A probe primer set, a kit and a detection method for detecting clematis virginiana

Abstract

The application discloses a probe primer group, a kit and a detection method for detecting European dodder, wherein the probe primer group comprises an upstream primer Ce-rbcL-F, a downstream primer Ce-rbcL-R and a TaqMan probe Ce-rbcL-P; the nucleotide sequence of the upstream primer Ce-rbcL-F is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer Ce-rbcL-R is shown as SEQ ID NO. 2; and the nucleotide sequence of the TaqMan probe Ce-rbcL-P is shown as SEQ ID NO. 3. The primer probe group is designed based on a specific region of the rbcL gene of European dodder, and specific identification of European dodder can be realized by combining an optimized real-time fluorescent PCR reaction system and conditions, namely, the application has the characteristics of rapidness, strong specificity, high identification accuracy, effectively solves the problem that near relatives are difficult to distinguish in traditional morphological identification, and avoids false positive misjudgment. Compared with traditional morphological identification and conventional PCR, the application also has the characteristics of high detection efficiency, short time consumption, simple operation and wide applicability, and can be applied to not only professional laboratory accurate identification, but also practical application of scenarios such as primary quarantine and field monitoring.

Description

Technical Field

[0001] This invention belongs to the field of identification of quarantine weeds, and specifically relates to a probe primer set, kit and detection method for detecting European dodder. Background Technology

[0002] European dodder ( Cuscuta europaea *Cuscuta chinensis* (L.) is a globally prevalent, highly parasitic weed that can infest various crops and horticultural plants, including soybeans, cotton, and vegetables. By absorbing nutrients from its host, it stunts crop growth, drastically reduces yields, and in severe cases, causes widespread crop failure or even total crop loss. It has been listed as a quarantine pest by many countries and regions. Rapid and accurate detection of *Cuscuta chinensis*, especially in scenarios such as port quarantine, field monitoring, and seed quarantine, is crucial for preventing its spread and ensuring agricultural production safety.

[0003] Traditional detection of dodder in Europe relies primarily on morphological identification. This method depends on the morphological characteristics of mature plants (such as flower, fruit, and seed morphology), which is not only time-consuming and labor-intensive but also requires a high level of expertise from the personnel involved. For early-infected seedlings, seeds, and trace amounts of dodder mixed in with crop seeds, morphological methods are insufficient for effective identification, easily leading to missed or false detections. With the development of molecular biology techniques, DNA sequence-based molecular detection methods have become the mainstream technology for dodder detection in Europe due to their advantages such as high specificity, high sensitivity, and lack of sample morphology limitations.

[0004] Currently reported molecular detection methods for dodder in Europe mainly include conventional PCR and real-time fluorescent PCR (qPCR). For example, the method disclosed in Reference 1 (Zhang Yujun, Liu Yueting, Liao Fang, et al. Molecular detection of European dodder based on rbcL gene sequence [J]. Plant Protection, 2009, 35(4):110-113.) has achieved the differentiation of European dodder from other closely related dodder species (such as southern dodder and field dodder) and can detect target DNA at the level of single seed. However, this method uses conventional PCR technology, which requires subsequent electrophoresis detection. The operation process is long and is prone to cross-contamination. Industry standards such as SN / T 4876.8-2020 "DNA Barcoding Methods Part 8: Dodder" recommend the use of ITS2+rbcL dual gene barcoding for the identification of dodder species. Although it can cover European dodder, this standard focuses on general identification at the genus level. The accuracy and efficiency of this specific detection need to be improved.

