CN109628614A - A kind of the PCR detection primer and detection method of Lee's Grapholita spp - Google Patents
A kind of the PCR detection primer and detection method of Lee's Grapholita spp Download PDFInfo
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- CN109628614A CN109628614A CN201910080627.5A CN201910080627A CN109628614A CN 109628614 A CN109628614 A CN 109628614A CN 201910080627 A CN201910080627 A CN 201910080627A CN 109628614 A CN109628614 A CN 109628614A
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- lee
- pcr detection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
Abstract
The present invention relates to the detection technique fields of Lee's Grapholita spp, specifically disclose a kind of PCR detection primer of Lee's Grapholita spp.The sequence of the detection primer are as follows: upstream primer Gf-346-F:5 '-CCTCTCCTCCAATATTGCCCA-3 '；Downstream primer Gf-528-R:5 '-TGAAAGTAGAAGTAATAAGG-3 '.The PCR detection primer for Lee's Grapholita spp that the present invention designs can quickly detect Lee's Grapholita spp from numerous relationship allied species and ecological allied species, the primer specificity is high, expanding effect is good, detection difficulty and testing cost are reduced, the specific aim and prevention and treatment efficiency of orchard pest control are substantially increased.
Description
Technical field
The present invention relates to a kind of PCR of the detection technique field of Lee's Grapholita spp more particularly to Lee's Grapholita spp detections to draw
Object and detection method.
Background technique
Lee's Grapholita spp is a kind of insect of Lepidoptera Tortricidae Grapholita, is distributed in northeast, North China, northwest etc.
Various regions, host have the various plants such as Lee, apricot, cherry, peach, strongly fragrant Lee, wherein it is aggrieved most heavy with Lee, before young wormy fruit on the face Chang Guo
Spinning netting, dwell in it is off the net start to gnaw pericarp eat into fruit, entering fruit hole in early days is black, has frass discharge after a few days, makes
It largely falls off at the big fruit of beans, is killed fruit later entering fruit hole and flows out a large amount of droplet shape pectin drop, Lee's Grapholita spp enters fruit
After can eat into food kernel or in length and breadth string food, and go here and there to carpopodium nearby sting bad conducting system, make fruit purpling red, be not easy to eat again,
Serious loss is caused to orchard.
There are also many relationship allied species and ecological allied species, including oriental fruit months, manchurian fruit, fiber crops for Lee's Grapholita spp
Grapholita spp, carpocapsa pononella, white Grapholita spp, dichocrocis punctiferalis, pear fruit borer, small heart-eating peach worm, all lepidopterous insects remove
Numb Grapholita spp be feeding hemp and rule grass type outside, other types be north of China rosaceae fruit tree (apple, pears, peach,
Apricot, Lee, hawthorn etc.) common moth fruit pest, and the formalness of heart-eating worm larva is quite similar, and it is difficult to increase traditional form identification
Degree, if the type to heart-eating worm is out of one's reckoning, does not know about its growth rhythm, then is easy to the control method using mistake,
Many environmental-friendly control methods have very strong specificity, these methods are only effective to a kind of control of insect, therefore use
Shi Bixu accurately identifies the type of pest.
At present to the identification of Grapholita, traditional morphological method and the DNA bar code based on COI are relied primarily on
Technology.However morphological method needs research background or abundant identification experience of the appraiser with Lepidoptera taxonomic identification,
And the more comprehensive materials of identification are grasped, such as identify that larva sample also needs to grasp the identification mark and data of larva, this is for general
Testing staff is difficult to, and the vulnerability of Lepidoptera sample more increases the difficulty of morphological method identification.DNA bar shaped
Code technology is strictly a kind of simple effective method, but this method not only needs to carry out the nucleic acid of extraction the expansion of bar shaped chip segment
Increase, also needs to be sequenced and compared, and examining order generally requires sequencing company completion, substantially prolongs detection cycle, it is subsequent
Comparison work be also required to obtain comprehensive and accurate database.
Summary of the invention
For the problems such as existing Lee's Grapholita spp detection difficulty is big, method is complicated, detection cycle is long, testing cost is high, originally
Invention provides a kind of PCR detection primer of Lee's Grapholita spp.
