Method for detecting blending homogeneity of formula tobacco by using fluorescent quantitative PCR (Polymerase Chain Reaction)

Abstract

The invention belongs to the technical field of tobacco processing detection, and particularly relates to a method for detecting formula tobacco blending homogeneity by using fluorescent quantitative PCR (Polymerase Chain Reaction), which comprises the following steps: selecting two tobacco varieties according to a cigarette formula, respectively screening out specific gene segments, and designing fluorescent quantitative PCR primers according to the specific gene segments; then determining the standard proportion of the DNAs of the two tobacco varieties in the cigarette formula, comparing the standard proportion with the DNA concentration of the tobacco shreds of the cigarette formula to be detected on the production line to determine whether the DNA concentration is within the standard range, and otherwise, judging that the blending is non-uniform. According to the technical scheme, the fluorescent quantitative PCR primer is used for detecting the mixing of two tobacco varieties at the same time, so that the detection accuracy is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco processing detection, and in particular relates to a method for detecting the homogeneity of formula tobacco blending by using fluorescent quantitative PCR. Background Art

[0002] Leaf group formula is usually made by mixing tobacco leaves of different varieties, origins, parts and grades in a specific proportion. In the whole process of cigarette production, there is a material mixing process through leaf beating and re-baking, leaf blending, loosening and moisture recovery, leaf shreds blending and other processes, which can easily cause fluctuations in the mixing ratio of tobacco materials and affect the quality of cigarette products. Therefore, the actual blending homogeneity of the varieties of cigarette formula has become an important evaluation indicator in the cigarette processing process.

[0003] Multiple tobacco varieties are usually used in cigarette formulas. Different varieties of tobacco leaves have great differences in chemical composition, physical properties and sensory quality. The reasonable combination of different varieties of tobacco leaves gives cigarette products unique style and quality characteristics. In the process of cigarette processing, different varieties of tobacco leaves are finally prepared into formula tobacco through many processes such as leaf beating, re-baking, blending, and shredding. In order to ensure the quality of cigarettes, it is necessary to accurately detect the actual proportion of the final formula tobacco varieties to ensure that it is consistent with the required proportion of the cigarette formula. However, due to the similar morphology of different varieties of tobacco leaves and tobacco leaves in the end, it is difficult to distinguish different varieties of tobacco leaves in the formula tobacco. At present, only a rough qualitative judgment can be made on the homogeneity of the blending through sensory evaluation. There is no quantitative evaluation method and indicator for the homogeneity of the blending of tobacco varieties in cigarette formulas.

[0004] There are already technologies that have been proposed to detect tobacco leaf varieties by using specific primers through PCR amplification, such as patent ZL 202010188464.5, a tobacco leaf specific primer and a method for identifying tobacco leaf varieties after curing, which mentions that the use of specific primers through PCR amplification can be used for the detection of Honghua Dajinyuan tobacco leaves, but this technology is mainly aimed at the detection of raw material varieties, and cannot achieve the actual proportion of formula tobacco varieties and the detection of mixed uniformity. Li Meng mentioned in his paper "Quantitative Detection of Honghua Dajinyuan in Mixed Tobacco Leaves by Fluorescence Quantitative PCR" that by designing Honghua Dajinyuan variety-specific primers and TaqMan MGB probes, the specificity and sensitivity of the primers and probes were tested, the reaction conditions of fluorescence quantitative PCR were determined, and on this basis, a standard curve was established to quantitatively detect the single variety of Honghua Dajinyuan in mixed tobacco leaves, but the document also did not involve the actual proportion of formula tobacco varieties and the detection of mixed uniformity. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting the homogeneity of blended formula tobacco using fluorescent quantitative PCR, so as to solve the problems existing in the above-mentioned background technology.

