figure 7 As can be seen from the fluorescence spectrum in (B), the SG signal value of healthy tobacco leaf a is very low, the SG intensity of severely diseased tobacco leaf c is the strongest, and the SG intensity of slightly diseased tobacco leaf b is the second. SG / I TAMRA The change in ratio enables quantitative detection of TMV.

[0040] 2. Field sample testing: A plot of land (about 6.5 mu) was selected from the Xuchang Science and Education Park of Henan Agricultural University for random sampling. The tobacco variety was Zhongyan 100, and 20 leaves were selected using the "Z-shaped sampling method". Figure 8 (A) is a top view of the plot. Figure 8 (B) Picture of 20 collected tobacco leaves. In order to verify the accuracy of the method, the tobacco leaf samples were measured using the system method and the gold standard method, and the results of the two methods were consistent. Therefore, the method has the advantages of high accuracy and strong sensitivity in detecting TMV.

[0041] Implementation effect analysis This application establishes a PCR-based ratiometric system for early detection of tobacco mosaic disease. SG is a non-toxic, low-background, highly sensitive asymmetric cyanine dye with excellent photophysical properties and temperature stability. It is low-cost and easy to use. It has become the most widely used real-time detection fluorescent dye to date. Compared with traditional fluorescence methods, ratiometric fluorescence sensors have the characteristics of high sensitivity and high accuracy. Under the optimal experimental conditions, within the range of 100 pmol / L to 1 amol / L, I SG / I TAMRA It has a good linear relationship with the TMV template concentration, and the fitted regression equation is I SG / I TAMRA =0.1743lgc + 0.4928 (R 2 =0.9965), the detection limit of the method was 0.89 aM. At the same time, in order to verify the value of this detection method in practical applications, artificially cultivated tobacco plants and field samples were selected for analysis, and satisfactory results were achieved. Therefore, the combination of fluorescent dual signal platform and PCR amplification technology can quickly and accurately detect tobacco mosaic disease at an early stage, and has a good application prospect in the detection of tobacco virus diseases.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A ratiometric fluorescence system based on PCR, characterized in that: It includes forward primer F, reverse primer R-TAMRA, nucleic acid dye SG working solution, 2×Taq Plus Master Mix and PBS buffer.

2. The PCR-based ratiometric fluorescence system according to claim 1, characterized in that: The nucleotide sequence of the forward primer F is shown in SEQ ID No. 1; the nucleotide sequence of the reverse primer R-TAMRA is shown in SEQ ID No. 2, and its 5' end is modified with a TAMRA group; wherein the volume ratio of the forward primer F to the reverse primer R-TAMRA is 1:

1.

3. The PCR-based ratiometric fluorescence system according to claim 1, characterized in that: The PBS buffer has a concentration of 10 mmol / L, a pH of 7.4, and a NaCl concentration of 100 mmol / L.

4. Use of the PCR-based ratiometric fluorescence system according to any one of claims 1 to 3 in early detection of TMV.

5. The use of the PCR-based ratiometric fluorescence system in early detection of TMV according to claim 4, characterized in that: Here are the steps: (1) Using the cDNA of the leaves of the sample to be tested as a template and the primer pair F and R-TAMRA as primers, PCR is performed to obtain a reaction product; (2) The reactants were mixed with the nucleic acid dye SG working solution, and the resulting reaction solution was diluted with PBS buffer, and then the fluorescence signal intensity was measured and compared. SG / I TAMRA The change in fluorescence intensity was substituted into the linear regression equation to calculate the cDNA concentration of TMV in the sample to be tested, thereby achieving quantitative detection of TMV.

6. The use of the PCR-based ratiometric fluorescence system in early detection of TMV according to claim 5, characterized in that: The volume ratio of the reactant to the nucleic acid dye SG working solution is 1:

3.

7. The use of the PCR-based ratiometric fluorescence system in early detection of TMV according to claim 6, characterized in that: The PCR amplification system is 50 μL, wherein the volume of the forward primer F and the reverse primer R-TAMRA are both 2 μL, the concentration is both 15 μmol / L, the volume of the sample to be tested is 5 μL, the volume of 2×Taq Plus Master Mix is ​​25 μL, and the volume of ddH2O is 16 μL.

8. The use of the PCR-based ratiometric fluorescence system in early detection of TMV according to claim 7, characterized in that: The PCR amplification program is: 95°C for 3 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, 35-45 cycles; 72°C for 7 min; and storage at 4°C.

9. The use of the PCR-based ratiometric fluorescence system in early detection of TMV according to claim 8, characterized in that: The linear regression equation is: SG / I TAMRA =0.1743 lgc + 0.4928, linear regression coefficient R 2 =0.9965, and the detection limit is 0.89 amol / L.

10. The use of the PCR-based ratiometric fluorescence system in early detection of TMV according to claim 9, characterized in that: The detection parameter of the fluorescence signal intensity is the wavelength interval △ λ=25 nm, slit width 5.0 nm, scanning speed 600 nm / min, wavelength scanning range 480 nm-640 nm.

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