[0005] Real-time fluorescence PCR (especially the TaqMan probe method) offers advantages such as closed-tube detection, rapid and efficient operation, and higher sensitivity. Therefore, this invention develops a highly specific and simple TaqMan real-time fluorescence PCR detection primer and probe set, kit, and detection method for European dodder. Summary of the Invention

[0006] To address the technical problems existing in the background art, the present invention aims to provide a probe primer set, reagent kit, and detection method for detecting European dodder. This solves the problems of insufficient specificity and cumbersome operation in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a probe primer set for detecting European dodder, comprising: an upstream primer Ce-rbcL-F and a downstream primer Ce-rbcL-R) and a TaqMan probe Ce-rbcL-P, wherein, The nucleotide sequence of the upstream primer Ce-rbcL-F is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer Ce-rbcL-R is shown in SEQ ID NO.2, and the nucleotide sequence of the TaqMan probe Ce-rbcL-P is shown in SEQ ID NO.3.

[0008] A second aspect of the present invention provides a kit for detecting European dodder, comprising the above-described probe and primer set.

[0009] Preferably, it also includes: 2×TaqMan Universal PCR Master Mix, a positive control template, and a negative control template; Preferably, the positive control template is a European dodder DNA template; the negative control template is selected from field dodder genomic DNA templates, Japanese dodder genomic DNA templates, Chinese dodder genomic DNA templates, southern dodder genomic DNA templates, five-pointed dodder genomic DNA templates, soybean genomic DNA templates, wheat genomic DNA templates, and barley genomic DNA templates.

[0010] A third aspect of the present invention provides a rapid detection method for European dodder, comprising the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Using the genomic DNA of the sample to be tested as a template, perform a TaqMan real-time fluorescence PCR reaction using the probe and primer set as described in claim 1; S3. After the reaction is complete, analyze the PCR amplification curve and the cycle threshold Ct value to determine whether the sample to be tested is European dodder.

[0011] Preferably, the PCR reaction system consists of: 12.5 μl of 2×TaqMan Universal PCR Master Mix, 0.5 μl of 10 μM upstream primer, 0.5 μl of 10 μM downstream primer, 0.5 μl of 10 μM probe, 1 μl of template DNA, and 10 μl of ddH2O, with a total system volume of 25 μl.

[0012] Preferably, the PCR reaction procedure is as follows: pre-denaturation at 95 °C for 10 min; followed by 40 cycles, each cycle including denaturation at 95 °C for 15 s, annealing / extension at 60 °C for 1 min, and acquisition of FAM channel fluorescence signal during the annealing / extension stage; and finally incubation at 4 °C for 30 min.

[0013] Preferably, the detection results fall into the following two categories: (1) If the sample to be tested shows a typical S-type amplification curve and the Ct value is <35, then the sample to be tested is determined to be European dodder; (2) If there is no typical amplification curve or the Ct value is ≥35, the sample to be tested is determined to be not European dodder.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) High specificity and high identification accuracy: This invention designs a combination of real-time fluorescent PCR primers and TaqMan probes with appropriate lengths (upstream primer 20 nt, downstream primer 19 nt, probe 22 nt) and upstream primer Ce-rbcL-F and downstream primer Ce-rbcL-R, and TaqMan probe Ce-rbcL-P, targeting the conserved specific region of the rbcL gene in *Cuscuta chinensis*. These primers and probes target specific segments of the gene sequence and achieve specific recognition of *Cuscuta chinensis* through sequence complementarity matching, thus avoiding cross-reactions with closely related species (such as *Cuscuta chinensis* and *Cuscuta japonica*) and host plants (such as soybean and wheat) at the molecular targeting level. Experimental results show that only European dodder samples showed specific amplification, while closely related species such as field dodder and Japanese dodder, as well as host plants such as soybean and wheat, did not show positive reactions. This effectively solves the problem of difficulty in distinguishing closely related species in traditional morphological identification and avoids false positives.

[0015] (2) High detection efficiency and short time consumption: Compared with traditional morphological identification (which requires several days of culture and observation, and takes a long time) and conventional PCR (which requires subsequent gel electrophoresis detection), the method of this invention integrates DNA extraction, amplification and result determination, and performs fluorescence signal acquisition and annealing / extension steps simultaneously. This ensures the specific binding of primers and probes and the efficiency of chain extension, simplifies the operation process, and improves the detection timeliness. The entire process can be completed within 3 hours, and no additional electrophoresis, staining and other steps are required, which greatly shortens the detection cycle and meets the needs of rapid screening.

