CN108342492B - Primer for rapidly identifying lugworm bugs, tea-wing bugs and stinkbug and identification method thereof - Google Patents
Primer for rapidly identifying lugworm bugs, tea-wing bugs and stinkbug and identification method thereof Download PDFInfo
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- CN108342492B CN108342492B CN201810424451.6A CN201810424451A CN108342492B CN 108342492 B CN108342492 B CN 108342492B CN 201810424451 A CN201810424451 A CN 201810424451A CN 108342492 B CN108342492 B CN 108342492B
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Abstract
The invention discloses a primer for rapidly identifying arma bug, tea wing bug and spot beard bug and an identification method thereof, wherein the primer consists of nucleotide sequences shown by SEQ ID No.1 and SEQ ID No.2, genome DNA of arma bug, tea wing bug and spot beard bug is used as a template, the primer is used for PCR amplification, restriction enzyme BamHI is used for enzyme digestion amplification products, after the enzyme digestion products are subjected to agarose gel electrophoresis, arma bug generates two bands with the sizes of 422bp and 146bp respectively, and the arma bug and spot beard bug only have one band with the size of 568bp, so that the arma bug, the tea wing bug and the spot beard bug can be rapidly identified according to the number and the size of the electrophoresis bands. The identification method is simple and quick, high in accuracy, short in identification period and low in cost.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to an identification method capable of rapidly distinguishing arma bug from agricultural and forestry pest tea bug and spot bugs, and a special primer for the identification method.
Background
Arma chinensis is a predatory insect, can prey on lepidoptera and coleoptera larvae, and is often used as a utilization object for biological control. The tea wing bugs and the stinkbug are insects of the stinkbug subfamily, often harm fruit trees and crops, and are common agricultural and forestry pests.
The three species all belong to the euschistus insects, are particularly similar in shape and are particularly difficult to distinguish in body type and body color. Therefore, in the actual production life, many people confuse the three insects, and the beneficial insects such as arma chinensis are usually killed when taking insect killing measures or predicting agricultural pest forecast.
Based on the current situation, the molecular method for rapidly identifying the lugworm is researched and invented, and has very important significance for production and scientific research.
Disclosure of Invention
The invention aims to provide a primer for rapidly identifying lugworm bugs, tea-wing bugs and stinkbugs and an identification method thereof, so as to effectively distinguish harmful beneficial insects.
In order to realize the purpose, the invention firstly provides a special primer for quickly identifying the lugworm bugs, the tea-wing bugs and the stinkbugs, wherein the primer is a primer pair consisting of a nucleotide sequence shown by SEQ ID NO.1 and a nucleotide sequence shown by SEQ ID NO. 2.
Specifically, primer-F represented by SEQ ID NO.1 is a nucleotide sequence consisting of 25 bases: ATAATTCAATGATGACGAGATATTA, respectively; primer-R represented by SEQ ID NO.2 is a nucleotide sequence consisting of the following 22 bases: GTCAATATCATGCTGCCGCTTC are provided.
Furthermore, the invention provides a method for rapidly identifying Arundinis armatus, Euschistus polyphylla and Euschistus marmoreus by using the primer pair, which is characterized in that genomic DNA of Arundinis armatus, Euschistus polyphylla and Euschistus marmoreus is used as a template, the primer pair consisting of nucleotide sequences of SEQ ID No.1 and SEQ ID No.2 is used for carrying out PCR amplification on the DNA template, restriction endonuclease is used for carrying out enzyme digestion on a PCR amplification product, and the property of the enzyme digestion product is identified according to the size of an electrophoresis band through agarose gel electrophoresis.
Specifically, the restriction enzyme of the present invention is BamHI.
The present invention can distinguish arma bug from tea bug and bug smut according to the number and size of electrophoresis bands. Because the PCR product of the lugworm DNA has BamHI enzyme cutting sites, two bands appear after the Armoracia chinensis DNA is subjected to enzyme cutting electrophoresis, and the sizes of the two bands are 422bp and 146bp respectively; and the PCR products of the DNA of the tea bug and the stinkbug have no BamHI enzyme cutting site, and only one band appears after enzyme cutting electrophoresis, and the size is 568 bp.
According to the differences of mitochondrial genome sequences of the Argus armatus, the Euschistus adelphocoris and the Euschistus punctatus, a specific region is selected for PCR amplification, a restriction endonuclease BamHI is further used for carrying out characteristic recognition enzyme digestion on GGATCC, the Argus armatus, the Euschistus punctatus and the Euschistus punctatus are distinguished through the number and size of bands generated by agarose gel electrophoresis, the identification accuracy is high, and the kit is used for identifying the Arschistus armatus, the Euschistus tomentosa and the Euschistus punctatus at any development stage, and the accuracy can reach 100%.
