Reagents, methods and applications of identifying Paris polyphylla by real-time fluorescence PCR

Abstract

The invention discloses a reagent, method and application for identifying Paris polyphylla by using real-time fluorescence PCR technology, relates to the technical field of plant identification, comprises a primer pair and a probe, the primer pair is composed of primers D2-F and D2-R, the sequence of the primer D2-F is shown in SEQ ID No.1, the sequence of the primer D2-R is shown in SEQ ID No.2; the sequence of the probe D2-probe is shown in SEQ ID No.3. The invention has excellent specificity and sensitivity, and can quickly and reliably identify Paris polyphylla.

Description

Technical Field

[0001] The invention belongs to the technical field of plant identification, and particularly relates to a reagent, a method and an application for identifying Paris polyphylla by adopting real-time fluorescence PCR technology. Background Art

[0002] Paris liana is a plant of the genus Paris of the family Melanophyllaceae. It is a perennial herb mainly distributed in Yunnan. It has the effects of clearing away heat and detoxifying, reducing swelling and relieving pain, cooling the liver and calming the nerves.

[0003] The traditional identification methods of Paris polyphylla are origin identification, trait identification, microscopic identification and physical and chemical identification. The theoretical basis of traditional identification methods is based on the analysis of trait characteristics of taxa, which are phenotypes closely related to the environment. From the perspective of molecular genetics, the differences in species phenotypes should be traced back to differences in genotypes, that is, differences in DNA sequences. Therefore, comparative studies on genome sequence differences undoubtedly provide an essential basis for plant classification and identification. With the continuous breakthroughs in life sciences, molecular biological identification methods have emerged. Such methods have achieved rapid development by using DNA molecular marker technology to identify Chinese medicinal raw plants and their medicinal materials and decoction pieces. There have been many reports on the application of DNA molecular marker identification in the identification of ginseng medicinal materials. RFLP, SSR, RAPD, AP-PCR, AFLP, ISSR, SNP and other technologies have been successively applied to the identification and identification of medicinal plants. However, in addition to PCR amplification, these technologies also require electrophoresis or sequencing technology to present identification results. They have the disadvantages of cumbersome operation steps, heavy workload, large human interference, low detection sensitivity, poor specificity and poor repeatability of experimental results. Especially in the identification of industrially processed and high-temperature dried medicinal materials, they are difficult to be applied in practice and gradually withdraw from the stage. With the development of molecular biology and the advent of the genomic era, new methods have been provided for the identification of some new medicinal plants.

[0004] Real-time fluorescence PCR technology is already a very mature and successful technology in species identification. It uses one or more sets of specific primer probes to achieve the purpose of identification by monitoring changes in fluorescence intensity. This method has the advantages of strong specificity, high sensitivity, simple operation, and high detection efficiency. It has good market application prospects and promotion and application value. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the identification methods in the prior art, such as poor reliability, complex operation, large workload, poor repeatability of experimental results, etc., and to provide a reagent, method and application for identifying Paris polyphylla using real-time fluorescence PCR technology.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A reagent for identifying Paris polyphylla by using real-time fluorescence PCR technology, comprising a primer pair and a probe, wherein the primer pair consists of primers D2-F and D2-R, the sequence of the primer D2-F is shown in SEQ ID No.1, the sequence of the primer D2-R is shown in SEQ ID No.2; the sequence of the probe D2-probe is shown in SEQ ID No.3.

[0008] The second aspect of the present invention provides a method for identifying Paris polyphylla by using real-time fluorescence PCR technology, comprising the following steps: using the genomic DNA of the sample to be identified as a template, using primers D2-F, D2-R and probe D2-probe to perform a real-time fluorescence quantitative PCR amplification reaction, and detecting that the CT value is less than 40, then it is identified as Paris polyphylla; the sequence of the primer D2-F is shown in SEQ ID No.1, the sequence of the primer D2-R is shown in SEQ ID No.2, and the sequence of the probe D2-probe is shown in SEQ ID No.3.

[0009] The sequences of the above primers and probes are shown in Table 1.

[0010] Specifically, the 5' end of the probe D2-probe is modified with VIC, and the 3' end of the probe D2-probe is modified with MGB.

[0011] Specifically, the minimum detection limit of the above template is 0.001 ng / μL.

[0012] Specifically, the reaction system of the real-time fluorescence quantitative PCR amplification is: 10 μL of 2xT5 Fast qPCR Mix, 0.7 μL of 10 uM upstream primer, 0.7 μL of 10 uM downstream primer, 0.6 μL of 10 uM probe, 1 μL of test template, and ddH2O is used to make up to 20 μL reaction system.

