A primer, a probe and a detection method thereof for the authenticity detection of American ginseng medicinal materials based on the second generation sequencing

Abstract

The present invention relates to primers, probes and a detection method developed based on next-generation sequencing for detecting and identifying the authenticity of American ginseng medicinal materials. The sequences of the upstream and downstream specific primers for detecting American ginseng are shown as SEQ ID NO.1 and 2, the probe sequence is shown as SEQ ID NO.3, the 5' end of the probe is modified with VIC, and the 3' end is modified with MGB. For the real-time fluorescence quantitative PCR detection of American ginseng, the present invention has the advantages of strong specificity and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology. Specifically, it relates to a primer, a probe, and a detection method for authenticating the genuineness of American ginseng medicinal materials developed based on next-generation sequencing. Background Art

[0002] American ginseng (Panax quinquefolius) is a perennial herbaceous plant of the Araliaceae family and the Panax genus. Its roots are fleshy, oval or spindle-shaped, with a light yellow outer surface, relatively delicate and smooth, growing vigorously, and the texture of the cross-section has a chrysanthemum shape; the stem is erect and cylindrical, smooth and hairless, green or dark purple-green, and the height of the stem varies with the age of the ginseng; the leaves are generally palmate compound leaves composed of 5 leaflets. The leaflets are obovate or ovate, the leaves are thinner, and the edges have irregular coarse serrations. Generally, a one-year-old American ginseng plant has only 1 compound leaf with 3 leaflets, a two-year-old one has 1 or 2 opposite compound leaves with 5 leaflets, and a 3-5-year-old one has 3-5 whorled compound leaves with 5 leaflets; it has an umbellate inflorescence composed of many small flowers; the berries are oblate in shape, distributed in pairs, and the color after maturity is bright red; the flowering period and the fruit maturity period are July and September respectively.

[0003] American ginseng is a "cool" ginseng. It is bitter, slightly sweet, and cool in nature, and has the effects of nourishing yin and replenishing qi, promoting fluid production and quenching thirst, relieving restlessness, clearing away deficiency-fire, strengthening healthy qi, and anti-fatigue. The active ingredient in American ginseng is ginsenoside, which has the effect of enhancing the body's resistance.

[0004] Real-time fluorescence PCR technology has been a very mature and successful technology in species identification. By using one or more sets of specific primer probes and monitoring the change in fluorescence intensity to achieve the purpose of identification, this methodology not only has the advantages of strong specificity, high sensitivity, and simple operation, but also has a high detection efficiency. Using real-time fluorescence PCR technology to identify Panax notoginseng not only has strong specificity, high sensitivity, accurate and rapid results, but also has good market application prospects and promotion application value. Summary of the Invention

[0005] In order to overcome the problems existing in the background art, the present invention provides a primer, a probe, and a detection method for authenticating the genuineness of American ginseng medicinal materials developed based on next-generation sequencing.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] A primer and a probe for American ginseng detection developed based on next-generation sequencing. The upstream and downstream primer sequences for American ginseng detection are shown in SEQ ID NO.1 and 2, and the probe sequence is shown in SEQ ID NO.3. The 5' end of the probe is modified with VIC, and the 3' end is modified with MGB.

[0008] The present invention also provides a detection method for primers and probes developed based on next-generation sequencing for the detection of American ginseng, comprising the following steps:

[0009] S1, Extract the genomic DNA of the sample to be tested using a plant genomic DNA extraction kit;

[0010] S2, Use the genomic DNA of the sample to be tested extracted in step S1 as the template to be tested, and perform a real-time fluorescence quantitative PCR amplification reaction on the template to be tested using the primers and probes as shown in claim 1;

[0011] S3, The method for judging whether the sample to be tested contains DNA fragments specific to the American ginseng genome is as follows: Judge whether the sample to be tested contains DNA fragments specific to the American ginseng genome based on the CT value. If the CT value is less than 40, it is judged as positive; if the CT value of the sample is greater than 40 or there is no value, it is judged as negative.

[0012] Further, the reaction system for real-time fluorescence quantitative PCR amplification in step S2 is: 10 μL of 2xT5 Fast qPCRMix, 0.7 μL of 10 μM upstream primer, 0.7 μL of 10 μM downstream primer, 0.6 μL of 10 μM probe, 1 μL of the template to be tested, and ddH2O is used to make up to 20 μL of the reaction system.