[0006] To achieve the above objectives, this application is implemented through the following technical solutions:

[0007] A method for detecting the homogeneity of formula tobacco blends using fluorescent quantitative PCR, comprising the following steps:

[0008] S1. Determine the tobacco varieties in the cigarette formula and the proportion of each tobacco variety in the cigarette formula, and after removing the tobacco variety with the highest proportion and the tobacco variety with the lowest proportion, select any two tobacco varieties from the remaining tobacco varieties, mark them as tobacco variety A1 and tobacco variety A2, and determine their percentage content in the cigarette formula, mark them as N1 and N2, respectively;

[0009] S2. Using molecular marker technology to screen out specific gene fragments M1 and M2 of tobacco variety A1 and tobacco variety A2, respectively, design fluorescent quantitative PCR primers, marked as K1 and K2, respectively, and confirm the fluorescent quantitative PCR primers using conventional PCR amplification technology;

[0010] S3, extracting genomic DNA from tobacco shreds of cigarette formula, and performing fluorescence quantitative PCR using K1 and K2, respectively, to obtain DNA concentration H1 of tobacco variety A1 and DNA concentration H2 of tobacco variety A2;

[0011] S4, according to the range of cigarette formula ratio, repeat step S1 to step S3 for n times, where n is a natural number greater than 2, to obtain n H1 and H2, and obtain the standard DNA concentration range nH1 of tobacco variety A1 in the cigarette formula and the standard DNA concentration range nH2 of tobacco variety A2 through statistics;

[0012] S5, sampling the tobacco shreds from the production line according to the sampling rules to obtain the tobacco shreds samples to be tested, and extracting the genomic DNA of the tobacco shreds to be tested according to step S3, and using K1 and K2 for fluorescence quantitative PCR to obtain the DNA concentration H10 of tobacco variety A1 and the DNA concentration H20 of tobacco variety A2 respectively;

[0013] S6. Determine whether H10 is within the range of nH1 and whether H20 is within the range of nH2. If so, it is determined that the blending of the tested formula tobacco is uniform. If any one of the items is not within the set range, it is determined that the blending of the tested formula tobacco is uneven.

[0014] Furthermore, in step S2, RAPD, SCAR or SSR molecular marker technology is used to screen specific gene fragments of tobacco varieties.

[0015] Furthermore, in step S2, the specific steps of confirming the fluorescent quantitative PCR primers using conventional PCR amplification technology are as follows:

[0016] In conventional PCR amplification, the 20 μL reaction system is:

[0017] DNA template, 50 ng / μL, 1-2 μL;

[0018] 2×PCR Buffer mix, 10 μL;

[0019] Upstream and downstream primers, 10 μmol / L, 0.3-0.5 μL each;

[0020] ddH2O, add to 20 μL;

[0021] For fluorescent PCR amplification, 0.4 µl of ROX Reference Dye should be added to 20 µL of reaction system, and the remaining components are the same as those for conventional PCR amplification;

[0022] The conventional PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 30 s, extension at 72°C for 20 s, 30 cycles; extension at 72°C for 5 min;

[0023] The fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 20 s, 40 cycles; extension at 72°C for 5 min; melting curve analysis: 65°C for 15 s.

[0024] Furthermore, the genomic DNA of tobacco shreds of cigarette formulations is extracted using an optimized CTAB method or a kit.

[0025] Furthermore, a detection kit comprising fluorescent quantitative PCR primers K1 and K2 of tobacco variety A1 and tobacco variety A2 is designed, and the detection kit is used in detecting the homogeneity of formulated tobacco blends.

[0026] Furthermore, step S3 also includes:

[0027] S31, extracting genomic DNA from tobacco variety A1, tobacco variety A2 and tobacco shreds of cigarette formula, respectively, and marking them as P1 and P2, respectively;

[0028] S32, diluting P1 and P2 to the percentage concentrations N1 and N2 of tobacco variety A1 and tobacco variety A2 in the cigarette formula to obtain P10 and P20, and then using K1 and K2 to perform fluorescence quantitative PCR on the genomic DNA of P10, P20, and tobacco shreds of the cigarette formula, respectively, to obtain the DNA concentration L1 of pure tobacco variety A1 and the DNA concentration L2 of pure tobacco variety A2 at the proportion of the cigarette formula;

[0029] S33, calculate the standard ratio F1 of the DNA of tobacco variety A1 = (L1 / H1) × 100%, and the standard ratio F2 of the DNA of tobacco variety A2 = (L2 / H2) × 100%;

[0030] S34. According to the range of cigarette formula ratio, after n repetitions, the standard ratio range nF1 of the DNA of tobacco variety A1 and the standard ratio range nF2 of the DNA of tobacco variety A2 are obtained.

