Detection method of tobacco mosaic virus nucleic acid interferon

Abstract

The invention discloses a tobacco mosaic virus nucleic acid interferon detection method, which comprises: S1, preparing a solution and a sample solution: weighing a proper amount of a tobacco mosaic virus nucleic acid interferon parent drug, and adding a diluent to dilute and dissolve the tobacco mosaic virus nucleic acid interferon parent drug; a standard sample solution: weighing a proper amount of a tobacco mosaic virus nucleic acid interferon standard sample, and adding a diluent for dilution and dissolution; s2, detecting, namely detecting by adopting a liquid chromatograph, sequentially injecting a standard sample solution, a sample solution, a sample solution and a standard sample solution, recording a chromatogram, and calculating according to an external standard method; chromatographic conditions are as follows: the detection wavelength is 260 nm; the flow rate is 0.5 to 1.0 mL / min; the column temperature is 25-50 DEG C; the sample injection volume is 5-30 [mu] L; a mobile phase is ultrapure water; a PDA detector; a chromatographic column is selected from one of a volume exclusion chromatographic column taking monodisperse porous silica gel as a matrix, a chemically bonded silica gel column of alkyl silane and an ion exchange column. The method has the advantages of good linear correlation, good specificity, good separation degree, high precision, high accuracy, good durability and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid pesticide analysis, and particularly relates to a detection method for tobacco mosaic virus nucleic acid interferon. Background Art

[0002] Tobacco mosaic virus is a single-stranded RNA virus with extremely strong infectivity and pathogenicity to plants. Tobacco mosaic virus is widely distributed in various tobacco-growing areas in China and is the main viral disease of tobacco. At present, the prevention and control of tobacco mosaic virus mainly uses chemical pesticides, but the drug resistance of the virus caused by chemical pesticides is becoming increasingly obvious. Correspondingly, the dosage of chemical pesticides has also increased year by year. This situation not only leads to an increase in the cost of virus prevention and control, but also causes a large amount of pesticide residues.

[0003] Tobacco mosaic virus nucleic acid interferon is a nucleic acid pesticide based on RNA interference technology, which only acts on tobacco mosaic virus and has excellent control effects and strong selectivity, and has no effect on organisms such as bees and earthworms and beneficial microorganisms such as photosynthetic bacteria and nitrogen-fixing bacteria in the soil. At present, tobacco mosaic virus nucleic acid interferon products have been successfully developed, such as the nucleic acid pesticide against tobacco mosaic virus disclosed in patent CN113717984A.

[0004] The tobacco mosaic virus nucleic acid interferon is double-stranded RNA. In the prior art, it is often detected by a gel imager, thin-layer chromatography, and a Q-PCR instrument. However, when using a gel imager for determination, only qualitative analysis can be performed by observing the brightness of the gel image, and the quantification is inaccurate; when using thin-layer chromatography for detection, only the presence or absence of substances can be judged, and quantification is impossible; when using a Q-PCR instrument for detection, transcription is required, the steps are relatively cumbersome, and a quantitative kit needs to be purchased. And the Nanodrop instrument for determining the content is only equipped with single-stranded RNA and DNA detection, without double-stranded RNA detection.

[0005] In summary, there is no literature report on the quantitative analysis method of tobacco mosaic virus nucleic acid interferon at present, and the existing detection methods cannot perform efficient and accurate quality detection on tobacco mosaic virus nucleic acid interferon, and it is difficult to control the quality of tobacco mosaic virus nucleic acid interferon products during use. Summary of the Invention

[0006] To solve the above problems existing in the prior art, the present invention provides a detection method for tobacco mosaic virus nucleic acid interferon with linearity, precision, and accuracy all meeting the requirements.