[0016] (3) The present invention uses “typical S-type amplification curve + Ct value < 35” as the positive judgment standard and “no S-type curve or Ct value ≥ 35” as the dual judgment basis for negative results, thus establishing a clear result judgment standard and providing a unified standard for the objectivity and repeatability of test results.

[0017] (4) Easy to operate and widely applicable: No complex parameter optimization is required, and operators can get started after simple training; the required equipment is a conventional real-time fluorescence PCR instrument and basic laboratory consumables, which is suitable for precise identification in professional laboratories and can also meet the practical application needs of grassroots quarantine, field monitoring and other scenarios. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results are the real-time fluorescence PCR amplification curves in the specificity experiment; where 1 represents European dodder, 2 represents field dodder, 3 represents Japanese dodder, 4 represents Chinese dodder, 5 represents southern dodder, 6 represents five-pointed dodder, 7 represents soybean, 8 represents wheat, 9 represents barley, and 10 represents blank control (water without nuclease). Detailed Implementation

[0020] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may be implemented in other embodiments without these specific details.

[0021] Example 1 1. Experimental Materials and Methods 1.1 Experimental Materials, Reagents, and Equipment European dodder ( Cuscuta europaeaThe following were used: field dodder, Japanese dodder, Chinese dodder, southern dodder, five-pointed dodder, soybean, wheat, and barley (all mature seeds ground into powder for specificity verification), nuclease-free water (blank control), 2x TaqMan Universal PCR Master Mix, upstream primer Ce-rbcL-F (10 μM), downstream primer Ce-rbcL-R (10 μM), TaqMan probe Ce-rbcL-P (10 μM), real-time fluorescence PCR instrument, and commercially available Tiangen plant tissue genomic DNA extraction kit.

[0022] 2. Experimental Methods 2.1 Design of probes and primers: Based on the specific DNA sequence of the rbcL gene (as a target gene) of European dodder published in the Genbank database, the core fragment (numbered to the original sequence position) sequence is shown in Table 1 below. This sequence is a conserved region unique to European dodder, and its homology with the rbcL gene sequences of other dodder species and common crops (soybean, wheat, barley) is less than 85%, ensuring the specificity of the detection.

[0023] Table 1. Core Fragment Sequence (Number corresponds to the position in the original sequence)

[0024] In this embodiment, a set of real-time fluorescent PCR primers were designed using primer design software for the specific DNA sequence of the European dodder rbcL gene. Specifically, the upstream primer Ce-rbcL-F and the downstream primer Ce-rbcL-R are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the TaqMan probe Ce-rbcL-P is shown in SEQ ID NO.3. The specific sequences are shown in Table 2 below.