[0031] Furthermore, it also includes calculating the actual proportion of DNA of tobacco variety A1 F10 = (L1 / H10) × 100%, and the standard proportion of DNA of tobacco variety A2 F20 = (L2 / H20) × 100%;

[0032] It is determined whether F10 is within the range of nF1 and whether F20 is within the range of nF2. If so, the determination of the mixing uniformity of the tested formula tobacco is accurate; if not, the determination of the mixing uniformity of the tested formula tobacco is inaccurate.

[0033] The beneficial effects of the present invention are:

[0034] The tobacco blending homogeneity test of the technical solution does not select tobacco varieties with large usage and tobacco varieties with the lowest tobacco usage, because through long-term experience, the tobacco variety with the largest usage cannot represent uniform mixing. On the other hand, the tobacco variety with the lowest usage usually has slight adjustments in certain aspects, and whether it is mixed evenly does not represent whether the entire cigarette formula is mixed evenly.

[0035] This technical solution uses fluorescent quantitative PCR primers to simultaneously detect the mixture of two tobacco varieties, thereby improving the accuracy of detection. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described in detail below through examples. The following examples are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, but cannot be construed as limiting the technical solution of the present invention.

[0037] The present application provides a method for detecting the homogeneity of blended tobacco using fluorescent quantitative PCR, comprising the following steps:

[0038] S1. Determine the tobacco varieties in the cigarette formula and the proportion of each tobacco variety in the cigarette formula, and after removing the tobacco variety with the highest proportion and the tobacco variety with the lowest proportion, select any two tobacco varieties from the remaining tobacco varieties. Here, a tobacco cut formula sample of the applicant is used as an example for explanation: the tobacco cut formula includes 5 varieties of tobacco cuts, specifically including Yunyan 87, Honghua Dajinyuan, K326, Zhongyan 100 and Longjiang 911. Of course, the tobacco cut formula also includes some stem shreds, thin slices, etc., which are not targeted at stem shreds and thin slices in this application. If fragments are also included, they are not considered. Only 5 varieties of tobacco cuts are used as an example for explanation. The dosage of these 5 varieties of tobacco cuts is different. In the following embodiments, the two varieties with the second and third dosage ratios are selected, which are Honghua Dajinyuan and Yunyan 87, respectively.

[0039] Honghua Dajinyuan is marked as tobacco variety A1 and Yunyan 87 is marked as tobacco variety A2, and their percentage content in the cigarette formula is determined and marked as N1 and N2 respectively; this part is for a certain cigarette formula, usually including multiple varieties, including different parts of the same variety. In this application, only the varieties are tested, and specific parts of the same variety cannot be tested.

[0040] S2. Molecular marker technology was used to screen out specific gene fragments M1 and M2 of tobacco variety A1 and tobacco variety A2, respectively, and fluorescent quantitative PCR primers were designed, marked as K1 and K2, respectively.

[0041] The specific primer sequence of K1 is as follows:

[0042] Downstream primer R: 5′-GATTGCCAAAAAAAAAAGAA-3′;

[0043] Upstream primer F: 5′-GGGCCACAATTATCACATCA-3′.

[0044] The specific primer sequence of K2 is as follows:

[0045] Downstream primer R: 5′- ACAGCAGTAACAGCAGGAACA-3′;

[0046] Upstream primer F: 5'-CCCTGGATTGGTCTGTCTTGG-3'.

[0047] And use the conventional PCR amplification technology to confirm the fluorescent quantitative PCR primers; in this application, the specific gene fragments of tobacco varieties are screened by RAPD, SCAR or SSR molecular marker technology. Specific to the specific gene fragments and fluorescent quantitative PCR primers, they can be designed with reference to the prior art. In the prior art, the identification method of the safflower Dajinyuan variety mentioned in the background technology section can be used. Of course, because the specific gene fragments of different tobacco varieties are different, the designed PCR primers must be different. Those skilled in the art design them according to the reference to the conventional technology, and no detailed description is given here.

