CN104673890B - Cotton black aphid PCR quick determination methods based on specific SS COI primers - Google Patents
Cotton black aphid PCR quick determination methods based on specific SS COI primers Download PDFInfo
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- 229920000742 Cotton Polymers 0.000 title claims abstract description 50
- 241001151957 Aphis aurantii Species 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 12
- 238000001514 detection method Methods 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 abstract description 20
- 230000003321 amplification Effects 0.000 abstract description 6
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 6
- 108020005196 Mitochondrial DNA Proteins 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract 1
- 241000219146 Gossypium Species 0.000 description 40
- 241001124076 Aphididae Species 0.000 description 6
- 241000238631 Hexapoda Species 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000013461 design Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241001600407 Aphis <genus> Species 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 101150087323 COI gene Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 240000002024 Gossypium herbaceum Species 0.000 description 1
- 235000004341 Gossypium herbaceum Nutrition 0.000 description 1
- 241000258937 Hemiptera Species 0.000 description 1
- 241001145009 Sophora alopecuroides Species 0.000 description 1
- 241000219793 Trifolium Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 244000062645 predators Species 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
Cotton black aphid PCR quick determination methods based on specific SS COI primers, it is according to the distinctive mtDNA COI sequences of cotton black aphid, devise specific COI primers Aa105 1F and the Aa105 1R of a pair of cotton black aphids, the primer only has specific amplification effect to cotton black aphid, amplified production size is 210bp, and DNA concentration limits are 0.192ng/ μ L.Determined for plant protection field cotton black aphid Dominant Natural Enemies and food link analysis, high specificity, accuracy rate height are convenient and swift.
Description
First, technical field
The invention belongs to biology field, is related to cotton black aphid PCR detection method, is specially that one kind is based on specificity
The PGR detection methods of COI primers.
2nd, background technology
Cotton black aphid (Aphis atrata Zhang) belongs to Homoptera, Aphidiadae.Also known as purple group aphid.It is distributed in Xinjiang, Ningxia, sweet
The ground such as respectful.Host plant has cotton, Sophora alopecuroide, on a small quantity on clover.
Cotton black aphid is cotton in Xinjiang seedling stage insect, and happiness, which is clustered in the tender head of cotton seedling, cotyledon, true leaf reverse side and sucks juice, causes harm,
Making cotton leaf shrinkage, growing point is withered to come off, and Axillary shoot proliferation, cotton plant downgrades deformity, stagnates cotton seedling mesoderm growing early stage, and bud is reduced,
Yield declines.
Using natural enemy to Field Pests carry out biological control, be plant protection work in agricultural production effective means it
One.Predator has obvious predation to aphid, and the method for studying the hostile aphid predation in day is a lot, traditional
Method is mainly directly observed using field, alimentary canal anatomic observation and biochemical method, such as ELISA (ELISA), same to work(
Enzyme electrophoresis, protein electrophoresis etc., but these methods the have limitation such as time-consuming, complex operation, expense height.At present, DNA
In terms of tracer technique is progressively applied to the food chain nutrition relationship and screening Dominant Natural Enemies of research field natural enemy and insect, it is
One of effective tool of food web between arthropod in the complicated Agro-ecological System of structure.
Species-Specific-COI PCR detection techniques are the spies to grow up on the basis of mtDNACOI technologies
Different sequence amplification zone marker, it is to utilize species specificity COI primers (SS-COI), is reflected according to the presence or absence of expected DNA bands
Regular inspection test sample sheet, without sequencing and sequence alignment, it has high sensitivity, high specificity, reproducible, quick, easy etc. excellent
Point.Cotten aphid specificity SS-COI primers disclosed in Chinese patent specification CN103436618A, the kit containing the primer and
Its detection method is the concrete application of this method.
But cotton black aphid, because distributed areas are limited, correlative study development is less, and cotton black aphid is not yet retrieved on Genbank
CO1 genetic fragments.Therefore this method is not yet applied in the context of detection of cotton black aphid.
3rd, the content of the invention
It is an object of the invention to provide a kind of cotton black aphid PCR quick determination methods based on specific SS-COI primers.