[0007] The technical solution adopted by the present invention includes the following steps:

[0008] A detection method for tobacco mosaic virus nucleic acid interferon includes the following steps:

[0009] S1: Prepare the solution

[0010] Sample solution: Weigh an appropriate amount of the mother drug of tobacco mosaic virus nucleic acid interferon, add a diluent and dilute and dissolve it to obtain the sample solution;

[0011] Standard sample solution: Weigh an appropriate amount of the standard sample of tobacco mosaic virus nucleic acid interferon, add a diluent and dilute and dissolve it to obtain the standard sample solution;

[0012] S2: Detection

[0013] Use a liquid chromatograph for detection. Inject samples in the order of the standard sample solution, the sample solution, the sample solution, and the standard sample solution in sequence, record the chromatogram, and calculate by the external standard method; The chromatographic conditions are as follows:

[0014] Detector: PDA detector; Detection wavelength: 260 nm; Flow rate: 0.5 - 1.0 mL / min; Column temperature: 25 - 50 °C; Injection volume: 5 - 30 μL; Mobile phase: ultrapure water;

[0015] The chromatographic column is selected from one of a size exclusion chromatographic column based on monodisperse porous silica gel, a chemically bonded silica gel column of alkylsilane, and an ion exchange column;

[0016] The diluent in step S1 is the same as the mobile phase in step S2.

[0017] Further, the pore size of the size exclusion chromatographic column based on monodisperse porous silica gel is 100 - 3000; The ion exchange column is DNA-STAT; The chemically bonded silica gel column of alkylsilane is DNAPacRP or DNACore1000C18; Preferably, the chromatographic column uses an SEC-1000 chromatographic column, and the chromatographic specification is 7.8×300 mm, 5 μm.

[0018] Further, the concentration range of the standard sample solution and the sample solution is 91.212 mg / L - 912.116 mg / L; Preferably, the concentration of the standard sample solution and the sample solution is 500 mg / L.

[0019] Further, the pH of the ultrapure water is adjusted to 7.0 - 9.0 with a sodium hydroxide solution; Preferably, the pH of the ultrapure water is adjusted to 8.3 with a sodium hydroxide solution.

[0020] Preferably, the injection volume is 5 μL.

[0021] Preferably, the column temperature is 30 °C.

[0022] Preferably, the flow rate is 0.8 mL / min.

[0023] The beneficial effects of the present invention are as follows:

[0024] 1. The present invention uses high performance liquid chromatography for the quantitative detection of tobacco mosaic virus nucleic acid interferon and determines its chromatographic conditions. This method can achieve effective elution in a relatively short time, avoid interference, and is easy to operate, realizing the efficient and accurate detection of tobacco mosaic virus nucleic acid interferon.

[0025] 2. The detection method of the present invention has the advantages of good linear correlation, good specificity, good resolution, high precision, high accuracy, and good durability. By using the detection method of the present invention, better quality control can be carried out during the popularization and use of tobacco mosaic virus nucleic acid interferon products. Description of the Drawings

[0026] Figure 1 It is the liquid chromatogram of the linear solution STD1 of the tobacco mosaic virus nucleic acid interferon standard sample.

[0027] Figure 2 It is the liquid chromatogram of the linear solution STD2 of the tobacco mosaic virus nucleic acid interferon standard sample.

[0028] Figure 3 It is the liquid chromatogram of the linear solution STD3 of the tobacco mosaic virus nucleic acid interferon standard sample.

[0029] Figure 4 It is the liquid chromatogram of the linear solution STD4 of the tobacco mosaic virus nucleic acid interferon standard sample.

[0030] Figure 5 It is the liquid chromatogram of the linear solution STD5 of the tobacco mosaic virus nucleic acid interferon standard sample.

[0031] Figure 6 It is the relationship diagram between the peak area and mass concentration of the tobacco mosaic virus nucleic acid interferon.

[0032] Figure 7 It is the liquid chromatogram of the precision solution TK-A1 of the tobacco mosaic virus nucleic acid interferon mother drug.

[0033] Figure 8 It is the liquid chromatogram of the precision solution TK-A2 of the tobacco mosaic virus nucleic acid interferon mother drug.

[0034] Figure 9 It is the liquid chromatogram of the precision solution TK-A3 of the tobacco mosaic virus nucleic acid interferon mother drug.

