Primer group for identifying phthorimaea operculella based on ddPCR and method for identifying phthorimaea operculella by detecting environmental DNA

Abstract

The invention discloses a primer group for identifying phthorimaea operculella based on ddPCR and a method for identifying phthorimaea operculella by detecting environmental DNA, and belongs to the technical field of pest detection. The primer group for identifying the phthorimaea operculella based on the ddPCR comprises a forward primer, a reverse primer and a specific fluorescent probe, the nucleic acid sequences of the forward primer, the reverse primer and the specific fluorescent probe are as shown in SEQ ID NO.1-SEQ ID NO.3; the C basic group at the 3'end of the forward primer, the A basic group at the 3 'end of the reverse primer, and the A basic groups and the T basic groups at the 12th site and the 13th site of the specific fluorescent probe are basic groups modified by locked nucleic acid. The primer group disclosed by the invention has very high specificity and sensitivity to the phthorimaea operculella, can be detected by taking eDNA as a template, can find the phthorimaea operculella earlier, and has an important application prospect in early detection and prevention of the phthorimaea operculella.

Description

Technical Field

[0001] The present invention belongs to the technical field of pest detection, and in particular relates to a primer set for identifying potato tuber moth based on ddPCR and a method for identifying potato tuber moth by detecting environmental DNA. Background Art

[0002] The potato tuber moth (Phthorimaea operculella (Zeller)), a member of the Lepidoptera order, Gelechiidae family, also known as the tobacco leafminer and potato wheat moth, is a oligophagous pest. Its primary host plants include potatoes, tobacco, eggplant, tomatoes, and peppers, but it is particularly fond of potatoes and tobacco. Its larvae bore into leaves, making it difficult to control, and it is listed as a key plant quarantine target in many countries, including my country. The tomato leafminer often co-occurs with the potato tuber moth, a species of the same family, and their morphology and feeding characteristics are similar, making field identification difficult. Due to the potato tuber moth's strong adaptability, high reproductive capacity, and significant economic losses, timely and early detection is an effective method for preventing its spread.

[0003] Currently, there are few detection technologies for the potato tuber moth. Traditional morphological identification methods and conventional PCR-based detection methods are commonly used, but conventional PCR has limited detection capacity and sensitivity. Therefore, these methods are difficult to detect in the early stages of an infestation and fail to meet the needs of early detection and prevention. Furthermore, most existing molecular biological detection methods require the collection of plant samples, causing irreversible damage to plants. Environmental DNA (eDNA) refers to DNA released from organisms into the natural environment, including DNA in cells and DNA molecules released after cell disruption. Molecular biological detection techniques such as PCR and sequencing can be used to qualitatively or quantify target products. In recent years, eDNA technology has been widely used in aquatic organism research. Compared to traditional sampling methods, eDNA is easier to obtain samples. Furthermore, because small DNA fragments can persist for long periods of time under dry, frozen, or dark conditions, eDNA technology has become a valuable tool in the field of detection and monitoring technology.

[0004] ddPCR, or droplet digital PCR, is a recently developed PCR technology. Its fluorescent signal generation principle is similar to that of qPCR. A droplet generator "divides" the fluorescent PCR reaction system containing nucleic acid molecules into tens of thousands of nanometer-sized droplets, where the nucleic acids are randomly distributed. Each droplet may contain no or at least one target nucleic acid molecule to be detected, and each droplet functions as an independent PCR reactor. Numerous experiments have demonstrated that ddPCR offers superior accuracy compared to qPCR. Currently, reports on ddPCR technology are primarily focused on disease diagnosis, pathogenic fungal detection, and aquatic organism detection. Its application in agricultural pest identification needs further expansion; no reports have been found using ddPCR to detect potato tuber moth. Summary of the Invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a primer set for identifying potato tuber moth based on ddPCR and a method for identifying potato tuber moth by detecting environmental DNA.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: A primer set for identifying potato tuber moth based on ddPCR, comprising a forward primer, a reverse primer, and a specific fluorescent probe; the nucleic acid sequence of the forward primer is shown in SEQ ID NO.1, the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.2, and the nucleic acid sequence of the specific fluorescent probe is shown in SEQ ID NO.3; The C base at the 3' end of the forward primer, the A base at the 3' end of the reverse primer, and the A base and T base at the 12th and 13th positions of the specific fluorescent probe are locked nucleic acid modified bases.

