PCR detection primers, detection method and application of Codling moth based on mtDNA Cytb gene

Abstract

This invention belongs to the field of gene detection technology, specifically relating to a PCR detection primer, method, and application for *Cootytis chinensis* based on the mtDNA Cytb gene. The primers are Cp-Cytb-F and Cp-Cytb-R, with the following nucleotide sequences: Cp-Cytb-F: 5'-GGTTCATTAATTGATTTACCATCTCCCTCT-3', Cp-Cytb-R: 5'-TACCTAATGGATTATTTGAACCTA-3'. The PCR primers, established reaction system, and reaction conditions of this invention can rapidly identify *Cootytis chinensis* without sequencing or sequence alignment, and have the advantages of high sensitivity, high specificity, speed, simplicity, and strong stability.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, and in particular relates to a PCR detection primer for Codling moth based on mtDNA Cytb gene, a detection method and an application thereof. Background Art

[0002] The main methods currently used to identify codling moth are: 1) morphological characteristics. In quarantine work, what is intercepted are often young larvae or eggs. The usual practice is to raise them indoors until they are mature larvae before identification, which is time-consuming, prolongs the customs clearance time for imported and exported goods, and increases the workload of quarantine personnel; 2) The mt DNA Cytb gene sequence has a high level of variation. At mt DNA COⅠ and mt DNACOⅡ; PCR technology, COⅠ and COⅡ technologies have been used to identify the codling moth and its related species, but the PCR products need to be recovered, purified, and sequenced, and the test results need to be judged based on sequence alignment and phylogenetic tree construction. There are too many operation steps, which also leads to a long time consumption.

[0003] In summary, the problems with existing methods for identifying codling moth are that they are time-consuming and slow in detection speed. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a PCR detection primer for codling moth based on mtDNA Cytb gene, a detection method and application. The primer, PCR reaction system and reaction conditions of the present invention can quickly identify codling moth without sequencing and sequence comparison, and have the advantages of high sensitivity, strong specificity, rapidity, simplicity and strong stability.

[0005] The present invention provides a PCR detection primer for Codling moth based on mtDNA Cytb gene, wherein the primers are Cp-Cytb-F and Cp-Cytb-R, and the nucleotide sequences are as follows:

[0006] Cp-Cytb-F:

[0007] 5'-GGTTCATTAATTGATTTACCATCTCCCTCT-3';

[0008] Cp-Cytb-R:

[0009] 5'-TACCTAATGGATTATTTGAACCTA-3'.

[0010] According to the results of our primer specificity test, the primers have clear and distinct detection results and can effectively distinguish peach borer, pear borer and codling moth.

[0011] The invention provides a detection kit, comprising PCR detection primers and PCR detection reagents for Codling moth based on mtDNA Cytb gene.

[0012] As described, the PCR detection reagents are 10×EasyTaq Buffer, High pure dNTPs and EasyTaq DNA Polymerase.

[0013] When using the above-mentioned detection kit to identify the codling moth, there is no need for sequencing, nor is there a need to judge the detection results based on sequence alignment and phylogenetic tree construction. The operation steps are simplified and the time is short. Compared with the existing technology, the detection speed is improved.

[0014] The present invention provides a PCR detection method for Codling moth, comprising the following steps:

[0015] Extracting genomic DNA from the sample to be tested;

[0016] Using the extracted genomic DNA as a template, PCR amplification is performed using the primers Cp-Cytb-F and Cp-Cytb-R;

[0017] The amplified product is analyzed by agarose gel electrophoresis. If the amplified product band is 586 bp, the sample to be tested is codling moth. If there is no 586 bp band, the sample to be tested is not codling moth.

[0018] Preferably, the PCR amplification reaction system is 25 μL, including: 0.5 μL each of primers Cp-Cytb-F and Cp-Cytb-R, 1 μL of genomic DNA, 2.5 μL of 10×EasyTaq Buffer, 2 μL of High pure dNTPs, 1 μL of EasyTaq DNA Polymerase, and ddH2O to make up to 25 μL.

[0019] Preferably, the amplification reaction conditions for PCR amplification are:

[0020] Pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 45°C-49°C for 30 s, extension at 72°C for 45 s, for a total of 40 cycles; extension at 72°C for 10 min.

[0021] Preferably, the annealing temperature is 45°C.

[0022] Preferably, the analysis is performed by 1% agarose gel electrophoresis at a constant voltage of 110-125 V for 15-20 min.