[0035] Figure 10 It is the liquid chromatogram of the precision solution TK-A4 of the tobacco mosaic virus nucleic acid interferon mother drug.

[0036] Figure 11 It is the liquid chromatogram of the precision solution TK-A5 of the tobacco mosaic virus nucleic acid interferon mother drug.

[0037] Figure 12 This is the liquid chromatogram of tobacco mosaic virus nucleic acid interferon mother drug accuracy solution TK-B1.

[0038] Figure 13 This is the liquid chromatogram of the tobacco mosaic virus nucleic acid interferon mother drug accuracy solution TK-B2.

[0039] Figure 14 This is the liquid chromatogram of tobacco mosaic virus nucleic acid interferon mother drug accuracy solution TK-B3.

[0040] Figure 15 This is the liquid chromatogram of tobacco mosaic virus nucleic acid interferon mother drug accuracy solution TK-B4.

[0041] Figure 16 This is a liquid chromatogram of the tobacco mosaic virus nucleic acid interferon mother drug accuracy solution TK-B5. DETAILED DESCRIPTION

[0042] The technical solution of the invention is further described in detail below with reference to specific embodiments in conjunction with the accompanying drawings. It should be understood that the following embodiments are only used to illustrate the invention rather than to limit the scope of the invention.

[0043] The sources of materials and main instrument specifications used in the following examples are as follows:

[0044] (1) Materials:

[0045] The tobacco mosaic virus nucleic acid interferon standard sample (Silicon Yi Technology (Shanghai) Co., Ltd., batch number: 20230409) has a mass fraction of 86.0%; the tobacco mosaic virus nucleic acid interferon parent drug (Silicon Yi Technology (Shanghai) Co., Ltd., batch number: 20230427) has a mass fraction to be tested.

[0046] The tobacco mosaic virus nucleic acid interferon described below is the anti-tobacco mosaic virus nucleic acid pesticide disclosed in CN113717984A "A nucleic acid pesticide against tobacco mosaic virus and its synthesis, purification and application".

[0047] (2) Main instruments: LC-20AD Shimadzu high performance liquid chromatograph, PDA detector.

[0048] Example 1

[0049] S1: Prepare solution

[0050] Sample solution: weigh 50 mg of tobacco mosaic virus interferon nucleic acid stock drug, add ultrapure water with a pH of 8.3 to dilute and dissolve, make up to 100 mL, and shake well;

[0051] Standard sample solution: Weigh 50 mg of the standard sample of tobacco mosaic virus nucleic acid interferon, add ultrapure water with a pH of 8.3 to dilute and dissolve it, make the volume up to 100 mL, and shake well to obtain it;

[0052] S2: Detection

[0053] Perform detection using a liquid chromatograph, and the chromatographic conditions are as follows:

[0054] Use a SEC-1000 chromatographic column; the specifications of the chromatographic column are 7.8 * 300 mm, 5 μm; detector: PDA detector; detection wavelength: 260 nm; flow rate: 0.8 mL / min; column temperature: 30 °C; injection volume: 5 μL; mobile phase: ultrapure water with a pH of 8.3.

[0055] Under the above chromatographic conditions, inject samples in the order of standard sample solution, sample solution, sample solution, and standard sample solution in sequence, record the chromatogram, and calculate by the external standard method.

[0056] Example 2

[0057] S1: Prepare the solution

[0058] Sample solution: Weigh 50 mg of the mother drug of tobacco mosaic virus nucleic acid interferon, add ultrapure water with a pH of 7.0 to dilute and dissolve it, make the volume up to 100 mL, and shake well to obtain it;

[0059] Standard sample solution: Weigh 50 mg of the standard sample of tobacco mosaic virus nucleic acid interferon, add ultrapure water with a pH of 7.0 to dilute and dissolve it, make the volume up to 100 mL, and shake well to obtain it;

[0060] S2: Detection

[0061] Use a DNA-STAT chromatographic column; detector: PDA detector; detection wavelength: 260 nm; flow rate: 0.5 mL / min; column temperature: 50 °C; injection volume: 15 μL; mobile phase: ultrapure water with a pH of 7.0. Under the above chromatographic conditions, inject samples in the order of standard sample solution, sample solution, sample solution, and standard sample solution in sequence, record the chromatogram, and calculate by the external standard method.