[0025] Table 2. Information on probes and PCR amplification primers

[0026] 2.2 Template DNA Extraction A commercial genomic DNA extraction kit (Cat. No. DP305-02) was used, and the procedure was strictly followed according to the kit instructions. The specific steps are as follows: (1) Sample pretreatment: Take about 50 mg of mature seed powder from each test sample, place it in a sterile grinding pestle, add 1 mL of liquid nitrogen and grind quickly until fine powder is formed (to avoid the sample absorbing moisture and clumping), and immediately transfer it to a pre-cooled 1.5 mL sterile centrifuge tube; (2) Lysis reaction: Add 600 μL of lysis buffer (preheated to 65 °C) to the centrifuge tube, vortex for 15 s to mix thoroughly, and incubate in a 65 °C constant temperature metal bath for 30 min, vortexing once every 10 min (10 s each time) to ensure that the seed powder is fully lysed. (3) Protein removal: Remove the centrifuge tube and cool it to room temperature. Add 200 μL of chloroform, vortex vigorously for 30 s, let it stand at room temperature for 5 min, and then centrifuge at 12000 r / min and 4 ℃ for 10 min. Carefully aspirate the upper aqueous phase (about 400 μL) and transfer it to a new sterile centrifuge tube. (4) Nucleic acid adsorption: Add an equal volume of anhydrous ethanol (kit-compatible or analytical grade) to the collected aqueous phase, gently invert the centrifuge tube 10 times to mix, slowly transfer the mixture to the adsorption column of the kit, centrifuge at 8000 r / min at room temperature for 1 min, and discard the waste liquid in the collection tube. (5) Washing and purification: Add 500 μL of washing buffer I (as provided with the kit) to the adsorption column, centrifuge at 8000 r / min for 1 min, and discard the waste liquid; then add 500 μL of washing buffer II (anhydrous ethanol needs to be added in advance), centrifuge at 8000 r / min for 1 min, and discard the waste liquid; put the adsorption column back into the collection tube, centrifuge at 12000 r / min for 2 min to completely remove the residual washing liquid and avoid affecting the subsequent PCR reaction; (6) Nucleic acid elution: Transfer the adsorption column to a new 1.5 mL sterile centrifuge tube, add 50 μL of nuclease-free water preheated to 65 °C to the center of the adsorption membrane, let stand at room temperature for 5 min, centrifuge at 12000 r / min for 2 min, and collect the elution buffer in the centrifuge tube, which is the extracted genomic DNA; (7) Purity and concentration detection: Take 2 μL of eluted DNA sample and test it with a nucleic acid concentration and purity detector. Record the OD260 / OD280 ratio and DNA concentration. The qualified standard is that the OD260 / OD280 ratio is between 1.8 and 2.0 (indicating that the DNA purity is good and there is no obvious protein or RNA contamination). According to the test results, adjust the DNA template concentration of each qualified sample to 5-50 ng / μL with nuclease-free water and store it in a -20 ℃ freezer for later use. Avoid repeated freeze-thaw cycles.

[0027] 2.3 PCR reaction 2.3.1 Preparation of PCR reaction system The following controls were set up: a positive control (genomic DNA of known European dodder), a negative control (genomic DNA of field dodder, Japanese dodder, Chinese dodder, southern dodder, five-pointed dodder, soybean, wheat, and barley), and a blank control (sterile deionized water to replace template DNA). Each group had three replicate wells to ensure the reliability of the results.

[0028] Preparation of the PCR reaction system: In a clean bench, add the components in the following proportions, vortex to mix, and centrifuge (3000 rpm, 1 min) to avoid bubble formation. The total volume of the PCR reaction system is 25 μL. The amounts of each component are as follows: 12.5 μL of 2×TaqMan Universal PCR Master Mix, 0.5 μL of 10 μM upstream primer Ce-rbcL-F, 0.5 μL of 10 μM downstream primer Ce-rbcL-R, 0.5 μL of 10 μM TaqMan probe Ce-rbcL-P, 1 μL of DNA template, and 10 μL of nuclease-free water.

[0029] 2.3.2 Reaction Program Setup Place the prepared PCR reaction system in a real-time fluorescence PCR instrument and set the following program: (1) Pre-denaturation: 95 ℃, 10 min; (2) Cyclic Phase (40 cycles): Denaturation: 95 ℃, 15 s; Annealing / Extension: 60℃, 1 min (primers specifically bind to the template and extend, while FAM channel fluorescence signals are collected simultaneously) Insulation: 4 ℃, 30 min.

[0030] (3) Fluorescence acquisition settings: After the annealing / extension stage of each cycle, the fluorescence signal is acquired. The fluorescence threshold is set to 10 times the average value of the fluorescence signal within 3-15 cycles, and the Ct value is automatically calculated.

[0031] 3. Result Judgment and Verification 3.1 Judgment criteria: Quality control requirements: Blank control (sterile deionized water) and negative control (field dodder, Japanese dodder, Chinese dodder, southern dodder, five-cornered dodder, soybean, wheat, barley) must meet the following requirements: no typical S-shaped amplification curve, Ct value showing "Undetermined" (not detected), and the coefficient of variation (CV) of Ct values ​​in 3 replicate wells <5% for the experiment to be valid; otherwise, the experiment is invalid and must be repeated.

