SS-COI PCR (Polymerase Chain Reaction) detection primer and detection method for malus micromalus
By designing SS-COI PCR detection primers for *Aphis chinensis* and using specific COI primers for PCR amplification, the problem of identifying *Aphis chinensis* eggs, larvae, and pupae was solved, achieving rapid and accurate detection results.
Patent Information
- Application Number
- CN202511578513.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2026-01-23
AI Technical Summary
Existing technologies make it difficult to quickly and accurately identify the eggs, larvae, and pupae of the apple slug, causing difficulties for border and forestry personnel in their quarantine work.
A primer for detecting SS-COI in apple gentian was designed. PCR amplification was performed using specific COI primers, and identification was performed using a 303bp amplification band. The sensitivity reached 3.427×10-2ng/μL.
It enables rapid and accurate detection of apple jewel beetles, exhibits good specificity, is suitable for detecting different insect stages and populations, and has high sensitivity.
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Figure CN121380356A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of molecular detection of Agrilus mali, and particularly relates to an A. mali SS-COI PCR detection primer and a detection method. BACKGROUND
[0002] Agrilus mali Matsumura belongs to the Coleoptera family Buprestidae and the Agrilus genus, and mainly harms plants of the Malus genus. A. mali is distributed in Xinjiang, Gansu, Qinghai, Inner Mongolia, Liaoning and other provinces in China, and has caused great influence on the growth of apple trees and the apple industry in the local area. In recent years, A. mali has occurred seriously in the wild fruit forest in the western Tianshan Mountains of Xinjiang, China, and has spread rapidly, and has become an important pest threatening the ecological construction of forestry and the healthy development of the fruit industry in Yili and even Xinjiang.
[0003] Since different buprestids have great differences in body size, body color, spots, reproductive organs and other aspects at the adult stage, the species identification of buprestid insects has always taken the characteristics of adults as the main identification basis. Studies have shown that the spreading and diffusing ability of A. mali adults is limited, and A. mali usually spreads and diffuses through seedlings transportation and trade exchanges with host plants in the form of eggs, larvae or pupae. However, it is difficult to accurately identify the species through the morphology of the eggs, larvae and pupae of buprestids, and it is difficult to meet the actual needs of port, customs and frontline forestry personnel to carry out quarantine work. Therefore, it is very important to develop a detection technology that can quickly identify A. mali.
[0004] At present, the Species-Specific COI (SS-COI) PCR detection technology has become an important means for rapid detection of a variety of pests. This technology uses species-specific COI primers for PCR amplification, and identifies the detection sample according to the presence or absence of the expected DNA band, without the need for sequencing and sequence alignment, and has the advantages of high sensitivity, strong specificity, good repeatability, rapidness, simplicity and the like. The key to this technology is to screen high-specificity and high-sensitivity PCR primers. Once the specific primers are obtained, rapid and accurate detection and identification of A. mali can be achieved. SUMMARY
[0005] In order to solve the above problems, the present application provides an A. mali SS-COI PCR detection primer and a detection method. The A. mali SS-COI PCR detection primer provided by the present application can specifically detect A. mali, and the sensitivity reaches 3.427 x 10 -2 ng / μL.
[0006] In order to achieve the above purpose, the present application provides the following technical solutions:
[0007] The application provides a Chrysopida SS-COI PCR detection primer, a nucleotide sequence of an upstream primer of the Chrysopida SS-COI PCR detection primer is shown in SEQ ID No. 1, and a nucleotide sequence of a downstream primer is shown in SEQ ID No. 2.
[0008] The application also provides application of the Chrysopida SS-COI PCR detection primer in detection of Chrysopida.
[0009] The application also provides a kit for detecting Chrysopida, wherein the kit contains the Chrysopida SS-COI PCR detection primer.
[0010] The application also provides a method for detecting Chrysopida, comprising the following steps:
[0011] 1) extracting DNA of a sample to be detected;
[0012] 2) performing PCR amplification on the DNA of the sample to be detected in step 1) by taking the Chrysopida SS-COI PCR detection primer as a template to obtain an amplification product;
[0013] 3) performing agarose gel electrophoresis on the amplification product in step 2), and when an amplification band of 303bp appears, the sample to be detected is Chrysopida.