The PCR amplification and enzyme digestion technology used by the invention is the traditional molecular biology technology, the operation is simple and rapid, the enzyme digestion identification is adopted, the time is saved compared with the traditional gene sequencing method, and the detection result can be obtained within one day. Meanwhile, the identification method is low in detection cost, and high detection cost of the traditional sequencing method is saved by skillfully utilizing gene sequence difference.
Drawings
FIG. 1 shows the result of PCR amplification of Armoracia chinensis and Euschistus palmatus.
FIG. 2 shows the results of the restriction enzyme identification of Armoracia chinensis, Argus polyphylla and Acorus lucorum.
Detailed Description
The following examples are only preferred embodiments of the present invention and are not intended to limit the present invention in any way. Various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The experimental methods used in the examples are all conventional methods unless otherwise specified. Materials, reagents and the like used in examples are commercially available unless otherwise specified.
Example 1.
Wild Armoracia chinensis, Euschistus sinensis and Euschistus tomentosa are collected. In the embodiment, the arma bug, the tea bug and the spot beard bug are collected in the Pongquan ditch national-level natural protection area of the Shanxi province in a net sweeping mode, and the species and the classification mark are determined after the collected specimens are subjected to morphological classification and identification by using a stereoscopic microscope.
Soaking the samples of the lugworm bugs, the tea-wing bugs and the stinkbugs marked in the classification way in 95% alcohol, and storing at 4 ℃ for later use.
Extraction of Genomic DNA A DNA extraction Kit (ONE-4-ALL Genomic DNA Mini-Preps Kit, product No.: B618503-0050, available from Shanghai Co., Ltd.) was selected and carried out in the following manner.
1. The 2% CTAB Buffer was preheated to 60 ℃.
2. Taking out the specimen soaked in 95% alcohol, removing alcohol on the surface of the specimen with absorbent paper, and soaking twice with 1 × TE Buffer for 20min each time.
3. Dissecting, taking individual breast muscle tissue, adding liquid nitrogen to rapidly freeze the muscle tissue, rapidly grinding the tissue into fine powder with a glass rod, adding preheated 600 μ L2% CTAB Buffer, and transferring into a 1.5mL centrifuge tube.
4. To a 1.5mL centrifuge tube was added preheated 600. mu.L of 2% CTAB extraction buffer (2% CTAB, 0.1mol/L Tris. Cl pH8.0, 0.02mol/L EDTA, 1.4mol/L NaCl), 4. mu.L of proteinase K at a final concentration of 20mg/mL, 2. mu.L of RNase at a final concentration of 50. mu.g/mL and 1.0. mu.L of beta-mercaptoethanol solution, and gently mixed.
5. Water bath is carried out at constant temperature of 60 ℃ for more than 3h, and the mixture is gently mixed from time to time.
6. Centrifuging at 7,000rpm at 4 deg.C for 3min, collecting supernatant, and removing impurity debris.
7. Adding chloroform/isoamyl alcohol (24: 1) mixed solution with the same volume, mixing uniformly, centrifuging at 7,000rpm for 10min, and taking supernatant fluid, measuring volume; this step was repeated once.
8. And taking the supernatant, adding 2 times of frozen absolute ethyl alcohol, and placing in a refrigerator at the temperature of 20 ℃ below zero for sedimentation of DNA for 2 hours.
9. The mixture was centrifuged at 12,000rpm for 20min at 4 ℃ and the supernatant was carefully discarded.
10. Adding 500 μ L70% ethanol (pre-cooled in refrigerator at 4 deg.C), washing the precipitate, centrifuging at 12,000rpm for 10min, and discarding the supernatant; this step was repeated once.
11. Air drying at room temperature or vacuumizing for 6 min.
12. The obtained genomic DNA was redissolved with 40. mu.L of double distilled water, the concentration of the genome was determined, and the resulting mixture was subpackaged at-20 ℃ for future use.
Example 2.
Carrying out multiple sequence comparison analysis on mitochondrial DNAs of the arma bug, the tea bug and the stinkbug by using DNAman software, designing primers by using Primer5.0 software, sending the designed primers to a large gene company for artificial synthesis, and synthesizing the designed primer sequences as follows.
primer-F：ATAATTCAATGATGACGAGATATTA。
primer-R：GTCAATATCATGCTGCCGCTTC。
Example 3.
PCR amplifies partial DNA fragments on the mitochondrial genomes of lugworm, tea-wing stinkbug and stinkbug.
The PCR amplification reaction system is as follows: mu.L of genomic DNA, 2.5. mu.L of buffer (10X), 1. mu.L of dNTP, 1. mu.L of each of the upstream and downstream primers, 0.5. mu.L of Taq enzyme, and make up 25. mu.L of water.