[0007] Based on the above scheme, the 5' end of the specific fluorescent probe is labeled with HEX, and the 3' end is labeled with BHQ1.

[0008] Application of the above-mentioned primer set for identifying potato tuber moth based on ddPCR in identifying potato tuber moth.

[0009] Based on the above scheme, potato tuber moth was identified by detecting environmental DNA.

[0010] A method for identifying potato tuber moth by detecting environmental DNA, using the above primer set for detection.

[0011] Based on the above scheme, the method for identifying potato tuber moth by detecting environmental DNA comprises the following steps: The extracted environmental DNA was used as a template, and ddPCR amplification was performed using the primers described in SEQ ID NO. 1 to SEQ ID NO. 3 and a specific fluorescent probe. After the reaction, droplet fluorescence data analysis was performed. When a fluorescent signal was present, it indicated that the sample to be tested was the potato tuber moth. When no fluorescent signal was present, it indicated that the sample to be tested was not the potato tuber moth.

[0012] Based on the above scheme, the environmental DNA is extracted from damaged parts or excrement collected from plants suspected of being damaged by potato tuber moth.

[0013] Based on the above scheme, the ddPCR reaction procedure is as follows: Pre-denaturation at 95°C for 10 min; 45 cycles of denaturation at 94°C for 30 s, annealing and extension at 49°C for 1 min; 98°C for 10 s; and finally storage at 4°C with a heating and cooling rate of 2°C / s.

[0014] Based on the above scheme, the ddPCR reaction system is as follows: 2 × ddPCR Supermix for Probes 10 μL, 1.6 μL of 10 μmol / μL forward primer, 1.6 μL of 10 μmol / μL reverse primer, 4 μL of 1 μmol / μL specific fluorescent probe, 1 μL DNA template, and ddH2O to 20 μL.

[0015] Advantages of the technical solution of the present invention: The present invention designs ddPCR primers and probes based on the mtCOI gene of the potato tuber moth, and establishes a method for detecting the potato tuber moth using ddPCR. The primers and probes of the present invention have high specificity and sensitivity for the potato tuber moth. This method for detecting the potato tuber moth based on ddPCR can detect the potato tuber moth using eDNA as a template, enabling earlier detection of the potato tuber moth than existing technologies, and has important application prospects in the early detection and prevention of the potato tuber moth. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 ddPCR amplification results at different annealing temperatures; Figure 2 Specificity detection results of the ddPCR-based method for detecting potato tuber moth; Figure 3 Sensitivity test results of the ddPCR-based method for detecting potato tuber moth; Figure 4 The effectiveness of ddPCR-based method for detecting potato tuber moth in eDNA samples. DETAILED DESCRIPTION

[0017] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. Below, in conjunction with specific examples, the present invention will be further described in detail with reference to data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0018] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. The experimental materials, reagents, and drugs used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0019] In the following examples, ddPCR reactions were performed using the Bio Rad QX200 ddPCR system; the primers and probes used were synthesized by Shandong Haichen Biotechnology Co., Ltd.

[0020] The specific primer set for detecting the potato tuber moth based on droplet digital polymerase chain reaction (ddPCR) is designed based on a 679 bp fragment on the COI gene of the potato tuber moth.

[0021] Example 1 A specific primer set for detecting potato tuber moth based on droplet digital polymerase chain reaction (ddPCR), comprising a forward primer, a reverse primer, and a specific fluorescent probe; the nucleic acid sequence of the forward primer is shown in SEQ ID NO.1, the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.2, and the nucleic acid sequence of the specific fluorescent probe is shown in SEQ ID NO.3.