[0048] S3. Extract genomic DNA from tobacco of cigarette formula, and perform fluorescence quantitative PCR using K1 and K2, respectively. The DNA concentration of tobacco variety A1 was 22.23 ng / μL, marked as H1, and the DNA concentration of tobacco variety A2 was 22.72 ng / μL, marked as H2.

[0049] In this application, step S3 also includes:

[0050] S31, extracting genomic DNA of tobacco variety A1 and tobacco variety A2 respectively, marking them as P1 and P2 respectively, and here the genome of pure tobacco variety A1 and pure tobacco variety A2 is to be determined.

[0051] Specifically, the extraction steps of tobacco genomic DNA for each sample are the same:

[0052] (1) Weigh 10 g of tobacco sample, grind it into powder, and pass it through a 100-mesh sieve.

[0053] (2) Accurately weigh 0.1 g of tobacco powder into a 2 mL centrifuge tube, add 800 µL of CTAB extraction solution, and lyse in a 70°C water bath for 40 min, shaking the tube upside down every 10 min.

[0054] (3) Add 800 µL of -20°C precooled chloroform and centrifuge at 10,000 rpm for 10 minutes.

[0055] (4) Aspirate the supernatant, add the same volume of CTAB and chloroform, and centrifuge again at 10,000 r / min for 10 minutes.

[0056] (5) Aspirate the supernatant, add 2 times the volume of the supernatant with CTAB precipitation solution and 1 times the volume of the supernatant with isopropanol, and let it stand at -20°C for 1 hour.

[0057] (6) After standing, the sample was centrifuged at 12,000 r / min for 10 min, the supernatant was discarded, and 500 µL 1.2 mol / L NaCl was added and allowed to stand for 10 min.

[0058] (7) Add 600 µL of -20°C precooled isopropanol and let stand for 40 min.

[0059] (8) Centrifuge at 12,000 r / min for 10 min, discard the liquid, add 500 µL of -20°C precooled 70% ethanol, wash twice, and open the lid to evaporate for 2-5 minutes.

[0060] (9) Add 50 µL ddH2O to dissolve the DNA and store at -20°C.

[0061] Through the above steps, the genomic DNA of tobacco variety A1 was obtained at 22.24 ng / μL, marked as P1, and the genomic DNA of tobacco variety A2 was obtained at 22.73 ng / μL, marked as P2.

[0062] S32. Dilute P1 and P2 to the percentage concentration N1 and N2 of tobacco varieties A1 and tobacco varieties A2 in the cigarette formula respectively to obtain P10 and P20. The proportion of Honghua Dajinyuan in the formula tobacco is 12%, and the proportion of Yunyan 87 in the formula tobacco is 10%. Therefore, P1 is diluted to 12% and marked as P10, and P2 is diluted to 10% and marked as P20.

[0063] This part takes into account the content of tobacco variety A1 and tobacco variety A2 in the entire formula of cigarette formula tobacco to ensure the accuracy of the test results. K1 and K2 are used to perform fluorescent quantitative PCR on the genomic DNA of P10, P20, and cigarette formula tobacco, respectively. The DNA concentration L1 of pure tobacco variety A1 is 2.67 ng / μL, and the DNA concentration L2 of pure tobacco variety A2 is 2.27 ng / μL under the proportion of cigarette formula.

[0064] S33. Calculate the standard proportion of DNA of tobacco variety A1: F1 = (L1 / H1) × 100% = 12.01% and the standard proportion of DNA of tobacco variety A2: F2 = (L2 / H2) × 100% = 9.99%.

[0065] S34. According to the range of cigarette formula ratios, after 5 repetitions, the standard ratio range nF1 of DNA of tobacco variety A1 is 11.87%-11.92%, and the standard ratio range nF2 of DNA of tobacco variety A2 is 9.89%-9.96%. This step takes into account that in any cigarette formula, the actual ratio of each tobacco variety is not a fixed point value, but a range. In this case, the standard ratio of DNA of the tobacco variety in the cigarette formula should not be a fixed point value, but a range to ensure that it is suitable for actual testing.