[0013] Further, the real-time fluorescence quantitative PCR amplification program in step S2 is: 95°C for 1 min, one cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles; collect the corresponding fluorescence for 1 min during the annealing and extension stage at 60°C.

[0014] The present invention also provides the application of the primers and probes for the detection of American ginseng in the preparation of an American ginseng detection kit.

[0015] Advantages of the present invention: Through sequence alignment and analysis of the chloroplast genome of American ginseng, the present invention screens out TaqMan-MGB fluorescence probes with certain specificity and sensitivity, enabling the probes to specifically detect the CT value of the target gene fragment, thereby realizing the rapid identification of American ginseng. Description of the Drawings

[0016] Figure 1 It is the standard curve and related amplification curve diagram of real-time fluorescence quantitative PCR in the present invention;

[0017] Figure 2 It is the amplification curve diagram of the primer and probe specificity verification test in the present invention;

[0018] Figure 3 It is the amplification curve diagram of the primer and probe sensitivity verification test in the present invention; Detailed Embodiments

[0019] To make the objectives, technical solutions and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below to facilitate understanding by those skilled in the art.

[0020] Example 1. Design of primer pairs and probes

[0021] Design: Based on the chloroplast genomic DNA of Panax quinquefolius, molecular biology software was used for the design of primers and probes.

[0022] The primer and probe sequences are shown in Table 1 below:

[0023] Table 1 Primer and probe sequences of Panax quinquefolius

[0024]

[0025] Reaction system:

[0026] Reaction system (20 μL) Added volume 2xT5 Fast qPCR Mix 10 μL Forward primer (10 uM) 0.7 μL Reverse primer (10 uM) 0.7 μL Probe (10 uM) 0.6 μL DNA template 1 μL <![CDATA[ddH2O up to]]> 20 μL

[0027] Amplification program:

[0028]

[0029] Example 2. Preparation of Panax quinquefolius standard plasmid and creation of probe standard curve

[0030] 1. The applicant commissioned a biological company to extract the genomic DNA of Panax quinquefolius. According to the primer sequences, reaction system and amplification program of Example 1, PCR amplification was carried out to obtain PCR products.

[0031] 2. Positive clone samples were prepared from the PCR products, and then sequenced. Positive clone samples with completely correct sequences in the sequencing results were selected, and plasmid extraction kits were used to complete the extraction of plasmids.

[0032] 3. Using the formula: C = A·B -1 ×6.02×10 14 (where A represents the plasmid concentration ng·μL -1 , B represents the molecular weight of plasmid DNA, and C represents copies·μL -1 ) to calculate the plasmid concentration copy number and obtain the mother liquor concentration: Panax quinquefolius standard plasmid (2.66*10 10 copies·μL -1 ), which will be used as the experimental plasmid standard.

[0033] 4. Dilute the Panax quinquefolius plasmid standard with a solvent to 2.66*10 8 copies·μL -1, and then dilute it to 5 gradients at 10-fold concentration. Respectively, use the diluted plasmid standard as a template and perform TaqMan RT-qPCR detection under the aforementioned reaction conditions. Each group of experiments is repeated 3 times to establish a standard curve. The detection results of the Panax quinquefolius plasmid standard are shown in Table 2, and the standard curve and related amplification curves are as Figure 1 shown.

[0034] Panax quinquefolius standard curve: y = -3.4303x + 45.069, R2 = 0.9999;

[0035] and the correlation coefficient (R2) of the Panax quinquefolius standard curve is greater than 0.98.

