CN103468677B - Specific SS-COI (Species-specific Cytochrome Oxidase I) primers of aphis craccivora, kit containing primers and detecting method of kit - Google Patents
Specific SS-COI (Species-specific Cytochrome Oxidase I) primers of aphis craccivora, kit containing primers and detecting method of kit Download PDFInfo
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- CN103468677B CN103468677B CN201310429628.9A CN201310429628A CN103468677B CN 103468677 B CN103468677 B CN 103468677B CN 201310429628 A CN201310429628 A CN 201310429628A CN 103468677 B CN103468677 B CN 103468677B
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Abstract
The invention relates to specific SS-COI (Species-specific Cytochrome Oxidase I) primers of aphis craccivora and a kit containing the primers. A pair of specific SS-COI primers LFAc and LFAcR (SEQ ID NO.1 and 2) of aphis craccivora is designed according to a special mtDNA sequence of aphis craccivora, the primers have specific amplification capacity only for aphis craccivora, and an amplification product has the size of 193bp, the plasmid concentration detection limit of 3.99E+4 and the DNA concentration detection limit of 0.413ng/muL. An SS-COI PCR (Polymerase Chain Reaction) technology is adopted in the invention, so that the detection accuracy is improved, the detection time is saved, and the primers are suitable for popularization and application in basement layers.
Description
Technical field
The present invention relates to biology field, specifically, relate to bean aphid specificity SS-COI primer, the test kit that contains this primer and detection method thereof.
Background technology
Bean aphid Aphis craccivora Koch, another name peanut aphid, aphis craccivora, belong to Hemiptera Aphidiadae, is the important pests of leguminous crop, and what tool was stronger migrates and diffusibility.Bean aphid harm host is often clustered in tender stem, young shoot, top tender leaf, lobus cardiacus, floral organ and pod place and draws juice.Be injured when serious, plant strain growth is bad, and blade is crispaturaed, and impact is blossomed and beared fruit.Bean aphid can also drain " honeydew " in a large number, causes plant sooty mold, thereby affects photosynthesis, causes bear pods minimizing, thousand seed weight of plant to decline.Bean aphid has multiple predator (as ladybug, Chrysopa, spider etc.) in field, the protection of natural enemy and utilization are the important measures of bean aphid control.How the control action kou of field different sorts natural enemy is carried out to reasonable assessment, so select that reproductivity is high, prey ability and searching host ability is strong, the obvious natural enemy resource of pest controlling effect, be basis and the core of carrying out natural enemy protection Reasonable Protection and utilization.
Species-Specific-COI PCR detection technique is the sequence-characterized amplified regionP growing up in mtDNA COI technical foundation, to utilize specific primer, have or not to judge detected result according to expection DNA band, without order-checking and sequence alignment, there is the advantages such as highly sensitive, high specificity, quick, easy and circulation ratio and stability is strong, for the predation of the hostile prey in accurate evaluation sky has been opened up new way.
Summary of the invention
The object of this invention is to provide bean aphid specificity SS-COI primer, the test kit that contains this primer and detection method thereof.
In order to realize the object of the invention, the present invention, according to the mtdna sequence of bean aphid, designs a pair of bean aphid specificity SS-COI primer, and it comprises:
Forward primer LFAcF:5'-CAAACAATATTGCTCATAATAAC-3 '
Reverse primer LFAcR:5 '-AAAATTAATAATATAGCTGTAATTAGG-3 '.
The present invention also provides the test kit for detection of bean aphid that contains primer LFAcF and LFAcR.Described test kit also comprises dNTPs, Taq archaeal dna polymerase, Mg
2+, at least one in PCR reaction buffer.Preferably, described test kit also comprises standard positive template.
The present invention also provides described primer LFAcF and LFAcR, and the test kit that contains primer LFAcF and LFAcR is in the application detecting in bean aphid.
The present invention also provides the specificity fast PCR detection method of a kind of bean aphid, comprises the following steps:
1) extract sample DNA;
2) DNA extracting taking step 1), as template, utilizes primer LFAcF and LFAcR described in claim 1 to carry out pcr amplification reaction;
3) analyze PCR product.