The PCR reaction program is: firstly, denaturation is carried out for 5min at 95 ℃; then extending for 1min at 95 ℃ for 40s, 50 ℃ for 40s and 72 ℃ for 35 cycles; finally, 10min at 72 ℃.
Taking 5 mu L of PCR amplification product, detecting by agarose gel with the concentration of 1.5%, carrying out electrophoresis at constant voltage of 150V for 20min, and observing whether a target band is amplified by a gel imager. The detection results are shown in figure 1, the arma bug ( spot wells 2, 3 and 4), the tea wing bug ( spot wells 5, 6 and 7) and the spotted bugs ( spot wells 8, 9 and 10) all amplify bright DNA bands, and the amplified DNA fragments are about 500bp in size and accord with 568bp of theoretical size through comparison analysis with DL 2000 DNA marker (spot well 1).
After detecting the target band, carrying out enzyme digestion identification by using a restriction enzyme BamHI.
And (3) PCR product enzyme digestion system: PCR product 10. mu.L, 10 XBuffer K2. mu.L, BamHI 1. mu.L, dd H2O7 mu L; the total volume was 20. mu.L. The reaction conditions are as follows: after digestion at 30 ℃ for 4h, 2. mu.L of 10 × Loading Buffer was added to terminate the reaction.
The enzyme digestion PCR product is subjected to electrophoresis detection by using agarose gel with the concentration of 1.5%, and the result is shown in figure 2, two bands appear after the arma bug ( spot wells 2, 3 and 4) is electrophoresed, and the sizes of the two bands are about 400bp and 100bp by comparing with DL 2000 DNA marker (spot well 1), and are consistent with the sizes of 422bp and 146bp which are theoretically obtained after BamHI enzyme digestion. And electrophoresis results of the tea bug ( spot wells 5, 6 and 7) and the spotted bugs ( spot wells 8, 9 and 10) only show one strip, which indicates that the amplified DNA fragment does not have enzyme cutting sites recognized by BamHI enzyme, and is consistent with the theoretically expected result.
Therefore, the arma bug, the tea bug and the stinkbug can be quickly and accurately identified by carrying out enzyme digestion analysis on the DNA fragments amplified by PCR.
SEQUENCE LISTING
<110> university of Shanxi agriculture, Tai Yuan Physician university
<120> primers for rapidly identifying lugworm bugs, tea-wing bugs and stinkbug and identification method thereof
<160> 5
<170> SIPO Sequence Listing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Synthesis
<220>
<223> primer
<400> 1
ATAATTCAAT GATGACGAGA TATTA 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Synthesis
<220>
<223> primer
<400> 2
GTCAATATCA TGCTGCCGCT TC 22
<210> 3
<211> 568
<212> DNA
<213> Arma bug (Arma customs, Fabrucius, 1794)
<400> 3
ATAATTCAAT GATGACGAGA TATTATTCGA GAAGCCACGT TTCAAGGTCA TCATACTATT 60
AAAGTTACTA ATGGATTAAA AATTGGAATA ATTTTATTTA TTATCTCAGA AGTATTCTTT 120
TTTATCTCAT TCTTCTGAGG ATTCTTCCAT AGAAGATTGG CCCCAACCAT TGAAATCGGA 180
ATATTATGAC CTCCTAAAGG AATTATAGTG TTTAACCCTA TAGAAATCCC TTTATTAAAT 240
ACTATAATCC TATTATGTTC AGGAATAACA GTTACATGAG CACACCATAG ATTAATAAAT 300
AACAATTATT CAGAAGTTAC AAAAAGATTG TCTTTAACTG TTATATTAGG TTTATATTTT 360
ACAGCCTTAC AAGCTTATGA ATATATGGAG GCAAGATTCT GTATTAGAGA TTCAGTTTAT 420
GGATCCTGCT TCTATATAGC AACAGGATTC CATGGTCTAC ACGTAATTAT TGGTACCATT 480
TTTTTAACAG TATGTTTAGT ACGACACATT AATTGTCACT TCTCAATAAA TCATCATTTT 540
GGATTTGAAG CGGCAGCATG ATATTGAC 568
<210> 4
<211> 568
<212> DNA
<213> tea wing bugs (Halyomorpha haiys (Stal, 1855))
<400> 4
ATAATTCAAT GATGACGTGA TATTATCCGA GAATCCACAT TTCAAGGAAA TCATACAATT 60
AAAGTTACAA ATGGATTAAA AATTGGCATA ATTTTATTTA TTGTTTCAGA AGTATTATTC 120