[0022] P. Operculella-F: 5'-CAGATATAGCTTTCCC+C-3' (SEQ ID NO. 1); P. Operculella-R: 5'-CAGCTCCATTTTCTAC+A-3' (SEQ ID NO. 2); P. Operculella-P: 5'-HEX-TGAAATTAATA+A+TGTAAGAGAGGGTGG-MGB-BHQ1-3' (SEQ ID NO. 3).

[0023] The "+" in the sequence indicates that the following bases are modified with locked nucleic acid (LNA). Specifically, the C base at the 3' end of the forward primer, the A base at the 3' end of the reverse primer, and the A and T bases at positions 12 and 13 of the specific fluorescent probe are bases modified with LNA.

[0024] Example 2 A method for identifying potato tuber moth (Phthorimaea operculella) by detecting environmental DNA (eDNA) based on droplet digital PCR (ddPCR) technology, including the following steps: (1) Extracting genomic DNA from the sample to be tested; The sample to be tested is the damaged part or excrement collected from the plant suspected to be damaged by the potato tuber moth.

[0025] The genomic DNA can be extracted using existing methods or a genomic DNA extraction kit, such as the TIANamp Micro DNA Kit (centrifugal column type). (2) Adding the prepared ddPCR reaction system and droplet generation oil to the droplet generation card, and placing it in a droplet generator to generate droplets; (3) The generated droplets are transferred to a 96-well ddPCR plate for amplification reaction. After the reaction is completed, the droplet fluorescence data is analyzed. When a fluorescent signal is present, it indicates that the sample to be tested is potato tuber moth. When no fluorescent signal is present, it indicates that the sample to be tested is not potato tuber moth.

[0026] The ddPCR reaction system is as follows: 2 × ddPCR Supermix for Probes 10 μL, 1.6 μL each of forward / reverse primers (10 μmol / μL), 4 μL of specific fluorescent probe (1 μmol / μL), 1 μL DNA template, and ddH2O to 20 μL; The nucleic acid sequence of the forward primer is shown in SEQ ID NO.1, the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.2, and the nucleic acid sequence of the specific fluorescent probe is shown in SEQ ID NO.3; The ddPCR reaction procedure is as follows: Pre-denaturation at 95°C for 10 min; 45 cycles of denaturation at 94°C for 30 s, annealing and extension at 49°C for 1 min; 98°C for 10 s; and finally storage at 4°C with a heating and cooling rate of 2°C / s.

[0027] Example 3 Effect of Annealing Temperature on ddPCR Detection of Potato Tuber Moth The extracted genomic DNA of the potato tuber moth was used as a template, and ddPCR detection was performed using the method of Example 2, wherein the annealing temperature in the ddPCR reaction was set to 45-55°C, with a total of 8 temperature gradients (from high to low: 55.0, 54.2, 52.9, 51.2, 48.9, 46.9, 45.7, 45.0); the ddPCR amplification results at different annealing temperatures were obtained, as shown in FIG. Figure 1 As shown. Figure 1 It can be seen that when the annealing temperature is 48.9°C, the ddPCR amplification efficiency is better.

[0028] Example 4 Specificity of the ddPCR-based method for detecting potato tuber moth The genomic DNA of potato tuber moth, tomato leafminer, pea leafminer, and American leafminer were extracted as templates, and ddH2O was used as a blank control. ddPCR detection was performed using the method of Example 2. The results are as follows: Figure 2 shown.

[0029] Depend on Figure 2 It can be seen that the method of Example 2 can successfully detect the potato tuber moth, and does not amplify the tomato leafminer, pea leafminer, and the American leafminer. It has strong specificity and can well meet the requirements of the potato tuber moth digital PCR detection.