Pcr amplification product is carried out to agarose gel electrophoresis detection, if occur, size is the DNA cloning band (SEQ ID No.3) of 193bp, judges that sample is as bean aphid.
The present invention is according to the bean aphid COI(JX559638 announcing in GenBank) a fragment gene sequence, design a pair of Auele Specific Primer, this primer specificity is strong, only bean aphid is had to amplification ability, and to the main insect in cotton field in cotton region, China North China without amplified production.Adopt method provided by the invention and primer to detect bean aphid, accuracy is high, can amplify the fragment of 193bp size.Adopt SS-COI round pcr to detect bean aphid, supplementing and improving RAPD technology, RFLP technology and mtDNACOI detection technique, SS-COI PCR detection technique is simple to operate, quick, efficient, generally can in 5 hours, complete detection, and there is higher sensitivity, plasmid concentration detects and is limited to 3.99E+4, and DNA concentration detects and is limited to 0.413ng/ μ L.
Brief description of the drawings
Fig. 1 is the PCR detected result of bean aphid Auele Specific Primer LFAcF/LFAcR in the embodiment of the present invention 1; Wherein, M:DNA molecular weight standard 2000bp ladder; 1-114 is the amplification of the corresponding insect of sequence number in table 1 ,-negative contrast.
Fig. 2 is the DNA concentration gradient detected result of bean aphid Auele Specific Primer LFAcF/LFAcR in the embodiment of the present invention 2; Wherein, M:DNA molecular weight standard 5000bp ladder; 1-8 is template DNA concentration 413.6ng/ μ L 10 times of weaker concn gradients successively;-negative contrast.
Fig. 3 is the plasmid concentration gradient detected result of bean aphid Auele Specific Primer LFAcF/LFAcR in the embodiment of the present invention 2; Wherein, M:DNA molecular weight standard 5000bp ladder; 1-11 is template plasmid concentration 3.99E+11 10 times of weaker concn gradients successively;-negative contrast.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is all according to normal experiment condition, as Sambrook equimolecular cloning experimentation handbook (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer specification sheets suggestion.
The expanding effect of embodiment 1 primer LFAcF/LFAcR to bean aphid
1) the genomic preparation of bean aphid
Single Aphids is put into 1.5ml centrifuge tube and ground, with TIANamp Genomic DNA Kit blood/cell/tissue genome DNA extracting reagent kit (centrifugal column type) (the biological company limited of day root, Beijing) extraction Single Aphids genome.DNA solution is stored in-20 DEG C for subsequent use, while carrying out pcr amplification, draw 1 μ L solution as DNA profiling.
(2) the synthetic specificity SS-COI primer that detects bean aphid
Specificity SS-COI primer sequence is as follows:
LFAcF:5'-CAAACAATATTGCTCATAATAAC-3'（SEQ?ID?No.1）
LFAcR:5'-AAAATTAATAATATAGCTGTAATTAGG-3'（SEQ?ID?No.2）
2) pcr amplification
Reaction system is 20 μ L, comprising: 10 × EasyTaq damping fluid, 2.0 μ L, dNTP(2.5mM) 0.4 μ L, EasyTaq archaeal dna polymerase (5U/ μ L) 0.2 μ L, the each 0.4 μ L of forward and reverse primer (10 μ M), template DNA 1.0 μ L, ddH
2o15.6 μ L.
3) pcr amplification program
94 DEG C of denaturation 4min; 94 DEG C of 30sec, 54 DEG C of 30sec, 72 DEG C of 30sec, 45 circulations; Last 72 DEG C are extended 1sec.
4) PCR Product Identification
Get 6 μ L pcr amplification products, with 1% agarose gel electrophoresis separation, after ethidium bromide staining, in gel imaging system, judge according to the size of amplified production.
5) result
Utilize primer LFAcF/LFAcR, taking bean aphid DNA as template, 59 kinds of other insects (in table 1) taking bean aphid same area carry out Species-Specific-COI pcr amplification as contrasting, result as shown in Figure 1,1, the bean aphid that 2 swimming lanes are corresponding has amplified the object band of 193bp, illustrates according to the high specificity of the Species-Specific-COI pcr amplification primer of bean aphid Mitochondrial DNA design.The object band that reclaims above-mentioned 193bp, checks order, and its sequence is as shown in SEQ ID No.3.