[0036] Table 2 Detection results of Panax quinquefolius plasmid standard

[0037] Standard plasmid Number of bases Molecular weight Concentration (ng / μL) Copy number (copies / μL) 2-P.quin 2030 1339800 59.259 2.66E+10 Dilution factor Gene Copy number Log value of copy number Average CT value 10*2 2-P.quin 2.66E+08 8.425310836 16.240 10*2 2-P.quin 2.66E+08 8.425310836 16.240 10*2 2-P.quin 2.66E+08 8.425310836 16.240 10*3 2-P.quin 2.66E+07 7.425310836 19.490 10*3 2-P.quin 2.66E+07 7.425310836 19.490 10*3 2-P.quin 2.66E+07 7.425310836 19.490 10*4 2-P.quin 2.66E+06 6.425310836 23.034 10*4 2-P.quin 2.66E+06 6.425310836 23.034 10*4 2-P.quin 2.66E+06 6.425310836 23.034 10*5 2-P.quin 2.66E+05 5.425310836 26.480 10*5 2-P.quin 2.66E+05 5.425310836 26.480 10*5 2-P.quin 2.66E+05 5.425310836 26.480 10*6 2-P.quin 2.66E+04 4.425310836 29.897 10*6 2-P.quin 2.66E+04 4.425310836 29.897 10*6 2-P.quin 2.66E+04 4.425310836 29.897

[0038] Example 3. Method for detecting Panax quinquefolius samples by real-time fluorescence PCR

[0039] 1. Prepare the reaction system and set the amplification program according to Example 1.

[0040] 2. During the detection process, in addition to the samples, set positive controls and negative controls. The positive control is: Panax quinquefolius plasmid standard, and the negative control is: ddH2O;

[0041] 3. When the results of the positive control and negative control are both normal, judge whether the test sample contains the specific DNA fragment of the Panax quinquefolius genome based on the CT value. If the CT value is less than 40, it is judged as positive; if the CT value of the sample is greater than 40 or there is no value, it is judged as negative.

[0042] Example 4. Verification of primer-probe specificity

[0043] Verification content: Amplify seven related species with specific primer-probes respectively, and each sample is repeated 3 times; the results are shown in Table 3, and the amplification curves are as Figure 2 shown.

[0044] Verification purpose: Verify the specificity of the primer-probe;

[0045] Table 3 Detection results of Panax quinquefoliu primer-probe specificity

[0046]

[0047] Results: As shown in Table 3 above, the Panax quinquefolius primer-probe only amplifies with Panax quinquefolius and has no amplification reaction with other species, with good specificity and close CT values for 3 repetitions, showing good repeatability.

[0048] Example 5: Verification of Primer Probe Sensitivity

[0049] Verification content: Corresponding to sample DNA concentrations of 0.00001 ng / μL, 0.0001 ng / μL, 0.001 ng / μL, 0.01 ng / μL, 0.1 ng / μL, and 1 ng / μL, the sensitivity of the corresponding primer probe was tested at 6 concentration gradients. Each sample was repeated three times, and a real-time fluorescence quantitative PCR instrument was used for determination; the detection results are shown in Table 4 and Figure 3 as follows.

[0050] Verification purpose: To verify the lowest detection limit, i.e., sensitivity, of the primer probe for Panax quinquefolius in the reaction system.

[0051] QuantStudio Dx Detection Results (Sensitivity)

[0052] Table 4: Sensitivity of the primer probe for Panax quinquefolius: 0.001 ng / μL

[0053] Sample Name Target Name Task Reporter Quencher Cт CтMean CтSD 1 2-P.quin UNKNOWN VIC NFQ-MGB 22.170 22.062 0.098 1 2-P.quin UNKNOWN VIC NFQ-MGB 22.035 22.062 0.098 1 2-P.quin UNKNOWN VIC NFQ-MGB 21.980 22.062 0.098 0.1 2-P.quin UNKNOWN VIC NFQ-MGB 25.696 25.705 0.010 0.1 2-P.quin UNKNOWN VIC NFQ-MGB 25.704 25.705 0.010 0.1 2-P.quin UNKNOWN VIC NFQ-MGB 25.715 25.705 0.010 0.01 2-P.quin UNKNOWN VIC NFQ-MGB 29.775 29.761 0.013 0.01 2-P.quin UNKNOWN VIC NFQ-MGB 29.758 29.761 0.013 0.01 2-P.quin UNKNOWN VIC NFQ-MGB 29.749 29.761 0.013 0.001 2-P.quin UNKNOWN VIC NFQ-MGB 33.529 33.746 0.188 0.001 2-P.quin UNKNOWN VIC NFQ-MGB 33.842 33.746 0.188 0.001 2-P.quin UNKNOWN VIC NFQ-MGB 33.866 33.746 0.188 0.0001 2-P.quin UNKNOWN VIC NFQ-MGB 38.013 37.936 0.108 0.0001 2-P.quin UNKNOWN VIC NFQ-MGB 37.860 37.936 0.108 0.0001 2-P.quin UNKNOWN VIC NFQ-MGB Undetermined 37.936 0.108 0.00001 2-P.quin UNKNOWN VIC NFQ-MGB Undetermined 0.00001 2-P.quin UNKNOWN VIC NFQ-MGB Undetermined 0.00001 2-P.quin UNKNOWN VIC NFQ-MGB Undetermined