[0032] (1) Determination of positive results: If the sample shows a typical S-shaped amplification curve and the cycle threshold (Ct value) is <35 (the critical value is set to 35 in this experiment), then the sample is determined to be European dodder. (2) Negative result determination: If the test sample does not show an S-type amplification curve, or the Ct value is ≥35, then the sample is determined not to be European dodder.

[0033] 2.4 Specificity test European dodder ( Cuscuta europaea Using *Cuscuta chinensis* as the standard sample, *Cuscuta chinensis* var. *chinensis*, *Cuscuta chinensis* var. *chinensis*, *Cuscuta chinensis* var. *chinensis*, *Cuscuta pentadactyla*, soybean, wheat, and barley were simultaneously used as reference samples (for specificity verification). DNA was extracted from each sample, and 2 μL of DNA was taken from each. Nuclease-free ddH2O was used as a blank control instead of the DNA template. PCR reactions were performed in a real-time fluorescence PCR instrument, and the real-time fluorescence PCR amplification curves are shown in the figure. Figure 1 .

[0034] Figure 1 The results showed that the standard sample of European dodder exhibited a typical S-shaped amplification curve and a Ct value < 35, meeting the criteria for a positive result. However, field dodder, Japanese dodder, Chinese dodder, southern dodder, five-pointed dodder, soybean, wheat, barley, and the blank control did not show S-shaped amplification curves, and the Ct values ​​were not detected. These results indicate that the detection method of the present invention can effectively distinguish European dodder from other closely related species and crops, avoiding false positive results caused by cross-reaction. In other words, this method can achieve specific identification of European dodder.

[0035] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. A probe primer set for detecting European dodder, characterized in that, include: The upstream primer is Ce-rbcL-F, the downstream primer is Ce-rbcL-R, and the TaqMan probe is Ce-rbcL-P. The nucleotide sequence of the upstream primer Ce-rbcL-F is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer Ce-rbcL-R is shown in SEQ ID NO.2, and the nucleotide sequence of the TaqMan probe Ce-rbcL-P is shown in SEQ ID NO.

3.

2. A kit for detecting European dodder, characterized in that, It includes the probe primer set as described in claim 1.

3. The reagent kit for detecting European dodder according to claim 2, characterized in that, Also includes: 2×TaqMan Universal PCR Master Mix, positive control template, and negative control template.

4. A rapid detection method for European dodder, characterized in that, Includes the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Using the genomic DNA of the sample to be tested as a template, perform a TaqMan real-time fluorescence PCR reaction using the probe and primer set as described in claim 1; S3. After the reaction is complete, analyze the PCR amplification curve and the cycle threshold Ct value to determine whether the sample to be tested is European dodder.

5. A rapid detection method for European dodder according to claim 4, characterized in that, The PCR reaction system consisted of: 12.5 μl of 2×TaqMan Universal PCR Master Mix, 0.5 μl of 10 μM upstream primer, 0.5 μl of 10 μM downstream primer, 0.5 μl of 10 μM probe, 1 μl of template DNA, and 10 μl of ddH2O, with a total volume of 25 μl.

6. The rapid detection method for European dodder according to claim 4, characterized in that, The PCR reaction procedure was as follows: pre-denaturation at 95 °C for 10 min; followed by 40 cycles, each cycle including denaturation at 95 °C for 15 s, annealing / extension at 60 °C for 1 min, with FAM channel fluorescence signal collected during the annealing / extension phase; and finally incubation at 4 °C for 30 min.

7. The rapid detection method for European dodder according to claim 4, characterized in that, The test results fall into the following two categories: (1) If the sample to be tested shows a typical S-type amplification curve and the Ct value is <35, then the sample to be tested is determined to be European dodder; (2) If there is no typical amplification curve or the Ct value is ≥35, the sample to be tested is determined to be not European dodder.

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