[0062] Example 3

[0063] S1: Prepare the solution

[0064] Sample solution: Weigh 50 mg of the mother drug of tobacco mosaic virus nucleic acid interferon, add ultrapure water with a pH of 9.0 to dilute and dissolve it, make the volume up to 100 mL, and shake well to obtain it;

[0065] Standard sample solution: Weigh 50 mg of the standard sample of tobacco mosaic virus nucleic acid interferon, add ultrapure water with a pH of 9.0 to dilute and dissolve it, make the volume up to 100 mL, and shake well to obtain it;

[0066] S2: Detection

[0067] Use a DNACore1000C18 chromatographic column; detector: PDA detector; detection wavelength: 260 nm; flow rate: 1.0 mL / min; column temperature: 25 °C; injection volume: 30 μL; mobile phase: ultrapure water with a pH of 9.0.

[0068] Under the above chromatographic conditions, inject samples in the order of standard solution, sample solution, sample solution, and standard solution in sequence, record the chromatogram, and calculate by the external standard method.

[0069] Example 4

[0070] S1: Prepare the solution

[0071] Sample solution: Weigh 50 mg of the mother drug of tobacco mosaic virus nucleic acid interferon, add ultrapure water with a pH of 8.3 to dilute and dissolve it, make up the volume to 100 mL, and shake well to obtain it;

[0072] Standard solution: Weigh 50 mg of the standard sample of tobacco mosaic virus nucleic acid interferon, add ultrapure water with a pH of 8.3 to dilute and dissolve it, make up the volume to 100 mL, and shake well to obtain it;

[0073] S2: Detection

[0074] Use a liquid chromatograph for detection, and its chromatographic conditions are as follows:

[0075] Use a DNAPacRP chromatographic column; detector: PDA detector; detection wavelength: 260 nm; flow rate: 0.8 mL / min; column temperature: 30 °C; injection volume: 5 μL; mobile phase: ultrapure water with a pH of 8.3.

[0076] Under the above chromatographic conditions, inject samples in the order of standard solution, sample solution, sample solution, and standard solution in sequence, record the chromatogram, and calculate by the external standard method.

[0077] Under the different chromatographic conditions of the above Examples 1-4 for detection, rapid elution, interference avoidance, and quantitative detection of tobacco mosaic virus nucleic acid interferon can be achieved. Among them, the elution effect and resolution of the detection results under the chromatographic conditions of Example 1 are the best. Therefore, the preferred chromatographic conditions of the present invention are as follows:

[0078] Chromatographic column: SEC-1000; specification of the chromatographic column: 7.8 * 300 mm, 5 μm; detector: PDA detector; detection wavelength: 260 nm; flow rate: 0.8 mL / min; column temperature: 30 °C; injection volume: 5 μL; mobile phase: ultrapure water with a pH of 8.3.

[0079] The linearity, precision and accuracy of the detection method in Example 1 of the methodological verification are adopted below. The instruments and chromatographic conditions in the following examples are the same as those in Example 1.

[0080] Example 5. Linearity test

[0081] Weigh 0.5303 g of the tobacco mosaic virus nucleic acid interferon standard sample, place it in a 100 mL volumetric flask, dissolve it with water and dilute it to the mark, shake well, and use it as the stock solution. Respectively transfer 0.2 mL, 0.5 mL, 1 mL, 1.5 mL, and 2 mL of the stock solution into 10 mL volumetric flasks, dilute them to the mark with water, shake well, and obtain the linear solutions of the tobacco mosaic virus nucleic acid interferon standard sample, which are respectively labeled as STD1 - STD5.