[0066] S4. According to the range of cigarette formula ratio, step S1 to step S3 are repeated 5 times to obtain 5 H1s and H2s. After statistics, the standard DNA concentration range of tobacco variety A1 in the cigarette formula is 22.18 ng / μL-22.23 ng / μL, and the standard DNA concentration range of tobacco variety A2 is 22.67 ng / μL-22.76 ng / μL.

[0067] S5, sampling the tobacco shreds from the production line according to the sampling rules to obtain the tobacco shreds samples to be tested, and extracting the genomic DNA of the tobacco shreds to be tested according to step S3, and using K1 and K2 for fluorescence quantitative PCR to obtain the DNA concentration H10 of tobacco variety A1 and the DNA concentration H20 of tobacco variety A2 respectively;

[0068] S6. Determine whether H10 is within the range of Nh1 and whether H20 is within the range of Nh2. If so, it is determined that the blending of the tested formula tobacco is uniform. If any one of the items is not within the set range, it is determined that the blending of the tested formula tobacco is uneven.

[0069] In this application, in step S2, the specific steps of confirming the fluorescent quantitative PCR primers using conventional PCR amplification technology are as follows:

[0070] In conventional PCR amplification, the 20 μL reaction system is:

[0071] DNA template, 50 ng / μL, 1-2 μL;

[0072] 2×PCR Buffer mix, 10 μL;

[0073] Upstream and downstream primers, 10 μmol / L, 0.3-0.5 μL each;

[0074] ddH2O, add to 20 μL;

[0075] For fluorescent PCR amplification, 0.4 µl of ROX Reference Dye should be added to 20 µL of reaction system, and the remaining components are the same as those for conventional PCR amplification;

[0076] The conventional PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 30 s, extension at 72°C for 20 s, 30 cycles; extension at 72°C for 5 min;

[0077] The fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 20 s, 40 cycles; extension at 72°C for 5 min; melting curve analysis: 65°C for 15 s.

[0078] In the present application, the optimized CTAB method or kit is used to extract the genomic DNA of the tobacco shreds of the cigarette formulation.

[0079] In the present application, a detection kit comprising fluorescent quantitative PCR primers K1 and K2 of tobacco variety A1 and tobacco variety A2 is designed, and the detection kit is used in detecting the homogeneity of formula tobacco blends.

[0080] The present application also includes calculating the actual DNA ratio F10 = (L1 / H10) × 100% of tobacco variety A1 and the standard DNA ratio F20 = (L2 / H20) × 100% of tobacco variety A2.

[0081] It is determined whether F10 is within the range of nF1 and whether F20 is within the range of nF2. If so, the determination of the mixing uniformity of the tested formula tobacco is accurate; if not, the determination of the mixing uniformity of the tested formula tobacco is inaccurate.

[0082] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A method for detecting the homogeneity of blended tobacco using fluorescent quantitative PCR, characterized in that: The following steps are involved: S1. Determine the tobacco varieties in the cigarette formula and the proportion of each tobacco variety in the cigarette formula, and after removing the tobacco variety with the highest proportion and the tobacco variety with the lowest proportion, select any two tobacco varieties from the remaining tobacco varieties, mark them as tobacco variety A1 and tobacco variety A2, and determine their percentage content in the cigarette formula and mark them as N1 and N2 respectively; S2. Using molecular marker technology to screen out specific gene fragments M1 and M2 of tobacco variety A1 and tobacco variety A2, respectively, design fluorescent quantitative PCR primers, marked as K1 and K2, respectively, and confirm the fluorescent quantitative PCR primers using conventional PCR amplification technology; S3, extracting genomic DNA from tobacco shreds of cigarette formula, and performing fluorescence quantitative PCR using K1 and K2, respectively, to obtain DNA concentrations H1 and H2 of tobacco variety A1 and tobacco variety A2, respectively; S4, according to the range of cigarette formula ratio, repeat step S1 to step S3 for n times, where n is a natural number greater than 2, to obtain n H1 and H2, and obtain the standard DNA concentration range nH1 of tobacco variety A1 in the cigarette formula and the standard DNA concentration range nH2 of tobacco variety A2 through statistics; S5, sampling the tobacco shreds from the production line according to the sampling rules to obtain the tobacco shreds samples to be tested, and extracting the genomic DNA of the tobacco shreds to be tested according to step S3, and using K1 and K2 for fluorescence quantitative PCR to obtain the DNA concentration H10 of tobacco variety A1 and the DNA concentration H20 of tobacco variety A2 respectively; S6. Determine whether H10 is within the range of nH1 and whether H20 is within the range of nH2. If so, it is determined that the blending of the tested formula tobacco is uniform. If any one of the items is not within the set range, it is determined that the blending of the tested formula tobacco is uneven.