[0054] As shown in Table 4 and Figure 3 as follows, the primer probe recorded in the present invention has relatively high sensitivity.

[0055] Example 6: Verification of Primer Probe Repeatability

[0056] Verification was carried out from the following two aspects respectively:

[0057] 1. Comparison between different batches of enzymes with the same experimenter and the same real-time fluorescence quantitative PCR instrument (QuantStudio Dx);

[0058] 2. Operation by different personnel with the same batch of enzymes and the same real-time fluorescence quantitative PCR instrument (QuantStudio Dx);

[0059] Verification content: Each sample was repeated 3 times, and repeatability evaluation was carried out by calculating the coefficient of variation (CV%). Calculation formula: cv = sd (standard deviation) / mean (average value) × 100%.

[0060] Verification purpose: To verify repeatability, personnel operability, and the stability of different batches of polymerases.

[0061] Table 5: Results of different batches of enzymes with the same experimenter and the same QPCR instrument (QuantStudio Dx)

[0062]

[0063]

[0064] Table 6: Operations by different personnel using the same batch of enzymes and the same QPCR instrument (QuantStudio Dx)

[0065]

[0066]

[0067] Results: As shown in Tables 5 - 6, comparisons were made between different batches of enzymes used by the same experimenter with the same real-time fluorescence quantitative PCR instrument (QuantStudio Dx); and operations by different personnel using the same batch of enzymes and the same real-time fluorescence quantitative PCR instrument (QuantStudio Dx). Experimental results: The repeatability CV% was less than 3%, indicating good experimental repeatability and excellent stability of different batches of polymerases.

[0068] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in terms of form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A primer and probe combination developed based on next-generation sequencing for the detection and identification of the authenticity of American ginseng medicinal materials, characterized by: The upstream and downstream primer sequences for American ginseng detection are shown in SEQ ID NO.1 and 2, and the probe sequence is shown in SEQ ID NO.

3. The 5' end of the probe is modified with VIC, and the 3' end is modified with MGB.

2. A method for identifying the authenticity of American ginseng medicinal materials by using the primer and probe combination as described in claim 1, characterized in that: Comprising the following steps: S1, Extract the genomic DNA of the sample to be tested using a plant genomic DNA extraction kit; S2, Use the genomic DNA of the sample to be tested extracted in step S1 as the template to be tested, and perform a real-time fluorescence quantitative PCR amplification reaction on the template to be tested using the primer and probe combination as shown in claim 1; S3, The method for judging whether the sample to be tested contains a DNA fragment specific to the American ginseng genome is as follows: Judge whether the sample to be tested contains a DNA fragment specific to the American ginseng genome based on the CT value. If the CT value is less than 40, it is judged as positive; if the CT value of the sample is greater than 40 or there is no value, it is judged as negative.

3. The method according to claim 2, wherein: The reaction system for real-time fluorescence quantitative PCR amplification in step S2 is: 10 μL of 2xT5 Fast qPCR Mix, 0.7 μL of 10 μM upstream primer, 0.7 μL of 10 μM downstream primer, 0.6 μL of 10 μM probe, 1 μL of the template to be tested, and make up to 20 μL of the reaction system with ddH2O.

4. The method according to claim 2, characterized in that: The real-time fluorescence quantitative PCR amplification program in step S2 is: 95°C for 1 min, one cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles; collect the corresponding fluorescence for 1 min during the annealing and extension stage at 60°C.

5. Use of the primer and probe combination as claimed in claim 1 in the preparation of an American ginseng detection kit.

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