TTTATTTCAT TCTTCTGAAG ATTTTTCCAT AGAAGACTAG CACCAACTGT AGAAATCGGT 180
ATAACATGAC CCCCAAAAGG GATTAAAGTA TTTAACCCTA TAGAAATCCC ATTATTAAAC 240
ACTATAATTC TTCTATGTTC AGGTATAACA GTAACATGAA CTCACCATAG AATAATAAAT 300
GGATTATACA ATGAAACAAA AAAAAGATTA ACAATAACTG TAATTTTAGG TATTTACTTT 360
ACCATGCTAC AAGCCTATGA ATATAAAGAA GCCAGATTTT GCATTAGAGA CTCAGTCTAT 420
GGATCTTGCT TCTTTATAGC AACAGGATTC CATGGATTCC ATGTCATCAT TGGTACAATA 480
TTTTTAATAG TTTGCCTTAT ACGACATATT AAATGCCACT TTTCAAAAAC CCATCACTTT 540
GGATTTGAAG CAGCAGCATG ATATTGAC 568
<210> 5
<211> 568
<212> DNA
<213> Sauropus androgynus (Dolycoris baccarum (Linnaeus, 1758))
<400> 5
ATAATTCAAT GATGACGGGA TATTATTCGA GAAGCCACAT TTCAAGGACA CCACACAATT 60
AAAGTAACTT CAGGCTTAAA AATTGGAATA ATTCTATTCA TTATTTCAGA AGTATTTTTT 120
TTCATTTCCT TTTTCTGAGG ATTTTTTCAC AGAAGTCTGG CGCCAACAGT AGAAATTGGA 180
ATAACTTGAC CCCCTAAGGG AATTAGTGTG TTTAATCCAA TAGAAATTCC GCTATTAAAT 240
ACTATAATCT TATTATGTTC AGGTATAACA GTAACATGAG CACACCATAG ACTTATAAGA 300
AATAATCATT CAGAAGCCAC TAAAAGTCTA ATTATAACTG TTACACTTGG ATTTTACTTC 360
ACAATATTAC AAGCCTACGA ATACTTAGAA GCAAGATTCT GTATTAGAGA CTCCATTTAC 420
GGAAGATGTT TTTTTATAGC AACAGGATTC CATGGACTAC ACGTTATTAT TGGAACACTA 480
TTTCTCATAG TATGTCTTAT ACGACATATT AATTGTCATT TCTCTATAAA CCATCATTTT 540
GGATTTGAAG CAGCAGCATG GTATTGAC 568
Claims (1)
1. A method for rapidly identifying Argy bugs, tea wing bugs and Saxifraga fortunei is characterized in that a primer pair consisting of a nucleotide sequence shown in SEQ ID No.1 and a nucleotide sequence shown in SEQ ID No.2 is used, and the Argy bugs, the tea wing bugs and the Saxifraga fortunei are rapidly identified according to the following method:
carrying out PCR amplification on the DNA template by using the genomic DNA of the arma bug, the tea wing bug and the spotted bugs as templates and using a primer pair consisting of the nucleotide sequences of SEQ ID NO.1 and SEQ ID NO. 2;
carrying out enzyme digestion on the PCR amplification product by using a restriction enzyme BamHI, and carrying out agarose gel electrophoresis on the enzyme digestion product;
after the DNA template PCR amplification product of the lugworm is subjected to enzyme digestion electrophoresis, two bands with the sizes of 422bp and 146bp respectively appear;
after enzyme digestion electrophoresis of DNA template PCR amplification products of the tea wing stinkbug and the dolichoris baccifera, only one strip appears, and the size of the strip is 568 bp;
the properties of the electrophoretic bands are discriminated according to their sizes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160115871A (en) * | 2016-08-11 | 2016-10-06 | 경상남도 | Pcr primers for determination of cultivars of pleurotus ostreatus, suhan-1ho, whasung-2ho and gimje-9ho |
| CN106978493A (en) * | 2017-04-18 | 2017-07-25 | 中国农业科学院茶叶研究所 | A kind of Rapid identification tea geometrid and the primer and its authentication method of grey tea geometrid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160115871A (en) * | 2016-08-11 | 2016-10-06 | 경상남도 | Pcr primers for determination of cultivars of pleurotus ostreatus, suhan-1ho, whasung-2ho and gimje-9ho |
| CN106978493A (en) * | 2017-04-18 | 2017-07-25 | 中国农业科学院茶叶研究所 | A kind of Rapid identification tea geometrid and the primer and its authentication method of grey tea geometrid |
Non-Patent Citations (2)
| Title |
|---|
| 五种真蝽（Pentatoma Olivier） mtDNA-COII基因序列分析及系统发育关系初探（半翅目:蝽科）;卜云等;《昆虫分类学报》;20051231;第27卷(第2期);第90-96页 * |
| 粘虫转录组中SSR位点的信息分析;胡艳华等;《山西农业大学学报(自然科学版)》;20151231;第35卷(第5期);第484-489页 * |
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