2. The method for detecting the homogeneity of blended tobacco using fluorescent quantitative PCR according to claim 1, characterized in that: In step S2, RAPD, SCAR or SSR molecular marker technology is used to screen specific gene fragments of tobacco varieties.

3. The method for detecting the homogeneity of blended tobacco using fluorescent quantitative PCR according to claim 1, characterized in that: In step S2, the specific steps of confirming the fluorescent quantitative PCR primers using conventional PCR amplification technology are as follows: In conventional PCR amplification, the 20 μL reaction system is: DNA template, 50 ng / μL, 1-2 μL; 2×PCR Buffer mix, 10 μL; Upstream and downstream primers, 10 μmol / L, 0.3-0.5 μL each; ddH2O, add to 20 μL; For fluorescent PCR amplification, 0.4 µl of ROX Reference Dye should be added to 20 µL of reaction system, and the remaining components are the same as those for conventional PCR amplification; The conventional PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 30 s, extension at 72°C for 20 s, 30 cycles; extension at 72°C for 5 min; The fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 20 s, 40 cycles; extension at 72°C for 5 min; melting curve analysis: 65°C for 15 s.

4. The method for detecting the homogeneity of blended tobacco using fluorescent quantitative PCR according to claim 1, characterized in that: The genomic DNA of tobacco shreds of cigarette formulations was extracted using the optimized CTAB method or kit.

5. The method for detecting the homogeneity of blended tobacco using fluorescent quantitative PCR according to claim 3, characterized in that: A detection kit comprising fluorescent quantitative PCR primers K1 and K2 of tobacco variety A1 and tobacco variety A2 is designed, and the detection kit is used in detecting the homogeneity of formula tobacco blends.

6. The method for detecting the homogeneity of blended tobacco using fluorescent quantitative PCR according to claim 1, characterized in that: Step S3 also includes: S31, extracting genomic DNA from tobacco variety A1, tobacco variety A2 and tobacco shreds of cigarette formula, respectively, and marking them as P1 and P2, respectively; S32, diluting P1 and P2 to the percentage concentrations N1 and N2 of tobacco variety A1 and tobacco variety A2 in the cigarette formula to obtain P10 and P20, and then using K1 and K2 to perform fluorescence quantitative PCR on the genomic DNA of P10, P20, and tobacco shreds of the cigarette formula, respectively, to obtain the DNA concentration L1 of pure tobacco variety A1 and the DNA concentration L2 of pure tobacco variety A2 at the proportion of the cigarette formula; S33, calculate the standard ratio F1 of the DNA of tobacco variety A1 = (L1 / H1) × 100%, and the standard ratio F2 of the DNA of tobacco variety A2 = (L2 / H2) × 100%; S34. According to the range of cigarette formula ratio, after n repetitions, the standard ratio range nF1 of the DNA of tobacco variety A1 and the standard ratio range nF2 of the DNA of tobacco variety A2 are obtained.

7. The method for detecting the homogeneity of blended tobacco using fluorescent quantitative PCR according to claim 6, characterized in that: It also includes calculating the actual proportion of DNA of tobacco variety A1, F10 = (L1 / H10) × 100%, and the standard proportion of DNA of tobacco variety A2, F20 = (L2 / H20) × 100%; It is determined whether F10 is within the range of nF1 and whether F20 is within the range of nF2. If so, the determination of the mixing uniformity of the tested formula tobacco is accurate; if not, the determination of the mixing uniformity of the tested formula tobacco is inaccurate.