[0023] The invention provides an application of PCR detection primers for codling moth based on mtDNA Cytb gene. The primers are used for identifying codling moth and distinguishing codling moth and its closely related species.

[0024] Preferably, the primers are used to distinguish between peach borer, pear borer and codling moth.

[0025] Preferably, the codling moth comes from Altay, Xinjiang Bole, Xinjiang Akto, Xinjiang Shihezi, Xinjiang, Zhangye, Gansu, Jinchang, Gansu, Zhangwu, Liaoning, Yingkou, Liaoning, Wafangdian, Liaoning, and Huludao, Liaoning.

[0026] Preferably, the minimum DNA concentration detection limit of the PCR is 3.125 ng / μL.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The PCR detection primers of the present invention utilize PCR to detect the genomic DNA of the sample to be tested, and determine the detection result according to the presence and size of the band after agarose electrophoresis, without the need for sequencing and sequence alignment, and have the advantages of high sensitivity, strong specificity, rapidity, simplicity and strong stability.

[0029] In addition, the PCR primers designed by the present invention and the established PCR reaction system and reaction conditions can achieve rapid, accurate and efficient identification of codling moth, and can be used for rapid identification of invasive pest codling moth in actual production. It can be used in practice for identification of agricultural pests and pests in inspection and quarantine, thereby providing a reference for formulating pest control strategies and quarantine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Agarose electrophoresis diagrams of PCR amplification effects of codling moth, peach borer, and pear borer at different annealing temperatures;

[0031] Lane M: Trans2K DNA Marker; Lane 1-Lane 3: Annealing temperature is 55.0℃ for peach borer, pear borer, and codling moth; Lane 4-Lane 6: Annealing temperature is 54.3℃ for peach borer, pear borer, and codling moth; Lane 7-Lane 9: Annealing temperature is 53.1℃ for peach borer, pear borer, and codling moth; Lane 10-Lane 12: Annealing temperature is 51.3℃ for peach borer, pear borer, and codling moth ; Lane 13-Lane 15: annealing temperature is 49.0℃ for peach borer, pear borer and codling moth; Lane 16-Lane 18: annealing temperature is 47.3℃ for peach borer, pear borer and codling moth; Lane 19-Lane 21: annealing temperature is 45.9℃ for peach borer, pear borer and codling moth; Lane 22-Lane 24: annealing temperature is 45.0℃ for peach borer, pear borer and codling moth.

[0032] Figure 2 This is the specificity detection diagram of PCR primers for codling moth;

[0033] Lane M: Trans2K DNA Marker; Lanes 1-3: Peach borer; Lanes 4-6: Pear borer; Lanes 7-9: Codling moth.

[0034] Figure 3 This is the agarose electrophoresis diagram for the sensitivity test of PCR primers for codling moth;

[0035] Lane M: Trans2K DNA Marker; Lane 1-Lane 8: 400ng / μL, 200ng / μL, 100ng / μL, 50ng / μL, 25ng / μL, 12.5ng / μL, 6.25ng / μL, 3.125ng / μL DNA; Lane 9: negative control.

[0036] Figure 4 Agarose electrophoresis test diagram for the identification of codling moth in different provinces of my country;

[0037] Lane M: Trans2K DNA Marker; Lane 1-Lane 10: Altay, Xinjiang, Bole, Xinjiang, Akto, Xinjiang, Shihezi, Xinjiang, Zhangye, Gansu, Jinchang, Gansu, Zhangwu, Liaoning, Yingkou, Liaoning, Wafangdian, Liaoning, Huludao, Liaoning; Lane 11: negative control.

[0038] Figure 5 is the agarose electrophoresis detection diagram of primer 1;

[0039] Lane M: Trans2K DNA Marker; Lanes 1-3: Peach borer; Lanes 4-6: Pear borer; Lanes 7-9: Codling moth.

[0040] Figure 6 is the agarose electrophoresis detection diagram of primer 2;

[0041] Lane M: Trans2K DNA Marker; Lanes 1-3: Peach borer; Lanes 4-6: Pear borer; Lanes 7-9: Codling moth. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0043] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0044] Example 1

[0045] A PCR detection primer for Codling moth based on mtDNA Cytb gene, the nucleotide sequences of which are shown in Cp-Cytb-F and Cp-Cytb-R in Table 1, corresponding to SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0046] The primers were designed based on the mitochondrial sequence of Codling moth and the length of the amplified fragment in the NCBI database and were artificially synthesized.