[0082] Under the operating conditions of Example 1, after the instrument is stable, measure the peak areas of the tobacco mosaic virus nucleic acid interferon in each solution in the order of STD1 - STD5. The liquid chromatogram is as Figures 1-5 shown. Take the average result of two measurements. Respectively plot the standard curve with the mass concentration of the tobacco mosaic virus nucleic acid interferon as the abscissa and the peak area as the ordinate. The test results are shown in Figure 6 and Table 1, where the mass fraction of the tobacco mosaic virus nucleic acid interferon standard sample is 86.0%.

[0083] Table 1. Determination of the linear range of the tobacco mosaic virus nucleic acid interferon - liquid chromatography

[0084]

[0085] It can be seen from Table 1 and Figure 6 that when the mass concentration of the tobacco mosaic virus nucleic acid interferon is between 91.212 mg / L - 912.116 mg / L, there is a good linear relationship between the mass concentration x (mg / L) of the tobacco mosaic virus nucleic acid interferon and the corresponding peak area y of the tobacco mosaic virus nucleic acid interferon. The regression equation is calculated as y = 3219.8x + 3260.2, and the correlation coefficient r = 0.9999, which fully meets the requirements of quantitative analysis.

[0086] Example 6. Precision test

[0087] Weigh 0.05 g (accurate to 0.0001 g) of the tobacco mosaic virus nucleic acid interferon standard sample, place it in a 100 mL volumetric flask, dissolve it with water and make up the volume to the mark, shake well, and use it as the standard sample solution, labeled as STD.

[0088] Weigh five portions of 0.06 g (accurate to 0.0001 g) of the mother drug of tobacco mosaic virus nucleic acid interferon, place them in 5 100-mL volumetric flasks respectively, dissolve with water and make up to the mark, shake well, and use them as the precision solutions of the mother drug of tobacco mosaic virus nucleic acid interferon, which are respectively labeled as TK-A1 to TK-A5.

[0089] Under the operating conditions of Example 1, after the instrument baseline is stable, carry out the determination in the order of the standard solution, precision solution, precision solution, and standard solution. The liquid chromatogram is as Figures 7-11 shown. The test results are shown in Table 2, in which the mass fraction of the tobacco mosaic virus nucleic acid interferon standard sample is 86.0%.

[0090] Table 2. Test results of the precision of the mother drug of tobacco mosaic virus nucleic acid interferon

[0091]

[0092] It can be seen from Table 2 that the mass fraction of the mother drug of tobacco mosaic virus nucleic acid interferon measured by the method of the present invention is 83.98%, and the RSD of the measurement result is 0.50%, which is less than the modified Horwitz formula 2(1 - 0.5logC)×0.67 = 1.38, indicating that the measurement result of the precision of the active ingredient analysis method meets the requirements.

[0093] Example 7. Accuracy test

[0094] Weigh 0.6320 g (accurate to 0.0001 g) of the tobacco mosaic virus nucleic acid interferon standard sample, place it in a 100-mL volumetric flask, dissolve with water and make up to the mark, ultrasonic oscillate for 20 min, cool to room temperature, and shake well to use as the mother liquor.

[0095] Weigh 0.05 g (accurate to 0.0001 g) of the tobacco mosaic virus nucleic acid interferon standard sample, place it in a 100-mL volumetric flask, dissolve with water and make up to the mark, shake well, and use it as the standard solution, which is labeled as STD.

[0096] Weigh five portions of 0.036 g (accurate to 0.00001 g) of the mother drug of tobacco mosaic nucleic acid interferon, place them in 100-mL volumetric flasks respectively, accurately pipette 1.0 mL of the mother liquor with a pipette and add it, dissolve with water and make up to the mark, shake well, and use them as the accuracy solutions of the mother drug of tobacco mosaic nucleic acid interferon, which are respectively labeled as TK-B1 to TK-B5. The specific sample masses are shown in Table 3 below.