The present invention designs the specific COI primer of a pair of cotton black aphids, had according to the mitochondrial DNA COI sequences of cotton black aphid
Body is
Forward primer Aa105-1F：5’-TCCCATAATAATAGGTTGTC-3’
Reverse primer Aa105-1R：5’-TACCTGCTAAATGAAGAGAG-3’.
The present invention detecting step be：
(1) sample total DNA is extracted；
(2) using the DNA of above-mentioned steps extraction as template, the primer Aa105-1F and Aa105- described in claim 1 are utilized
1R carries out pcr amplification reaction；Reaction system is calculated as with 20 μ L：
Reaction condition is：94 DEG C 3 minutes, 94 DEG C 30 seconds, 50 DEG C 30 seconds, 72 DEG C 1 minute, totally 45 circulation；72 DEG C afterwards
10 minutes.
(3) enter row agarose gel electrophoresis detection to pcr amplification product, the DNA bands that size is 210bp such as occur, then
It is cotton black aphid to show sample.
The present invention also provides the kit for being used to detect cotton black aphid containing primer Aa105-1F and Aa105-1R, the examination
Agent box also includes cotton black aphid genomic DNA standard positive template.
The present invention is by the universal primer in DNA bar code technology
LepF1：(5 '-ATTCAACCAATCATAAAGATATTGG-3 ') and
LepR1：(5′-AAACTTCTGGATGTCCAAAAAATCA-3’)
The mitochondrial COI gene of cotton black aphid is expanded, is compared through sequencing, and the COI sequences of the common aphid in other crop fields,
A plurality of primer is designed, therefrom filters out a pair of special primers.The primer specificity is strong, and only cotton black aphid can amplify one clearly
Clear, single length is 210bp specific band, and to other insects in cotton field without expanding effect.Examined using the technology
Cotton black aphid is surveyed, accuracy is high, and simple to operate, quick, the DNA concentration limits of cotton black aphid are 0.192ng/ μ L.
4th, illustrate
Fig. 1 is primer Aa105-1F/Aa105-1R of the present invention to cotton black aphid specific amplification design sketch.Wherein, M：100bp
DNA Iadder；1-64 is the amplification of insect corresponding to sequence number in table 1 ,-it is negative control.
Fig. 2 is cotton black aphid specific primer Aa105-1F/Aa105-1R of the present invention DNA concentration gradient testing result figure.
Wherein, M：100bp DNA Iadder；1-5 is the concentration ladder that cotton black aphid DNA profiling concentration 49.1ng/ μ L dilute 4 times successively
Degree ,-it is negative control.
5th, embodiment
Following instance is carried out according to conventional molecular biological experiment condition, and according to the condition of product description suggestion
Operation.
Part I, amplifications of the primer Aa105-1F/Aa105-1R to cotton black aphid
(1) preparation of cotton black aphid STb gene
Cotton black aphid is gathered in cotton field, Single Aphids are put into 1.5mL centrifuge tubes and ground, with OMEGA Insect
Genomic DNA Kit extracts kits (OMEGA companies, the U.S.) extract the STb gene of cotton black aphid.DNA solution is stored in -20
It is DEG C standby, 1 μ L solution is drawn as DNA profiling when entering performing PCR amplification.
(2) specific COI primer of synthesis detection cotton black aphid
Specific COI primer sequence is as follows：
Aa105-1F：5′-TCCCATAATAATAGGTTGTC-3’(SEQ ID No.1)
Aa105-1R：5′-TACCTGCTAAATGAAGAGAG-3’(SEQ ID No.2)
(3) PCR is expanded
Reaction system is 20 μ L, including：μ L of 10 × Taq buffer solutions 2.0, μ L of dNTP (2.5mM) 1.6, Taq archaeal dna polymerases
(5U/ μ L) 0.2 μ L, each 0.3 μ L of forward and reverse primer (20nM), the μ L of template DNA 1.0, ddH2O 14.6 μ L.
PCR amplification programs：94 DEG C of pre-degenerations 3 minutes；94 DEG C 30 seconds, 50 DEG C 30 seconds, 72 DEG C 1 minute, totally 45 circulation；Most
72 DEG C extend 10 minutes afterwards.
(4) PCR primer is identified
8 μ L PCR primers are taken, is separated with 1% agarose gel electrophoresis, after ethidium bromide staining, is put in gel imager
In, there are the DNA bands (SEQ ID No.3) that size is 210bp.
