CN106244673A - CPASA is for the method detecting Aphis gossypiipopulation allelic mutation frequency - Google Patents
CPASA is for the method detecting Aphis gossypiipopulation allelic mutation frequency Download PDFInfo
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- CN106244673A CN106244673A CN201510313713.8A CN201510313713A CN106244673A CN 106244673 A CN106244673 A CN 106244673A CN 201510313713 A CN201510313713 A CN 201510313713A CN 106244673 A CN106244673 A CN 106244673A
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Abstract
A kind of cPASA method for cotten aphid nAChR beta1 allelic mutation frequency detecting, during specificity cPASA design of primers, downstream primer 3 ' end is fixed at mutational site, the base pairing with 242 sites or mispairing.The present invention designs a competitive downstream primer by improving in the outside of specific primer so that it is be applicable to the detection of mutational site G/C.Detection to Aphis gossypiipopulation allelic mutation frequency, the method for the present invention is reliable, and stability is high, the cycle is short, highly sensitive, easy to operation, and experimental expenses is cheap.
Description
Technical field
The present invention relates to biology field, specifically, refer to competitive allele specific PCR (cPASA) the technology for detection cotten aphid of one
The method of population nAChR (nAChR) allelic mutation frequency.
Background technology
Cotten aphid is a kind of worldwide cotton-plant pest-insects, and breeding is fast, and harm weight, wherein chemical prevention is maximally effective control measures.Anabasine insecticide
It is an efficient medicament of class of prevention and control of aphides, but is as a large amount of uses of anabasine insecticide, the cotten aphid Drug resistance to which creating higher level.
Therefore, cotten aphid is extremely urgent to the research of anabasine insecticide resistance.
Cotten aphid is concentrated mainly on metabolic resistance and target resistance to anabasine insecticide Resistence research, and the action target of anabasine insecticide is nicotine
Type acetylcholinergic receptor (nAChR).Research shows, cotten aphid produces the beta1 Asia that the main cause of resistance is nAChR to anabasine insecticide
The sudden change (R81T) of base the 81st amino acids.Based on this point, set up a kind of resistance molecule detection technique, quickly detect this site of Aphis gossypiipopulation
Mutation frequency.On a molecular scale, the resistance of monitoring cotton Aphid, provide theoretical direction for resistance management.
The present invention is directed to the gene order of the beta1 subunit of nAChR and known mutational site, by 3 ' ends and the mutational site base of downstream primer
Pairing, according to sudden change and two specific Down Stream primers (3R/3S) of unmutated design, with common 5 ' forward primer (cPASA-F) amplification 184bp
The purpose fragment of size.In view of this gene point mutation is the sudden change of G-C, only by primer 3 ' end one base mispairing distinguishes to go out sudden change with not
Sudden change, then redesigns the downstream primer (cPASA-X) of a fragment that can amplify 586bp mesh with 5 ' forward primer (cPASA-F), rises
To Competition.Because competitive downstream primer cPASA-X mates completely with template, amplification efficiency is higher than Allele Specific Primers (3R/3S),
Improve the specificity that Allele Specific Primers (3R/3S) expands, it is thus possible to distinguish sudden change and unmutated individuality simultaneously.The spy that the present invention provides
Specific primer has commonly used value to the detection whether cotten aphid nAChR beta1 subunit amino acid 81 site (R81T) undergos mutation, permissible
It is widely applied and the detection of Aphis gossypiipopulation nAChR beta1 gene mutation frequency.
Summary of the invention
Present invention solves the technical problem that it is to design and develop 4 specific primers detection cotten aphid nAChR beta1 gene 242 site allele to be
No undergo mutation, and be homozygote or heterozygote.These four primers are respectively designated as cPASA-F, cPASA-3R, cPASA-3S by applicant
And cPASA-X.
Concrete, the nucleotide sequence of 4 specific primers that the present invention designs and develops is as follows:
General forward primer cPASA-F sequence is: 5 ' ACGAGAAGCGTCTGGTTAG 3 '.
Specificity unmutated downstream primer cPASA-3S sequence is 5 ' TGATAGTCCCTCCATACAAGTC 3 '.
Specific mutations downstream primer cPASA-3R sequence is 5 ' TGATAGTCCCTCCATACAAGTG 3 '.
Competitive downstream primer cPASA-X sequence is 5 ' GTCCGTCTGGGTAGGTGA 3 '.
The present invention is by the following technical solutions: according to the cotten aphid nAChR beta1 gene mRNA coding region sequence announced, and applies Primer
The amplification of Primer5.0 software design comprises the primer that a segment length is 586bp aim sequence in beta1 gene 242 site, further according to mutational site
(G-C), 3 ' ends of specific primer are fixed at mutational site, and single goal band can be amplified with general forward primer.These four primers
Specificity is high, and expanding effect is good, can detect whether cotten aphid beta1 subunit amino acid 81 site undergos mutation, and allele be homozygote also
It is heterozygote, and then detection Aphis gossypiipopulation allelic mutation frequency.
Accompanying drawing explanation
4 cPASA amplimers of design in Fig. 1 embodiment 1；
Fig. 2 embodiment 1 carries out cPASA amplification with 3R and 3S primer scheme and distinguishes the electrophoretogram of mutational site R81T gene type.
Detailed description of the invention
According to the cotten aphid nAChR beta1 gene mRNA coding region sequence announced, and it has been reported that the sudden change (G-C) in 242 sites,
Application Primer Primer5.0 software design comprises this mutational site, and product length is the pair of primers of 586bp, and designs according to mutational site
Can distinguish sudden change and two unmutated Allele Specific downstream primers, these two primers are in addition to 3 ' ends are for mutating alkali yl, and other base is the most identical.