[0014] Preferably, a system for the PCR amplification in step 2) is as follows: 2x Taq Plus PCR Master Mix 12.5 μL, the upstream primer with a concentration of 10 μmol / L 1 μL, the downstream primer with a concentration of 10 μmol / L 1 μL, genomic DNA template 2 μL and ddH2O 8.5 μL.
[0015] Preferably, a program for the PCR amplification in step 2) is as follows: 94℃ pre-denaturation for 2min; 94℃ denaturation for 30s, 55℃, 57℃, 59℃ or 61℃ annealing for 30s, 72℃ extension for 30s, a total of 30 cycles; and finally 72℃ extension for 2min.
[0016] The application has the following beneficial effects:
[0017] Specific detection results show that the primer provided by the application has good specificity and can specifically amplify Chrysopida, and cannot specifically amplify Chrysoprata formosa, Agrilus planipennis and Agrilus subfuscatus and other close species, which indicates that the Chrysopida SS-COI PCR detection primer and the detection method provided by the application have good specificity and are suitable for rapid and accurate detection of Chrysopida.
[0018] The application is respectively subjected to PCR amplification at 8 annealing temperatures, and by comparing the amplification results, only the sample of Agrilus mali can produce an amplification band and its close relatives cannot produce an amplification band when the annealing temperature is 55 DEG C, 57 DEG C, 59 DEG C and 61 DEG C. In order to ensure the PCR amplification efficiency, 55 DEG C is taken as the optimal annealing temperature of the application, and subsequent related experiments are all carried out at 55 DEG C as the annealing temperature.
[0019] The sensitivity detection result shows that the detection limit of the Agrilus mali SS-COI PCR detection method provided by the application is 3.427 x 10 -2 ng / μL, which shows that the detection sensitivity is high. BRIEF DESCRIPTION OF DRAWINGS
[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below.
[0021] Figure 1 The electrophoresis detection diagram of the amplification results of Agrilus mali and other three kinds of narrow beetles by using universal primers LCO1490 / HCO2198 (M: DNA marker; 1: Agrilus mali; 2: Agrilus viduus; 3: Agrilus fleischeri; 4: Agrilus planipennis; NTC: negative control).
[0022] Figure 2 The schematic diagram of designing primers according to part of the sequence in the complete COI gene of Agrilus mali.
[0023] Figure 3 The electrophoresis detection diagram of the amplification results of Agrilus mali and other three kinds of narrow beetles by using different primers (M: DNA marker; 1: Agrilus mali; 2: Agrilus viduus; 3: Agrilus fleischeri; 4: Agrilus planipennis; NTC: negative control).
[0024] Figure 4 The PCR amplification annealing temperature screening of SS-COI primers AMSSCOI-F / AMSSCOI-R (M: DNA marker; 1: Agrilus mali; 2: Agrilus viduus; 3: Agrilus fleischeri; 4: Agrilus planipennis; NTC: negative control).
[0025] Figure 5 SS-COI primer AMSSCOI-F / AMSSCOI-R amplified electrophoretic detection map of apple small jewel beetle larvae, pupae and adults (M: DNA marker; 1: apple small jewel beetle larvae / Fuxin, Liaoning; 2: apple small jewel beetle larvae / Balian, Xinjiang; 3: apple small jewel beetle pupae / Fuxin, Liaoning; 4: apple small jewel beetle pupae / Balian, Xinjiang; 5: apple small jewel beetle adults / Fuxin, Liaoning; 6: apple small jewel beetle adults / Balian, Xinjiang; NTC: negative control).
[0026] Figure 6 SS-COI primer AMSSCOI-F / AMSSCOI-R amplified electrophoretic detection map of 9 different geographical populations of apple small jewel beetle (M: DNA marker; 1: Chaoyang, Liaoning; 2: Fuxin, Liaoning; 3: Nileke, Xinjiang; 4: Heba, Xinjiang; 5: Balian, Xinjiang; 6: Tuergen, Xinjiang; 7: Guide, Qinghai; 8: Pingliang, Gansu; 9: Chifeng, Inner Mongolia; NTC: negative control).