[0047] Table 1 Primer sequences

[0048]

[0049]

[0050] Example 2

[0051] A PCR detection method for identifying Codling moth, comprising the following steps:

[0052] a. Extract genomic DNA from the sample to be tested;

[0053] The genomic DNA extraction in step a was performed using a DNA extraction kit purchased from Beijing Tiangen Biochemical Co., Ltd., DP304. The main steps of genomic DNA extraction are as follows:

[0054] (1) Remove the alcohol from the sample to be tested and air dry it;

[0055] (2) Place the sample to be tested into a sterilized 1.5 mL centrifuge tube, freeze it quickly with liquid nitrogen, and grind it with a grinding rod;

[0056] (3) Add 200 μL buffer GA and shake until thoroughly suspended;

[0057] (4) Add 20 μL of Proteinase K solution and mix well;

[0058] (5) Heating in a metal bath at 56°C overnight;

[0059] (6) After the metal bath is finished, take it out and centrifuge it to remove the water droplets on the inner wall;

[0060] (7) Add 200 μL of buffer GB, invert to mix, and place in a metal bath at 70°C for 10 min.

[0061] (8) Centrifuge at 5000 rpm for 1 min and transfer the supernatant into a new 1.5 mL centrifuge tube;

[0062] (9) Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds;

[0063] (10) Centrifuge at 5000 rpm for 1 min;

[0064] (11) Add all the solution obtained in the previous step to the adsorption column CB3, and place the adsorption column in a collection tube;

[0065] (12) Centrifuge at 12000 rpm for 30 s, pour out the liquid in the collection tube, and put the adsorption column back into the collection tube;

[0066] (13) Add 500 μL of buffer GD to the adsorption column;

[0067] (14) Centrifuge at 12000 rpm for 30 s, pour out the liquid in the collection tube, and put the adsorption column back into the collection tube;

[0068] (15) Add 600 μL of rinse solution PW to the adsorption column;

[0069] (16) Centrifuge at 12000 rpm for 30 s, pour out the liquid in the collection tube, and put the adsorption column back into the collection tube;

[0070] (17) Repeat steps (15)-(16);

[0071] (18) Place the adsorption column back onto the emptied collection tube and centrifuge at 12,000 rpm for 2 min.

[0072] (19) Open the adsorption column and let it air dry at room temperature for 10 min to completely dry the rinse solution PW on the adsorption column film to avoid affecting the DNA concentration and purity;

[0073] (20) Place the dried adsorption column in a clean 1.5 mL centrifuge tube and drop 50–200 μL of elution buffer TE onto the film in the middle of the adsorption column;

[0074] (21) Leave it at room temperature for 5 minutes;

[0075] (22) Centrifuge at 12000 rpm for 2 min. The liquid collected in the centrifuge tube is the DNA of the sample;

[0076] b. Using the extracted genomic DNA as a template, PCR amplification was performed using the codling moth-specific primers Cp-Cytb-F and Cp-Cytb-R;

[0077] The PCR amplification reaction system was 25 μL, including: 0.5 μL of primers Cp-Cytb-F and Cp-Cytb-R, 1 μL of genomic DNA, 2.5 μL of 10×EasyTaq Buffer, 2 μL of High pure dNTPs, 1 μL of EasyTaq DNA Polymerase, and ddH2O to 25 μL. The nucleotide sequences of Cp-Cytb-F and Cp-Cytb-R are shown in Table 1.

[0078] Among them, 10×EasyTaq Buffer, High pure dNTPs, and EasyTaq DNA Polymerase were all purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0079] The amplification reaction conditions for PCR amplification are:

[0080] Pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 45°C for 30 s, extension at 72°C for 45 s, for a total of 40 cycles; extension at 72°C for 10 min.

[0081] c. The amplified products were analyzed by agarose gel electrophoresis, and the presence or absence of amplified product bands was used to identify whether it was codling moth.

[0082] The agarose gel electrophoresis analysis in step c is specifically:

[0083] 1% agarose gel electrophoresis analysis, constant voltage 110-125V, 15-20min. In the specific test of this embodiment, the specific voltage used is 120V, 15min.

[0084] In the step c, whether it is codling moth is identified based on the presence or absence of the amplified product band, specifically: if the amplified product band is 586bp, the sample to be detected is codling moth.