To achieve the above object of the invention, the embodiment of the present invention uses the following technical solution:
A kind of PCR detection primer of Lee's Grapholita spp, the sequence of detection primer are as follows:
Upstream primer Gf-346-F:5 '-CCTCTCCTCCAATATTGCCCA-3 '；
Downstream primer Gf-528-R:5 '-TGAAAGTAGAAGTAATAAGG-3 '.
Compared with the existing technology, the PCR detection primer specificity of Lee's Grapholita spp provided by the invention is high, is drawn with the detection
8 kind relationship allied species and ecological allied species (oriental fruit months, the apple small food heart of the object to Lee's Grapholita spp and Lee's Grapholita spp
Worm, numb Grapholita spp, carpocapsa pononella, white Grapholita spp, dichocrocis punctiferalis, pear fruit borer and small heart-eating peach worm) genomic DNA into
The detection of row PCR amplification, the PCR detection primer can only obtain amplified production as template using the genomic DNA of Lee's Grapholita spp, other
Relationship allied species and ecological allied species are all unable to get amplified production, i.e., just can determine whether by the gel electrophoresis result of amplified production
It whether is out Lee's Grapholita spp, therefore the PCR detection primer of Lee's Grapholita spp designed through the invention can be fast and accurately
Lee's Grapholita spp is detected from all relationship allied species and ecological allied species, and detection method is simple and fast, at low cost.
Further, the present invention also provides the method for carrying out Lee's Grapholita spp PCR detection using the primer, the uses
Method, comprising the following steps:
A, the genomic DNA of insect to be detected is extracted；
B, using the genomic DNA in step a as template, PCR detection is carried out with detection primer；
C, detected through gel electrophoresis is carried out to the pcr amplification product in step b.
Preferably, DNA extraction is carried out using larva abdominal tissues in the step a.
Preferably, the extraction of the genomic DNA is extracted using CTAB method or genome extraction kit is extracted.
Preferably, the genomic DNA of extraction carries out PCR detection with COI universal primer, is extracted according to amplified production judgement
The accuracy of genomic DNA.
Preferably, the COI universal primer are as follows:
COI-F:5 '-GGTCAACAATCATAAAGATATTGG-3 '；
COI-R:5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 '.
Preferably, the amplification system that PCR is detected in the step b is 25 μ l, comprising: 2 × Premix Ex of 12.5 μ l
The deionized water of Taq, the Gf-346-F of 1 μ l, the Gf-528-R of 1 μ l, the DNA profiling of 1 μ l and 9.5 μ l.
Preferably, the concentration of the Gf-346-F and Gf-528-R is 10 μM.
Preferably, the PCR amplification condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 51 DEG C are moved back 30s, and 72 DEG C are prolonged
30s is stretched, 30 circulations are run；72 DEG C of extension 10min.
Detailed description of the invention
Fig. 1 is to be carried out in the embodiment of the present invention 2 with amplified production of the PCR detection primer to the genomic DNA of 9 kinds of insects
Agarose gel electrophoresis figure；Wherein, the DNA Marker band of the leftmost side from bottom to top size be followed successively by 100bp, 200bp,
300bp、400bp、500bp、600bp。
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
The design of the PCR detection primer of Lee's Grapholita spp: by the COI sequence (SEQ IDNO:1) and NCBI of Lee's Grapholita spp
The COI sequence of all kinds compares in the Grapholita category that can be downloaded in database, according to the COI of Lee's Grapholita spp
Sequence designs to obtain the PCR detection primer of Lee's Grapholita spp, the primer designed relative to the otherness site of comparison sequence
By Shanghai, bioengineering limited liability company is synthesized, and the particular sequence of PCR detection primer is as follows:
Gf-346-F:5 '-CCTCTCCTCCAATATTGCCCA-3 '；
Gf-528-R:5 '-TGAAAGTAGAAGTAATAAGG-3 '.