[0027] Figure 7 SS-COI primer AMSSCOI-F / AMSSCOI-R amplified electrophoretic detection map of 10-fold diluted apple small jewel beetle DNA sample (M: DNA marker; 1: 342.7 ng / μL; 2: 34.27 ng / μL; 3: 3.427 ng / μL; 4: 3.427×10 -1 ng / μL; 5: 3.427×10 -2 ng / μL; 6: 3.427×10 -3 ng / μL; 7: 3.427×10 -4 ng / μL; 8: 3.427×10 -5 ng / μL; 9: 3.427×10 -6 ng / μL; NTC: negative control) DETAILED DESCRIPTION
[0028] The application provides an apple small jewel beetle SS-COI PCR detection primer, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID No. 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2.
[0029] SEQ ID No. 1: 5'-GTAGGAACCGCGCTTAGTTTAT-3';
[0030] SEQ ID No. 2: 5'-GTAAACAGTTCACCCTGTACCA-3'.
[0031] The application also provides application of the apple small jewel beetle SS-COI PCR detection primer in detection of the apple small jewel beetle.
[0032] The application also provides a kit for detecting the apple small jewel beetle, which contains the apple small jewel beetle SS-COI PCR detection primer.
[0033] The application also provides a method for detecting the apple small jewel beetle, which comprises the following steps:
[0034] 1) extracting DNA of a sample to be detected;
[0035] 2) taking the DNA of the sample to be detected as a template, and performing PCR amplification on the apple small jewel beetle SS-COI PCR detection primer to obtain an amplification product;
[0036] 3) performing agarose gel electrophoresis on the amplification product, and when an amplification band of 303 bp appears, the sample to be detected is the apple small jewel beetle.
[0037] The method for extracting DNA of the sample to be detected is not particularly limited, and a person skilled in the art can adopt a conventional method.
[0038] In the application, the PCR amplification system is preferably 2x TaqPlus PCR Master Mix 12.5 μL, 1 μL of the upstream primer with a concentration of 10 μmol / L, 1 μL of the downstream primer with a concentration of 10 μmol / L, 2 μL of the genomic DNA template, and 8.5 μL of ddH2O.
[0039] In the application, the PCR amplification program is preferably 94 ℃ pre-denaturation for 2 min, 30 cycles of 94 ℃ denaturation for 30 s, 55 ℃, 57 ℃, 59 ℃ or 61 ℃ annealing for 30 s, 72 ℃ extension for 30 s, and finally 72 ℃ extension for 2 min.
[0040] In order to further illustrate the application, the application is described in detail in combination with examples below, but they should not be understood as limiting the protection scope of the application.
[0041] Example 1
[0042] Extraction of total DNA of the apple small jewel beetle and related species and screening of specific primers
[0043] 1 Total DNA extraction of Agrilus sinuatus and related species
[0044] The test samples (see Table 1) were ground into powder with liquid nitrogen, and the sample DNA was extracted using a blood / cell / tissue genomic DNA extraction kit (TIANGEN Company).
[0045] Table 1 Specific information of samples
[0046]
[0047]
[0048] The above sample DNA was used as a template, and the mitochondrial COI gene fragment was amplified using universal primers LCO1490 / HCO2198 to test whether the sample DNA was available. The PCR amplification system was 25 μL: 2x TaqPlus PCR Master Mix (Beijing Ruiboxingke Biotechnology Co., Ltd.) 12.5 μL, upper and lower stream primers LCO1490 / HCO2198 (10 μmol / L) each 1 μL, DNA template 2 μL, and ultrapure water added to 25 μL. The PCR amplification program was: 94°C pre-denaturation for 2 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, a total of 30 cycles, and finally 72°C extension for 2 min. After PCR amplification, all samples were subjected to 1.2% agarose gel electrophoresis, and all showed bright and clear amplification bands, indicating that the sample DNA could be used for subsequent detection. Figure 1
[0049] 2 Screening of Agrilus sinuatus specific primers
[0050] According to the part of the complete COI gene of Agrilus sinuatus that is significantly different from the insects of the genus Agrilus (Agrilus planipennis, Agrilus planipennis, Agrilus liragus, Agrilus smaragdifrons, Agrilus roscidus, Agrilus osburni, Agrilus ribbei), primers were designed Figure 2 ). After sequence alignment, five pairs of primers were designed for PCR amplification of A. mali, A. iris, A. fabii and A. planipennis, respectively (Table 2). The PCR amplification system was 25 μL: 2 × TaqPlus PCR Master Mix (Beijing Rebiya Xingke Biotechnology Co., Ltd.) 12.5 μL, 1 μL of each upstream and downstream primer (10 μmol / L), 2 μL of DNA template, and ultra-pure water to 25 μL. The PCR amplification program was 2 min of pre-denaturation at 94°C, 30 s of denaturation at 94°C, 30 s of annealing at 55°C, 30 s of extension at 72°C, for a total of 30 cycles, and finally 2 min of extension at 72°C. After the PCR reaction, agarose gel electrophoresis was performed. The results showed that only primer No. 2 had an amplification band in the A. mali sample, and no amplification band in other closely related species, so primer No. 2 had high specificity Figure 3
[0051] Table 2 PCR amplification primer information
[0052]
[0053] The 303 bp fragment amplified by primer No. 2 was named AMSSCOI-F / AMSSCOI-R, and the specific information is as follows:
[0054] Upstream primer AMSSCOI-F: 5'-GTAGGAACCGCGCTTAGTTTAT-3' (SEQ ID No. 1);
[0055] Downstream primer AMSSCOI-R: 5'-GTAAACAGTTCACCCTGTACCA-3' (SEQ ID No. 2).