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

[0014] The third aspect of the present invention provides the use of the above reagent in identifying Paris polyphylla.

[0015] Table 1 Primer and probe sequences of Paris polyphylla

[0016]

[0017] The beneficial effects of the present invention are:

[0018] The identification method and reagent provided by the present invention have excellent specificity and sensitivity, and can accurately identify or detect Paris polyphylla in the genus Paris polyphylla by judging the detection CT value, thereby realizing rapid identification of Paris polyphylla. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the standard curve of Example 2 of the present invention;

[0020] Figure 2 This is the amplification map of Example 2 of the present invention;

[0021] Figure 3 This is the amplification map of Example 3 of the present invention;

[0022] Figure 4 This is the amplification map of Example 4 of the present invention;

[0023] Figure 5 Amplification profile for the first batch of enzyme verification;

[0024] Figure 6 Amplification profile for the second batch of enzyme verification;

[0025] Figure 7 This is the amplification map verified by experimenter A;

[0026] Figure 8 This is the amplification map of the operation verification performed by experimenter B. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0028] Example 1 PCR amplification and identification of target plant samples

[0029] The extracted genomic DNA of Paris polyphylla was used as a template, and primers D2-F and D2-R were used for PCR amplification, and probe D2-probe was used for real-time fluorescence quantitative PCR detection. The sequence of primer D2-F was ATGATACGTAAGCAAGGTATTTCAGG, the sequence of primer D2-R was AAATACAAGAGTATTGTTCCTCACCG, the sequence of probe D2-probe was TTTATAGAGAAAACAGAT, the 5' end of probe D2-probe was modified with VIC, and the 3' end of probe D2-probe was modified with MGB. The amplification reaction system is shown in Table 1.1, the amplification procedure is shown in Table 1.2, and the identification results and PCR products are obtained after amplification.

[0030] Table 1.1 Reaction system

[0031]

[0032] Table 1.2 Amplification procedures

[0033]

[0034] Example 2 Preparation of Paris polyphylla standard plasmid and creation of probe standard curve

[0035] Positive clone samples were prepared according to the PCR products obtained in Example 1, and then sequenced. Positive clone samples with completely correct sequences in the sequencing results were selected, and plasmids were extracted using a plasmid extraction kit.

[0036] Calculate the plasmid concentration copy number as follows:

[0037] C=A•B -1 ×6.02×10 14 ,

[0038] In the formula, A is the plasmid concentration (ng / μL), B is the molecular weight of the plasmid DNA, and C is copies•μL -1 Calculate the concentration of the mother solution: Paris polyphylla standard plasmid (1.05*10 10 copies•μL -1 ), used as experimental plasmid standard.

[0039] Use solvent to dilute the Paris polyphylla plasmid standard to 1.05*10 10 copies•μL -1, and then diluted it into 5 gradients at 10 times the concentration, and then used the diluted gradient plasmid standards as templates to perform real-time fluorescence quantitative PCR detection under the reaction conditions of Example 1. Each group of experiments was repeated 3 times to develop a standard curve. The detection results of the Paris polyphylla plasmid standard are shown in Table 2, and the standard curve is shown in Table 2. Figure 1 The relevant amplification patterns are shown in Figure 2 shown.

[0040] The standard curve equation of Paris polyphylla is: y = -4.321x + 52.101; R² = 0.9997; R² is greater than 0.98, which meets the standard requirements of real-time fluorescence quantitative PCR.

[0041] Table 2 Results of testing for Paris polyphylla plasmid standards

[0042]

[0043] Example 3 Specificity verification of identifying Paris polyphylla

[0044] The present embodiment provides a method for identifying Paris polyphylla, comprising the following steps: using genomic DNA of Paris polyphylla, Paris polyphylla, Paris polyphylla multiflora, Paris polyphylla gaopingensis, Paris polyphylla hainanensis and other 19 closely related species as templates, and setting up a negative control group (ddH2O), using the primer pairs, probes, reaction systems and amplification procedures of the first embodiment to amplify the templates, repeating each sample 3 times, and the detection results are shown in Tables 3.1, 3.2 and 3.3, and the amplification patterns are shown in Tables 3.1, 3.2 and 3.3. Figure 3 shown.

[0045] Table 3.1 Specificity test results

[0046]

[0047] Table 3.2 Specificity test results

[0048]

[0049] Table 3.3 Specificity test results

[0050]

[0051] As can be seen from Tables 3.1, 3.2 and 3.3, the primer pairs and probes provided by the present invention have amplification reactions and fluorescence phenomena only with Paris polyphylla, with good specificity, and the CT values ​​of every three repetitions are similar, with good repeatability.