Embodiment 2
The primer pair Lee Grapholita spp and its 8 kinds of relationship allied species and ecological allied species (the small food of Lee designed using embodiment 1
Heart worm, oriental fruit months, manchurian fruit, numb Grapholita spp, carpocapsa pononella, white Grapholita spp, dichocrocis punctiferalis, pear fruit borer and
Small heart-eating peach worm) it is detected, detection method is as follows:
A, it extracts the genomic DNA of insect to be detected: taking Lee's Grapholita spp, oriental fruit months, manchurian fruit, fiber crops respectively
The larva abdominal tissues progress of Grapholita spp, carpocapsa pononella, white Grapholita spp, dichocrocis punctiferalis, pear fruit borer, small heart-eating peach worm
Abdominal tissues are respectively placed in 1.5ml centrifuge tube by the extraction of DNA, and liquid feeding nitrogen is fully ground, using Tiangeng biochemical technology (north
Capital) company blood/cell/tissue genome DNA extracting reagent kit, to specifications method extract genomic DNA, extraction
Genomic DNA carries out the detection of DNA concentration with NanoDrop 2000C spectrophotometer, and by the DNA sample of extraction in -20 DEG C
Refrigerator saves backup, and the testing result of DNA concentration is as shown in table 1.
Table 1
By the dilution of extract 9 kinds of genomic DNAs or it is concentrated as 50ng/ μ l, it is limited using Shanghai bioengineering share
The COI universal primer of company's synthesis carries out amplification verifying to 9 kinds of genome DNA samples of extraction, and PCR instrument is public using Whatman
The grads PCR instrument for taking charge of the model BIO-RAD T100Thermal Cycler of production is tied according to amplified production size and sequencing
Fruit verifies the accuracy of genome DNA sample.Wherein COI universal primer are as follows:
COI-F:5 '-GGTCAACAATCATAAAGATATTGG-3 '；
COI-R:5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 '.
The system for carrying out PCR amplification verifying to the genome DNA sample of extraction is 50 μ l, comprising:
Amplification condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 51 DEG C of annealing 30s, 72 DEG C of extension 30s, operation 30
A circulation；72 DEG C of extension 10min.
9 kinds of obtained amplified productions are sequenced, sequencing result is as shown in SEQ ID NO:1-SEQ ID NO:9, root
It is compared according to sequencing result, only manchurian fruit sequencing result is the type of COI sequence not having in database, but the small food heart of apple
The COI sequence of worm is allied species relationship with other kinds are belonged to, therefore also indicates that extracting genome DNA success.
B, respectively using the 9 kinds of genomic DNAs extracted in step a as template, with embodiment 1 design PCR detection primer into
Row PCR detection, PCR amplification system are 25 μ l, comprising:
PCR amplification condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 51 DEG C of annealing 30s, 72 DEG C of extension 30s, operation
30 circulations；72 DEG C of extension 10min.
C, the PCR detection primer designed using embodiment 1 carries out agar to the amplified production of the genomic DNA of 9 kinds of insects
Sugared gel electrophoresis, electrophoresis result is as shown in Figure 1, in 9 kinds of insects, and only Lee's Grapholita spp amplifies the segment that size is 202bp,
Other samples do not amplify the segment of 202bp, sequencing company will be sent to be sequenced after segment recycling, obtained sequencing result