[0030] Example 5 Sensitivity of the ddPCR-based method for detecting potato tuber moth The concentration of genomic DNA from the potato tuber moth was measured. The concentration of the template stock solution was 53.8 ng / μL. 4 μL of the template stock solution was added to 36 μL of ddH2O and diluted 10-fold. A total of 6 concentration gradients were set (53.8 ng / μL, 53.8×10 -1 ng / μL、53.8×10 -2 ng / μL、53.8×10 -3 ng / μL、53.8×10 -4 ng / μL、53.8×10 -5 ng / μL、53.8×10 -6 ng / μL), and ddH2O was used as a blank control template. The method of Example 2 was used for detection. The results are shown in Figure 2. Figure 3 As shown in the figure, the detection limit of digital PCR for the concentration of potato tuber moth DNA was 5.38×10 -4 ng / μL.

[0031] Example 6: Effect of ddPCR method on detecting potato tuber moth in eDNA samples The eDNA of potato tuber moth, tomato leafminer, pea leafminer, and American leafminer were extracted respectively. The DNA of potato tuber moth was diluted 1000 times as a positive control, the eDNA of tomato leafminer, pea leafminer, American leafminer and healthy tomato leaf DNA were used as negative controls, and ddH2O was used as a blank control. ddPCR detection was performed using the method of Example 2. The results are as follows: Figure 4 shown.

[0032] Depend on Figure 4 It can be seen that the method of Example 2 can successfully detect the DNA and eDNA of potato tuber moth, and does not amplify the eDNA of tomato leafminer, pea leafminer, and leafminer and the DNA of healthy tomato leaves. It has strong specificity and can well meet the requirements of the ddPCR method for detecting potato tuber moth in eDNA samples.

[0033] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A primer set for identifying potato tuber moth based on ddPCR, characterized in that: It comprises a forward primer, a reverse primer and a specific fluorescent probe; the nucleic acid sequence of the forward primer is shown in SEQ ID NO.1, the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.2, and the nucleic acid sequence of the specific fluorescent probe is shown in SEQ ID NO.3; The C base at the 3' end of the forward primer, the A base at the 3' end of the reverse primer, and the A base and T base at the 12th and 13th positions of the specific fluorescent probe are locked nucleic acid modified bases.

2. The primer set for identifying potato tuber moth based on ddPCR according to claim 1, characterized in that The 5' end of the specific fluorescent probe is labeled with HEX, and the 3' end is labeled with BHQ1.

3. Use of the primer set for identifying potato tuber moth based on ddPCR according to claim 1 or claim 2 in identifying potato tuber moth.

4. The use according to claim 3, characterized in that Identification of potato tuber moth by detecting environmental DNA.

5. A method for identifying potato tuber moth by detecting environmental DNA, characterized in that: Detection is performed using the primer set described in claim 1 or 2.

6. The method for identifying potato tuber moth by detecting environmental DNA according to claim 5, characterized in that: Here are the steps: The extracted environmental DNA was used as a template, and ddPCR amplification was performed using the primers described in SEQ ID NO. 1 to SEQ ID NO. 3 and a specific fluorescent probe. After the reaction, droplet fluorescence data analysis was performed. When a fluorescent signal was present, it indicated that the sample to be tested was the potato tuber moth. When no fluorescent signal was present, it indicated that the sample to be tested was not the potato tuber moth.

7. The method for identifying potato tuber moth by detecting environmental DNA according to claim 6, characterized in that: The environmental DNA is extracted from damaged parts or excrement collected from plants suspected of being damaged by potato tuber moth feeding.

8. The method for identifying potato tuber moth by detecting environmental DNA according to claim 7, characterized in that: The ddPCR reaction procedure is as follows: Pre-denaturation at 95°C for 10 min; 45 cycles of denaturation at 94°C for 30 s, annealing and extension at 49°C for 1 min; and 10 s at 98°C. Finally, storage at 4°C with a heating and cooling rate of 2°C / s was performed.

9. The method for identifying potato tuber moth by detecting environmental DNA according to claim 7, characterized in that: The reaction system of the ddPCR is as follows: 2 × ddPCR Supermix for Probes 10 μL, 1.6 μL of 10 μmol / μL forward primer, 1.6 μL of 10 μmol / μL reverse primer, 4 μL of 1 μmol / μL specific fluorescent probe, 1 μL DNA template, and ddH2O to 20 μL.

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