Part II, specific amplification effects of the primer Aa105-1F/Aa105-1R to cotton black aphid
Using primer Aa105-1F/Aa105-1R, using cotton black aphid DNA as template, with 32 kinds of cotton black aphid same area in table 1 its
His insect is control progress COI PCR amplifications, as a result as shown in figure 1, cotton black aphid has amplified 210bp's corresponding to 3,4 sequence numbers
Purpose band, illustrate according to cotton black aphid mitochondrial DNA COI genes design COI primer specificities it is strong, its 210bp sequence such as SEQ
Shown in ID No.3.
Involved caste in the detection of the primer specificity of table 1
Measure of the Part III primer Aa105-1F/Aa105-1R to cotton black aphid limit of identification
Single head cotton black aphid STb gene is extracted by Part I method, then by original template solution 49.1ng/ μ L with 4 times of progress
Successively decrease and be diluted to detection not shaping band, the template for taking 1 μ L to be detected as PCR, enter performing PCR reaction, its reaction system is the same as first
Described in point.
The measure of limit of identification is done using primer Aa105-1F/Aa105-1R, the DNA concentration limits of cotton black aphid are
0.192ng/ μ L, as a result as shown in Figure 2.
Sequence table
SEQ ID No.1
Aa105-1F：5′-TCCCATAATAATAGGTTGTC-3’
SEQ ID No.2
Aa105-1R：5’-TACCTGCTAAATGAAGAGAG-3’
SEQ ID No.3
Claims (2)
1. a kind of cotton black aphid PCR quick determination methods based on specific SS-COI primers, it is characterised in that described primer is
Forward primer Aa105-1F：5’-TCCCATAATAATAGGTTGTC-3’
Reverse primer Aa105-1R：5’-TACCTGCTAAATGAAGAGAG-3’
Detecting step is：
A. sample total DNA is extracted；
B. using the DNA of above-mentioned steps extraction as template, it is anti-to enter performing PCR amplification using above-mentioned primer Aa105-1F and Aa105-1R
Should；Reaction system is calculated as with 20 μ L：
Reaction condition is：94 DEG C 3 minutes, 94 DEG C 30 seconds, 50 DEG C 30 seconds, 72 DEG C 1 minute, totally 45 circulation；72 DEG C 10 points afterwards
Clock；
C. enter row agarose gel electrophoresis detection to pcr amplification product, the DNA bands that size is 210bp such as occur, then show sample
Product are cotton black aphid.
2. the kit for being used to detect cotton black aphid containing primer described in claim 1, it is characterised in that the kit also wraps
Include cotton black aphid genomic DNA standard positive template.
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| CN106609306A (en) * | 2016-06-17 | 2017-05-03 | 新疆农业科学院植物保护研究所 | Rapid chrysopa formosa brauer PCR detection based on specific SS-COI primers |
| CN110656186B (en) * | 2019-10-31 | 2023-07-14 | 新疆农业科学院植物保护研究所 | Kit and method for rapidly identifying walnut black spot aphids |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436618A (en) * | 2013-08-23 | 2013-12-11 | 中国农业科学院植物保护研究所 | Aphis gossypii Glover specificity SS-COI primers, kit containing primer and detection method of kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436618A (en) * | 2013-08-23 | 2013-12-11 | 中国农业科学院植物保护研究所 | Aphis gossypii Glover specificity SS-COI primers, kit containing primer and detection method of kit |
Non-Patent Citations (2)
| Title |
|---|
| "DNA Barcoding and the Associated PhylAphidB@se Website for the Identification of European Aphids (Insecta Hemiptera Aphididae)";Armelle Coeur dˊacier et al.;《PLOS ONE》;20160604;第9卷(第6期);摘要和第2页右栏 DNA Extraction, Amplification and Sequencing * |
| "Use of a mitochondrial COI sequence to identify species of the subtribe Aphidina (Hemiptera, Aphididae)";Jian Feng Zhang et al.;《ZooKeys》;20110811;第1-17页 * |
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