As shown in SEQ ID NO:10, which is one section of base sequence in Lee's Grapholita spp COI gene order.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement or improvement etc., should all be included in the protection scope of the present invention within mind and principle.
SEQUENCE LISTING
<110>Technology Center Of Hebei Import and Export Inspection and Quarantine Bureau
<120>the PCR detection primer and detection method of a kind of Lee's Grapholita spp
<130> 2019
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 708
<212> DNA
<213>Lee's Grapholita spp (Grapholita funebrana)
<400> 1
ggtcaacaat cataaagata ttggaacact atattttatt ttcggaattt gagctggaat 60
agtaggaact tcattaagtt tacttattcg agcagaatta ggaaaccccg gatctttaat 120
tggcgatgat caaatttata atactattgt tacagcacat gcttttatta taattttttt 180
tatagtaatg cccattataa ttggaggatt tggtaattga ttagttcccc taatattagg 240
agccccagat atagctttcc cccgaataaa taatatgaga ttttgattat taccaccttc 300
tattatatta ttaatttcaa gaagaattgt agaaaatgga gcaggaacag gatgaactgt 360
ataccccccc ctctcctcca atattgccca tagaggaaga tcagttgact tagcaatttt 420
ttctctgcac ttagctggta tttcatcaat tttaggagct gttaatttta ttacaactat 480
cattaatata cgaccaaata atatatcact agatcaaata ccattatttg tatgagctgt 540
tggtattaca gccttattac ttctactttc attacccgta ttagcaggtg ctattactat 600
acttttaacc gatcgaaatc ttaatacctc attttttgat cctgctggag ggggtgatcc 660
cattttatac caacacttat tttgattttt tggtcaccct gaagttta 708
<210> 2
<211> 708
<212> DNA
<213>oriental fruit months (Grapholita molesta)
<400> 2
ggtcaacaat cataaagata ttggaacatt atattttatt tttggaattt gagctggtat 60
agtaggaact tctttaagat tattaattcg agcagaatta ggtaatccag gttctttaat 120
tggagatgat caaatttata atactattgt tactgcacat gcttttatta taattttttt 180
tatagtaata cctattataa ttggaggatt tggaaattga ttagtaccat taatattagg 240
tgccccagat atagctttcc cccgaataaa taatataaga ttttgattat tacctccttc 300
tattttatta ttaatctcca gtagaattgt ggaaaatgga gcaggaacag gatgaactgt 360
gtacccccca ctatcatcta atattgctca tagaggaagc tcagtagact tagcaatttt 420
ttctttacat ttagcaggta tttcttcaat tttaggagct attaatttta ttacaactat 480
tattaatata cgaccaaata atatatcttt agatcaaata ccattatttg tttgagctgt 540
tggtattaca gctcttttat tactactttc actaccagta ttagctggtg ctattactat 600
acttttaaca gatcgaaatc ttaatacttc attttttgac cctgcaggag gaggagatcc 660
tattctttat caacatttat tttgattttt tggtcaccct gaagttta 708
<210> 3
<211> 708
<212> DNA
<213>manchurian fruit (Grapholita inopinata)
<400> 3
ggtcaacaat cataaagata ttggaacatt atattttatt tttggtattt gagctggaat 60
aattggaact tcattaagtt tattaattcg agcagaatta ggaaatccag gttctttaat 120
tggagatgat caaatttata acactattgt taccgctcat gcctttatta taattttttt 180
tatagtaata ccaattataa ttggaggatt tggtaattga ttagtccccc ttatgttagg 240
agcacccgat atagctttcc cccgaataaa taacataaga ttttgactat tacccccttc 300
tattatatta ttaatttcaa gaagaattgt agaaaatgga gcaggaacag gatgaacagt 360
ttacccccca ctctcatcca atattgctca tagaggaaga tctgtagatt tagcaatttt 420
ttccttacat ttagctggta tttcttcaat tttaggagct gttaatttta ttacaactat 480
tattaatata cgacctaata acatatcatt agaccaaata cctttatttg tatgagctgt 540
tggtattaca gctcttctac tcctcctttc tttaccagta ttagcaggtg ctattactat 600
actcttaact gatcgaaatc ttaatacttc cttttttgac cctgcaggtg gtggtgatcc 660
tattttatat caacacttat tctgattttt tggtcaccct gaagttta 708
<210> 4
<211> 708
<212> DNA
<213>numb Grapholita spp (Grapholita delineana)
<400> 4
ggtcaacaat cataaagata ttggaacatt atattttatt tttggtattt gagcaggaat 60
agttggaact tctttaagtc ttattattcg tgcagaatta ggaaatcccg gatcattaat 120
tggagatgat caaatttata atactattgt aacagctcat gcatttatta taattttttt 180
tatagtaata ccaattataa ttggaggatt cggaaactga ttagtacctt taatattagg 240
agccccagat atagcatttc cccgaataaa taatataaga ttctgactat tacctccgtc 300