[0056] Example 2
[0057] Annealing temperature screening of primer pair AMSSCOI-F / AMSSCOI-R PCR amplification
[0058] In order to determine the optimal annealing temperature of the primer pair AMSSCOI-F / AMSSCOI-R in PCR amplification, 8 annealing temperatures were tested, which were 47℃, 49℃, 51℃, 53℃, 55℃, 57℃, 59℃ and 61℃. The PCR amplification system was 25 μL: 2x Taq Plus PCR MasterMix (Beijing Ruiboxingke Biotechnology Co., Ltd.) 12.5 μL, upstream and downstream primers AMSSCOI-F / AMSSCOI-R (10 μmol / L) 1 μL each, DNA template 2 μL, and ultrapure water to 25 μL. The PCR amplification program was: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, a total of 30 cycles, and finally 72℃ extension for 2 min, only the annealing temperature was changed. The samples in Table 1 were tested as an example.
[0059] After all samples were subjected to 1.2% agarose gel electrophoresis, the amplification results were as shown in Figure 4 Four samples were subjected to PCR amplification at 8 annealing temperatures in turn, and by comparing the amplification results, when the annealing temperature was 55℃, 57℃, 59℃ and 61℃, only the Ageladomorpha sample would produce an amplification band and its close relatives would not produce an amplification band. In order to ensure the PCR amplification efficiency, 55℃ was used as the optimal annealing temperature of the present application, and 55℃ was used as the annealing temperature in subsequent related experiments.
[0060] Example 3
[0061] Stability and sensitivity test of primer pair AMSSCOI-F / AMSSCOI-R
[0062] 1. Primer stability test
[0063] The test sample (see Tables 3 and 4) was ground into powder with liquid nitrogen, and the sample DNA was extracted with a blood / cell / tissue genomic DNA extraction kit (TIANGEN Co., Ltd.).
[0064] Table 3 Specific information of Ageladomorpha samples in different insect stages
[0065]
[0066] Table 4 Specific information of Ageladomorpha samples of different populations
[0067] Number Species name Place Number (head) 1 Chrysoteuchia orichalcea adult Chaoyang, Liaoning 1 2 Chrysoteuchia orichalcea adult Fuxin, Liaoning 1 3 Chrysoteuchia orichalcea adult Chifeng, Inner Mongolia 1 4 Chrysoteuchia orichalcea adult Pingliang, Gansu 1 5 Chrysoteuchia orichalcea adult Guide, Qinghai 1 6 Chrysoteuchia orichalcea adult Nileke County, Yili Prefecture, Xinjiang 1 7 Chrysoteuchia orichalcea adult Turgeng, Xinyuan County, Yili Prefecture, Xinjiang 1 8 Chrysoteuchia orichalcea adult Kebake Village, Gulei County, Yili Prefecture, Xinjiang 1 9 Chrysoteuchia orichalcea adult 72nd Team, Yili Prefecture, Xinjiang 1
[0068] The sample DNA was used as a template, and specific primers AMSSCOI-F / AMSSCOI-R were used for PCR amplification. The PCR amplification system was 25 μL: 2x Taq Plus PCR Master Mix (Beijing Ruiboxingke Biotechnology Co., Ltd.) 12.5 μL, 1 μL of each of the upper and lower primers AMSSCOI-F / AMSSCOI-R (10 μmol / L), 2 μL of DNA template, and ultra-pure water to 25 μL. The PCR amplification program was: 94°C pre-denaturation for 2 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, a total of 30 cycles, and finally 72°C extension for 2 min.