The mutation frequency of application following step detection Aphis gossypiipopulation nAChR beta1 gene.
The extraction of 1.RNA
(1) take single head cotten aphid to put in 1.5mL centrifuge tube.
(2) it is initially charged 100 μ L lysates, grinds with tissue grinder, then supply 350 μ L lysates.
(3) adding 350 μ L 70% ethanol in lapping liquid one step up, fully mixing (moves liquid to inhale and beat, be not centrifuged).
(4) by 700 μ L sample (comprising all precipitations), proceeding in adsorption column, 10000rpm is centrifuged 15s, discards filtrate.
(5) adding 700 μ L RW1 buffer in adsorption column, 10000rpm is centrifuged 15s, abandons filtrate.Pillar it is carefully removed from after Li Xin, it is ensured that
Do not contact and abandon liquid.
(6) adding 500 μ L RPE buffer in post, 10000rpm is centrifuged 15s, abandons filtrate.
(7) adding 500 μ L RPE buffer again in post, 10000rpm is centrifuged 2min, abandons filtrate.Adsorption column is put into a new 2mL receive
In collector, build lid, then 10000rpm is centrifuged 1min
(8) adsorption column is put in a new 1.5mL centrifuge tube.Directly to the unsettled addition in adsorption column intermediate coat position 30-50 μ L RNase-free
Water.Build lid 10000rpm and be centrifuged 1min, wash RNA.
2. reverse transcription, obtains cDNA
(1) the removing reaction of genomic DNA
By 10 μ L systems, hatch 2min for 42 DEG C.
(2) reverse transcription reaction
By above-mentioned 20 μ L systems, react by following program: 37 DEG C of 15min, 85 DEG C of 5s.
3.PCR expands
Reaction system is as follows: 10 × PCR Buffer (Mg2+Plus) 2.5 μ L, dNTP Mixture (each 2.5mM) 2 μ L, general forward primer
(cPASA-F), competitive downstream primer (cPASA-X), each 1 μ L of specific Down Stream primer cPASA-3R or cPASA-3S, Taq DNA
Polymerase 0.3 μ L, template DNA 2 μ L, cumulative volume 25 μ L.
Amplification condition is as follows:
94℃4min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, 35 circulations；72℃7min.
3.PCR product detects
Taking 5 μ LPCR products and carry out electrophoresis with 2% agarose gel, electrophoretic buffer is 1 × TAE, 150V constant voltage electrophoresis 20min.
PCR primer stripe size is identified with ultraviolet gel imaging system.
4. population allelic mutation frequency detecting
Adopt back 3-4 Aphis gossypiipopulation, each population picking 50-100 head cotten aphid from field, in aforementioned manners its allele is detected, according to quick
Sense gene frequency (%)=(sensitive homozygote number × 2+ resistance heterozygote number) ÷ 2 ÷ total individual number × 100；R81T mutation allele
Frequency (%)=(resistance homozygote number × 2+ resistance heterozygote number) ÷ 2 ÷ total individual number × 100, it is thus achieved that saltation frequency.
Claims (4)
1. one kind for cotten aphid mutation of allelic gene point detection competitive allele specific PCR method (cPASA), include successively specific primer design,
Regular-PCR amplification, agarose gel electrophoresis, determine the step whether allele suddenlys change according to positive reaction, it is characterised in that: described specificity
In cPASA design of primers, two specific Down Stream primer 3 ' ends are designed at mutational site, share a forward primer, only by primer 3 '
The mispairing of end G/C, PCR amplification efficiency is the same, can not distinguish and suddenlys change and unmutated, therefore, adds one in the outside of specific primer
Competitive downstream primer, makes the primer amplification level of mispairing reduce.
2. the method as described in right 1, it is characterised in that: primer 3 ' end is fixed at mutational site, and mispairing mode is the mispairing of G/C, specific primer
Outside there is also competitive primer.
3. the method as described in right 1, it is characterised in that: described cPASA primer is two pairs of primers, shares forward primer, and two downstream primers are 3 '
End and mutational site Mismatching and mispairing, an other competitive downstream primer is according to the forward primer design shared.
4. the method one of as described in claim 1-3 is in the application of detection cotten aphid nAChR betal subunit 24 2 site allelic mutation.
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| CN201510313713.8A CN106244673A (en) | 2015-06-10 | 2015-06-10 | CPASA is for the method detecting Aphis gossypiipopulation allelic mutation frequency |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974518A (en) * | 2010-11-05 | 2011-02-16 | 南京农业大学 | Primer and PCR amplification of specificalleles (PASA) method for simultaneously detecting mutations of AChE genes and Na+ channel genes of potato beetles |
| CN104164498A (en) * | 2014-07-28 | 2014-11-26 | 电子科技大学附属医院·四川省人民医院 | Glaucoma screening kit |
-
2015
- 2015-06-10 CN CN201510313713.8A patent/CN106244673A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974518A (en) * | 2010-11-05 | 2011-02-16 | 南京农业大学 | Primer and PCR amplification of specificalleles (PASA) method for simultaneously detecting mutations of AChE genes and Na+ channel genes of potato beetles |
| CN104164498A (en) * | 2014-07-28 | 2014-11-26 | 电子科技大学附属医院·四川省人民医院 | Glaucoma screening kit |
Non-Patent Citations (1)
| Title |
|---|
| JING ZHANG等: "Frequency detection of imidacloprid resistance allele in Aphis gossypii field populations by real-time PCR amplification of specific-allele (rtPASA)", 《PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY》 * |
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