[0085] We used the method of Example 2 to test a variety of samples. The results are shown in Figure 1-Figure 4 . Figure 1 Agarose electrophoresis diagrams of PCR amplification effects of codling moth, peach borer, and pear borer at different annealing temperatures. Figure 1 The samples of codling moth were collected from an apple orchard in Yingkou, Liaoning Province, and the samples of peach borer and pear borer were collected from an apple orchard in Shenyang, Liaoning Province. Figure 1 Except for the annealing temperature, which is different from that in Example 2, the rest of the operations are the same as those in Example 2. Figure 1Lane M: Trans2K DNA Marker; Lane 1-Lane 3: Annealing temperature is 55.0℃ for peach borer, pear borer, and codling moth; Lane 4-Lane 6: Annealing temperature is 54.3℃ for peach borer, pear borer, and codling moth; Lane 7-Lane 9: Annealing temperature is 53.1℃ for peach borer, pear borer, and codling moth; Lane 10-Lane 12: Annealing temperature is 51.3℃ for peach borer, pear borer, and codling moth Moth; Lane 13-Lane 15: annealing temperature is 49.0℃ for peach borer, pear borer, and codling moth; Lane 16-Lane 18: annealing temperature is 47.3℃ for peach borer, pear borer, and codling moth; Lane 19-Lane 21: annealing temperature is 45.9℃ for peach borer, pear borer, and codling moth; Lane 22-Lane 24: annealing temperature is 45.0℃ for peach borer, pear borer, and codling moth. The external features of young larvae of peach borer, pear borer, and codling moth are very similar, and it is difficult to accurately identify and identify species based on the morphological features of the larvae. The method of the present invention can quickly and accurately identify peach borer, pear borer, and codling moth.

[0086] Figure 2 This is a graph of the specificity detection of PCR primers for codling moth, lane M: Trans2K DNA Marker; lanes 1-3: peach borer; lanes 4-6: pear borer; lanes 7-9: codling moth. Figure 2 The detection method is completely consistent with that in Example 2, and the results show that the method can significantly distinguish peach borer, pear borer and codling moth.

[0087] Figure 3 This is the agarose electrophoresis diagram of the sensitivity test of PCR primers for codling moth. Figure 3 Except for the difference in DNA concentration from Example 2, the rest of the operations were the same as in Example 2. Figure 3 Lane M: Trans2K DNA Marker; Lanes 1 to 8 represent DNA concentrations of 400ng / μL, 200ng / μL, 100ng / μL, 50ng / μL, 25ng / μL, 12.5ng / μL, 6.25ng / μL, and 3.125ng / μL DNA, respectively; Lane 9: negative control. Figure 3 It can be seen that the minimum DNA concentration detection limit is 3.125 ng / μL, and the primer sensitivity is good.

[0088] Figure 4 The agarose electrophoresis detection diagram is for the identification of codling moth in different provinces of my country. The detection method is consistent with that in Example 2. Figure 4Lane M: Trans2K DNA Marker; Lanes 1-10: Xinjiang Altay, Xinjiang Bole, Xinjiang Akto, Xinjiang Shihezi, Gansu Zhangye, Gansu Jinchang, Liaoning Zhangwu, Liaoning Yingkou, Liaoning Wafangdian, Liaoning Huludao; Lane 11: Negative control. Figure 4 It can be seen that the method of the present invention can effectively detect codling moth from multiple sources, has wide applicability and strong stability.

[0089] We also conducted specificity experiments of different primer sequences, and designed other primers to identify codling moth according to the primer design principle, but the specificity effects were not as good as the primers provided in Table 1 of the present invention. The experimental steps and experimental results are as follows.

[0090] Primer 1 test and results:

[0091] The sequence of primer 1 is control F1: 5'-CCATCATTACGTTTATTATACTTATTAG-3', as shown in SEQ ID NO.3, and control R1: 5'-TCTACTTTTACCCCTAATGAAGGAATTGT-3', as shown in SEQ ID NO.4. The conditions of Example 2 were referred to for operation and electrophoresis, except that the primer was replaced with primer 1. The genomic DNA of the sample to be tested was extracted as in Example 2. The extracted genomic DNA was used as a template, and PCR amplification was performed using primers F1 and control R1. The amplified product was analyzed by agarose gel electrophoresis. The electrophoresis results are shown in FIG. Figure 5 As shown, lane M: Trans2K DNA Marker; lanes 1-3: peach borer; lanes 4-6: pear borer; lanes 7-9: codling moth. Both peach borer and codling moth show bright bands and cannot be distinguished. Primer 1 cannot specifically distinguish codling moth.