[0069] The amplification results of the primer pair AMSSCOI-F / AMSSCOI-R in different insect stages and different populations of apple leaf-roller are shown in Figure 5 and Figure 6 According to the amplification results of Figure 5 , the larva, pupa and adult samples of apple leaf-roller all have amplification bands, indicating that this primer and PCR program can stably detect apple leaf-roller and are suitable for detection of apple leaf-roller larvae, pupae and adults. According to the amplification results of Figure 6 , the adult samples of apple leaf-roller in 9 populations of Xinjiang, Inner Mongolia, Liaoning, Gansu and Qinghai all have amplification bands, indicating that the specific primer AMSSCOI-F / AMSSCOI-R can be used for detection of apple leaf-roller in different populations.
[0070] 2 Sensitivity test of primers
[0071] Taking the sample DNA of No. 6 (apple leaf-roller pupae in Yili 72nd Team 8th Company, Xinjiang) in Table 3 as an example, 10-fold gradient dilution was performed for sensitivity limit test. The DNA concentration was measured by NanoDrop ND-100 spectrophotometer (USA NanoDrop Technology Company) to be 1 No. 342.7 ng / μL, and the DNA was diluted to 2 No. 34.27 ng / μL, 3 No. 3.427 ng / μL, 4 No. 3.427x10 -1 ng / μL, 5 No. 3.427x10 -2 ng / μL, 6 No. 3.427x10 -3 ng / μL, 7 No. 3.427x10 -4 ng / μL, 8 No. 3.427x10 -5 ng / μL, 9 No. 3.427x10 -6 ng / μL, respectively. The samples 1-9 were used as templates, and the primer pair AMSSCOI-F / AMSSCOI-R was used for PCR amplification (the PCR amplification system and the PCR amplification program were the same as above).
[0072] The amplification results of the diluted DNA samples are shown in Fig. 2. Figure 7 As shown in Fig. 2, when the DNA concentration is 5# 3.427 x 10 Figure 7 ng / μL, a very shallow band is produced, and when the DNA concentration is less than 5# 3.427 x 10 - 2 ng / μL and the blank control (using sterile water as template) sample, no band is produced. Therefore, the detection limit of the AMSSCOI-F / AMSSCOI-R specific primer pair for DNA concentration is 3.427 x 10 -2 ng / μL. -2
[0073] Although the above embodiment has described the present application in detail, it is only a part of the embodiments of the present application, but not all the embodiments. Other embodiments can be obtained according to the present embodiment without creativity, which are all within the protection scope of the present application.
Claims
1. A primer for detecting apple gentian SS-COI PCR, characterized in that, The nucleotide sequence of the upstream primer of the Apple-Gilead SS-COI PCR detection primer is shown in SEQ ID No. 1, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.
2.
2. The application of the SS-COI PCR detection primers for chloranthus as described in claim 1 in the detection of chloranthus.
3. A reagent kit for detecting apple gentian, characterized in that, The kit contains the apple succinate SS-COI PCR detection primers as described in claim 1.
4. A method for detecting apple gentian, characterized in that, Includes the following steps: 1) Extract DNA from the sample to be tested; 2) Using the DNA of the sample to be tested described in step 1) as a template, perform PCR amplification using the apple SS-COI PCR detection primers described in claim 1 to obtain the amplification product; 3) Perform agarose gel electrophoresis on the amplification product described in step 2). When a 303bp amplification band appears, the sample to be tested is apple dichotoma.
5. The method according to claim 4, characterized in that, The PCR amplification system in step 2) is as follows: 12.5 μL of 2×TaqPlus PCR Master Mix, 1 μL of upstream primer with a concentration of 10 μmol / L, 1 μL of downstream primer with a concentration of 10 μmol / L, 2 μL of genomic DNA template, and 8.5 μL of ddH2O.
6. The method according to claim 4, characterized in that, The PCR amplification program in step 2) is as follows: pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55℃, 57℃, 59℃ or 61℃ for 30 s, extension at 72℃ for 30 s, for a total of 30 cycles; and finally extension at 72℃ for 2 min.