[0097] Under the operating conditions of Example 1 above, after the instrument baseline is stable, carry out the determination in the order of the standard solution, accuracy solution, accuracy solution, and standard solution. The liquid chromatogram is as Figures 12-16As shown in the figure, the test results are shown in Table 3. Among them, the mass fraction of the tobacco mosaic virus nucleic acid interferon standard sample is 86%, and the mass fraction of the tobacco mosaic virus nucleic acid interferon mother drug is 83.98%.

[0098] Table 3. Test results of the recovery rate of the tobacco mosaic virus nucleic acid interferon mother drug

[0099]

[0100]

[0101] It can be seen from Table 3 that the average recovery rate of the tobacco mosaic virus nucleic acid interferon mother drug is 99.65%, indicating good accuracy.

[0102] In summary, the detection method of the tobacco mosaic virus nucleic acid interferon provided by the present invention has the advantages of good linear correlation, good resolution, high precision and high accuracy, effectively solving the problem of inability to control the quality of the tobacco mosaic virus nucleic acid interferon.

[0103] It should be understood that the above specific embodiments of the present invention are only used to illustrate or explain the technical solutions of the present invention, rather than limiting the present invention. Therefore, any modifications, equivalent replacements or improvements made without departing from the spirit and scope of the present invention shall be included in the protection scope of the present invention.

Claims

1. A detection method for tobacco mosaic virus nucleic acid interferon, characterized in that, it comprises the following steps: S1: Prepare solutions Sample solution: Weigh an appropriate amount of the mother drug of tobacco mosaic virus nucleic acid interferon, and add a diluent to dilute and dissolve it to obtain; Standard sample solution: Weigh an appropriate amount of the standard sample of tobacco mosaic virus nucleic acid interferon, and add a diluent to dilute and dissolve it to obtain; S2: Detection Use a liquid chromatograph for detection. Inject samples in the order of the standard sample solution, the sample solution, the sample solution, and the standard sample solution in sequence, record the chromatogram, and calculate by the external standard method; The chromatographic conditions are as follows: Detector: PDA detector; Detection wavelength: 260 nm; Flow rate: 0.5 - 1.0 mL / min; Column temperature: 25 - 50 °C; Injection volume: 5 - 30 μL; Mobile phase: ultrapure water; The chromatographic column is selected from one of a size exclusion chromatographic column based on monodisperse porous silica gel, a chemically bonded silica gel column of alkylsilane, and an ion exchange column; The diluent in step S1 is the same as the mobile phase in step S2.

2. The detection method for tobacco mosaic virus nucleic acid interferon according to claim 1, characterized in that, the pore size of the size exclusion chromatographic column is 100 - 3000; the ion exchange column is DNA-STAT; the silica gel column is DNAPacRP or DNACore1000C18.

3. The detection method for tobacco mosaic virus nucleic acid interferon according to claim 1, characterized in that, the concentration range of the standard sample solution and the sample solution is 91.212 mg / L - 912.116 mg / L.

4. The detection method for tobacco mosaic virus nucleic acid interferon according to claim 1, characterized in that, the pH of the ultrapure water is adjusted to 7.0 - 9.0 with a sodium hydroxide solution.

5. The detection method for tobacco mosaic virus nucleic acid interferon according to claim 1, characterized in that, the concentration of the standard sample solution and the sample solution is 500 mg / L.

6. The detection method for tobacco mosaic virus nucleic acid interferon according to claim 1, characterized in that, the pH of the ultrapure water is adjusted to 8.3 with a sodium hydroxide solution.

7. The detection method for tobacco mosaic virus nucleic acid interferon according to claim 1, characterized in that, the chromatographic column uses an SEC-1000 chromatographic column, and the chromatographic specifications are 7.8×300 mm, 5 μm.

8. The detection method for tobacco mosaic virus nucleic acid interferon according to claim 1, characterized in that, the injection volume is 5 μL.

9. The detection method for tobacco mosaic virus nucleic acid interferon according to claim 1, characterized in that, the column temperature is 30 °C.

10. The detection method for tobacco mosaic virus nucleic acid interferon according to claim 1, characterized in that, the flow rate is 0.8 mL / min.

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