[0052] Example 4: Sensitivity Verification of Primer Probes

[0053] The DNA concentrations of the Paris polyphylla samples used in Example 3 were set to seven concentration gradients of 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, 0.001 ng / μL, 0.0001 ng / μL, 0.00001 ng / μL, and 0.000001 ng / μL, respectively. The samples were amplified using the primer pairs, probes, reaction systems, and amplification procedures of Example 1 to test the sensitivity of the primer pairs and probes. Each sample was repeated three times and measured using a real-time fluorescence quantitative PCR instrument. The test results are shown in Tables 4 and 5. Figure 4 shown.

[0054] Table 4 Primer probe sensitivity test results

[0055]

[0056] As can be seen from Table 4, the primer pairs and probes of the present invention have high sensitivity to Paris polyphylla, with the lowest detection limit of 0.001 ng / μL and the average CT value of 38.138.

[0057] Example 5 Repeatability Verification

[0058] 1. Repeatability verification between different batches of enzymes by the same experimenter and the same real-time fluorescence quantitative PCR instrument (QuantStudio Dx);

[0059] 2. Repeatability verification between different operators using the same batch of enzyme and the same real-time fluorescence quantitative PCR instrument (QuantStudio Dx).

[0060] The samples were collected in the field and confirmed to be two known samples of Paris polyphylla by morphology and chloroplast genome comparison. The primer pairs, probes, reaction systems and amplification procedures of Example 1 were used. Each sample was repeated 3 times, and the repeatability evaluation was performed by calculating the coefficient of variation (CV%) value. The calculation formula for the coefficient of variation value is:

[0061] CV=SD (standard deviation) / Mean (mean) × 100%.

[0062] The validation results of different batches of enzymes by the same experimenter and the same QPCR instrument are shown in Table 5.1 and Table 5.2; the amplification profile of the first batch of enzyme validation is shown in Figure 5 The amplification profile of the second batch of enzyme verification is as follows Figure 6 The verification results of the same batch of enzymes and the same QPCR instrument operated by different personnel are shown in Tables 5.3 and 5.4; the amplification map of the verification of the operation of experimenter A is shown in Figure 7 The amplification map verified by experimenter B is as follows: Figure 8 .

[0063] Table 5.1 Validation results of the first batch of enzymes by the same experimenter and QPCR instrument

[0064]

[0065] Table 5.2 Verification results of the second batch of enzymes by the same experimenter and QPCR instrument

[0066]

[0067] Table 5.3 Verification results of the same batch of enzyme and QPCR instrument operated by experimenter A

[0068]

[0069] Table 5.4 Verification results of the same batch of enzyme and QPCR instrument operated by experimenter B

[0070]

[0071] It can be seen from Tables 5.1, 5.2, 5.3 and 5.4 that the coefficient of variation of the repeatability verification results is less than 3% whether it is between different batches of enzymes of the same experimenter and the same QPCR instrument, or between different operators of the same batch of enzymes and the same QPCR instrument. The experimental repeatability is good, the stability of different batches of polymerase is excellent, and the reaction system is stable.

[0072] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0073] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A reagent for identifying Paris polyphylla by real-time fluorescence PCR technology, characterized in that: It contains a primer pair and a probe, wherein the primer pair consists of primers D2-F and D2-R, the sequence of the primer D2-F is shown in SEQ ID No.1, the sequence of the primer D2-R is shown in SEQ ID No.2; the sequence of the probe D2-probe is shown in SEQ ID No.

3.

2. A method for identifying Paris polyphylla using real-time fluorescence PCR technology, characterized in that: The method comprises the following steps: using the genomic DNA of the sample to be identified as a template, using the primers D2-F, D2-R and the probe D2-probe in claim 1 to perform a real-time fluorescent PCR amplification reaction, and detecting that if the CT value is less than 40, the sample is identified as Paris polyphylla.

3. The method for identifying Paris polyphylla according to claim 2, characterized in that: The 5' end of the probe D2-probe is modified with VIC, and the 3' end of the probe D2-probe is modified with MGB.

4. The method for identifying Paris polyphylla according to claim 2, characterized in that: The minimum detection limit of the template is 0.001 ng / μL.

5. The method for identifying Paris polyphylla according to claim 2, characterized in that: The reaction system of the real-time fluorescent PCR amplification is: 10 μL of 2×T5 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 test template, and ddH2O is used to make up to 20 μL reaction system.

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

7. Use of the reagent according to claim 1 in identifying Paris polyphylla.

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