Related caste during table 1 primer specificity detects
The mensuration of embodiment 2 primer LFAcF/LFAcR to bean aphid limit of identification
1) preparation of bean aphid templet gene group
Extract single head bean aphid genomic dna by embodiment 1, then original template solution, with 10 times of gradient dilutions that successively decrease to can not detecting band, is got the template of 1 μ L as pcr amplification, is directly added in PCR reaction system, and reaction system is with the description in embodiment 1.
Utilize primer LFAcF/LFAcR to do the mensuration of limit of identification, carry out pcr amplification taking the bean aphid genomic dna of different extension rates as template, DNA concentration detects and is limited to 0.413ng/ μ L(Fig. 2).
2) preparation of bean aphid plasmid
By case study on implementation 1, bean aphid genomic dna is increased, after amplification, detect with 1.5% agarose gel electrophoresis.Utilize Axygen sepharose DNA purification kit to reclaim PCR product, PCR product reclaims rear clone through glue and (press test kit description operation) on pEasy-T3 carrier, recombinant plasmid transformed Trans1-T1 competent cell, then coat on the LB flat board that contains Ampicillin/X-gal/IPTG, be inverted for 37 DEG C and cultivate 15 hours.After blue hickie screening, 10 positive colonies of picking are containing overnight incubation in the LB liquid nutrient medium of Amp, within second day, extract plasmid with Axygen plasmid extraction kit, then the plasmid extracting extremely can not detect band with 10 times of gradient dilutions that successively decrease, get the template of 1 μ L as pcr amplification, directly be added in PCR reaction system, reaction system is with the description in embodiment 1.
Utilize primer LFAcF/LFAcR to do the mensuration of limit of identification, carry out pcr amplification with the bean aphid plasmid concentration of different extension rates, template plasmid concentration detection limit (copy number) is 3.99E+4(Fig. 3).
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (8)
1. bean aphid specificity SS-COI primer, is characterized in that, it comprises:
Forward primer LFAcF:5'-CAAACAATATTGCTCATAATAAC-3 '
Reverse primer LFAcR:5 '-AAAATTAATAATATAGCTGTAATTAGG-3 '.
2. contain the test kit for detection of bean aphid of primer described in claim 1.
3. test kit according to claim 2, is characterized in that, described test kit also comprises dNTPs, Taq archaeal dna polymerase, Mg
2+, at least one in PCR reaction buffer.
4. according to the test kit described in claim 2 or 3, it is characterized in that, described test kit also comprises standard positive template.
5. the application of test kit in detection bean aphid described in primer or claim 2-4 any one described in claim 1.
6. the PCR method for detecting specificity of bean aphid, is characterized in that, comprises the following steps:
1) extract sample DNA;
2) DNA extracting taking step 1), as template, utilizes primer LFAcF and LFAcR described in claim 1 to carry out pcr amplification reaction;
3) analyze PCR product.
7. method according to claim 6, is characterized in that, PCR reaction system is counted with 20 μ l:
8. according to the method described in claim 6 or 7, it is characterized in that, PCR reaction conditions is: 94 DEG C 4 minutes; 94 DEG C 30 seconds, 54 DEG C 30 seconds, 72 DEG C 30 seconds, totally 45 circulations; 72 DEG C 1 second.
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| CN201310429628.9A CN103468677B (en) | 2013-09-18 | 2013-09-18 | Specific SS-COI (Species-specific Cytochrome Oxidase I) primers of aphis craccivora, kit containing primers and detecting method of kit |
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| CN201310429628.9A CN103468677B (en) | 2013-09-18 | 2013-09-18 | Specific SS-COI (Species-specific Cytochrome Oxidase I) primers of aphis craccivora, kit containing primers and detecting method of kit |
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| CN110343769A (en) * | 2019-07-11 | 2019-10-18 | 山东省花生研究所 | A kind of COI primer and its application for peanut aphid PCR detection |
| CN110257531A (en) * | 2019-07-11 | 2019-09-20 | 山东省花生研究所 | A method of for detecting prey Dominant Natural Enemies |
| 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 |
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