[0092] Primer 2 test and results:

[0093] The sequence of primer 2 is control F2: 5'-TCTATAAACCTTTACGAAAAACCCACCCT-3', as shown in SEQ ID NO.5, and control R2: 5'-TTGCAAGAATAAATGGAAGAAGA-3', as shown in SEQ ID NO.6. The conditions of Example 2 were referred to for operation and electrophoresis, except that the primer was replaced with primer 1. The genomic DNA of the sample to be tested was extracted as in Example 2. The extracted genomic DNA was used as a template, and PCR amplification was performed using primers F2 and control R2. The amplified product was analyzed by agarose gel electrophoresis. The electrophoresis results are shown in FIG. Figure 6As shown, lane M: Trans2K DNA Marker; lanes 1-3: peach borer; lanes 4-6: pear borer; lanes 7-9: codling moth. The results showed that peach borer and pear borer had multiple bands, and amplified bands appeared at a size close to that of codling moth. The agarose electrophoresis detection results of primer 2 were also clear and definite, just like the Cytb primer of codling moth.

[0094] Comprehensively compare the agarose electrophoresis test results of the above-mentioned primers 1, primer 2 and Cytb primers of codling moth. When the primer sequences are different, even if the genomic DNA extraction method is the same, the PCR system and PCR amplification conditions are the same, and the agarose detection conditions are the same, different test results with significant differences will still be obtained. Even if Primer 1, Primer 2 and Cytb primers of codling moth are primer sequences designed according to the primer design principle, the test results are still significantly different, indicating that the effects of different primer sequences designed according to the primer design principle are not equivalent, and there is no regularity to follow for the quality of the agarose electrophoresis test results. The present invention first discovered that Cytb primers of codling moth can clearly and clearly identify and distinguish peach borer, pear borer and codling moth.

[0095] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0096] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A PCR detection primer for Codling moth based on mtDNA Cytb gene, characterized in that: Cp-Cytb-F and Cp-Cytb-R, the nucleotide sequences are as follows: Cp-Cytb-F: 5'-GGTTCATTAATTGATTTACCATCTCCCTCT-3'; Cp-Cytb-R: 5'-TACCTAATGGATTATTTGAACCTA-3'.

2. A detection kit, characterized in that: The method comprises the primers and PCR detection reagents described in claim 1.

3. A PCR detection method for detecting Codiaeella pumila, characterized in that: The following steps are involved: Extracting genomic DNA from the sample to be tested; Using the extracted genomic DNA as a template, PCR amplification is performed using the primers described in claim 1; The amplified product is analyzed by agarose gel electrophoresis. If the amplified product band is 586 bp, the sample to be tested is codling moth. If there is no 586 bp band, the sample to be tested is not codling moth.

4. The PCR detection method for detecting Codiaeella pylori according to claim 3, characterized in that: The PCR amplification reaction system was 25 μL, including: 0.5 μL each of primers Cp-Cytb-F and Cp-Cytb-R, 1 μL of genomic DNA, 2.5 μL of 10×EasyTaq Buffer, 2 μL of High pure dNTPs, 1 μL of EasyTaq DNA Polymerase, and ddH2O to make up to 25 μL.

5. The PCR detection method for detecting Codiaeum apple moth according to claim 4, characterized in that: The amplification reaction conditions of PCR are: Pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 45°C-55°C for 30 s, extension at 72°C for 45 s, for a total of 40 cycles; extension at 72°C for 10 min.

6. The PCR detection method for detecting Codiaeum apple moth according to claim 5, characterized in that: Analyze by 1% agarose gel electrophoresis at a constant voltage of 110-125V for 15-20min.

7. The use of primers for PCR detection of Codiaeella malariae based on mtDNACytb gene according to claim 1, characterized in that: The primers are used for identifying the codling moth and distinguishing the codling moth and its closely related species.

8. The use of PCR detection primers for Codling moth based on mtDNA Cytb gene according to claim 7, characterized in that: The primers are used to distinguish peach borer, pear borer and codling moth.

9. The use of PCR detection primers for Codling moth based on mtDNA Cytb gene according to claim 7, characterized in that: The codling moth comes from Altay, Bole, Akto, Shihezi, Zhangye, Jinchang, Zhangwu, Yingkou, Wafangdian, and Huludao, Liaoning.

10. The use of PCR detection primers for Codling moth based on mtDNACytb gene according to claim 7, characterized in that: The minimum DNA concentration detection limit of the PCR was 3.125 ng / μL.

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