tattatgtta ttaatttcaa gtagaattgt agaaaatgga gcaggaacag gatgaacagt 360
ttatccccca ctttcatcta atattgctca tagaggaaga tctgtagatt tagctatttt 420
ttctttacat ttagcgggta tttcttctat tttaggagct gttaatttta ttacaactat 480
tattaatata cgacctaata atatatcatt agaccaaata ccattatttg tttgagctgt 540
tggaattaca gctcttttat tattattatc attaccagta ttagcgggag ctattacaat 600
acttttaaca gatcgaaacc ttaatacttc attttttgat cctgctggag gaggagatcc 660
tattttatat caacatttat tttgattttt tggtcaccct gaagttta 708
<210> 5
<211> 708
<212> DNA
<213>carpocapsa pononella (Laspeyresia pomonella)
<400> 5
ggtcaacaat cataaagata ttggaacatt atattttatt tttggtattt gagccggaat 60
agtaggaact tctctaagat tacttattcg agcagaatta ggaaatccag gatctttaat 120
tggtgatgat caaatttata atactattgt aactgctcat gcttttatta taattttttt 180
tatagtaata cctattataa ttggtggatt tggtaattga ttagtaccac taatattagg 240
agctcctgat atagcttttc ctcgaataaa taatataaga ttttgattat tacctccatc 300
tattatactt ttaatttcaa gcagaatcgt tgaaaatgga gcaggaacag gatgaacagt 360
gtacccccca ctttcttcta atattgccca tagaggaaga tctgttgatt tagctatttt 420
ttctctgcat ttagcaggta tttcttctat tttaggagct gttaatttta ttacaactat 480
tattaatata cgacctaata atatatcatt agatcaaata ccattatttg tttgagctgt 540
aggaattaca gctcttttac ttcttttatc attaccagta ttagcaggtg ctattactat 600
gcttcttaca gatcgaaatc ttaatacatc attttttgac cctgctggtg gaggtgatcc 660
tattctctac caacacttat tttgattttt tggtcaccct gaagttta 708
<210> 6
<211> 708
<212> DNA
<213>white Grapholita spp (Spilonota albicana)
<400> 6
ggtcaacaat cataaagata ttggaacatt atattttatt tttggtattt gagcaggaat 60
agttgggact tctttaagat tattaattcg agctgaatta ggaaatcctg gatatttaat 120
tggagacgat caaatttata acactattgt tactgcacat gcctttatta taattttttt 180
tatagtaata ccaattataa ttggaggatt tggaaattga ttagtgcctt taatattagg 240
agctccagat atagctttcc cgcgaataaa taatataaga ttttgattac tacctccatc 300
tattatactc ttaatttcaa gaagaattgt agaaaatgga gccggtacag gatgaacagt 360
atacccccca ctttcatcta atatcgctca tagaggcagt tccgtagact tagctatttt 420
ttctcttcac ttagcaggaa tttcttctat tttaggtgct gttaatttta ttactactat 480
tattaatata cggcctaata atataacttt agatcaaata ccattatttg tctgagctgt 540
cggaattact gctcttcttc ttcttctttc tttaccagta ttagcgggag ctattacaat 600
acttctcaca gatcgaaatc ttaatacatc attttttgat cctgctggag gaggggatcc 660
tattttatat caacatttat tttgattttt tggtcaccct gaagttta 708
<210> 7
<211> 708
<212> DNA
<213>dichocrocis punctiferalis (Conogethes punctiferalis)
<400> 7
ggtcaacaat cataaagata ttggaacttt atattttatt tttggaattt gagctggaat 60
agtgggaact tccttaagtt tattaattcg agctgaatta ggtaatccag gatcattaat 120
tggagatgat caaatttata atacaattgt aacagctcat gcttttatta taattttttt 180
tatggtaata cctattataa ttggaggttt tggaaattga ttagttcctc taatattagg 240
ggccccagat atagctttcc ctcgaataaa taatataaga ttttgattac ttcccccttc 300
actaactctt ttaatttcca gaagaattgt tgaaaatgga gctggaacag gatgaacagt 360
atacccccct ctttcatcta atattgcaca tggtggaaga tctgttgatc ttgctatttt 420
ttcccttcat ttagcgggaa tttcttctat tttaggagcg attaatttca ttacaacaat 480
tatcaatata cgaattaatg gattatcatt tgatcaaata cctcttttta tttgagctgt 540
aggaattaca gctttattac ttcttctatc tctgccagta ttagcgggtg ctattactat 600
acttttaaca gatcgtaatc ttaatacttc attttttgat ccggctggag ggggagatcc 660
tattttatat caacatttat tttgattttt tggtcaccct gaagttta 708
<210> 8
<211> 708
<212> DNA
<213>pear fruit borer (Acrobasis pirivorella)
<400> 8
ggtcaacaat cataaagata ttggaacttt atattttatt ttcggaattt gatcaggaat 60
actaggaaca tctttaagac ttttaattcg agctgaatta ggtactccag gatctttaat 120
tggagatgat caaatttata atactattgt tacaggtcat gcttttatta taattttttt 180
tatagttata cctattataa ttggaggatt tggaaattga ttagttcctt taatgttagg 240
agcccccgat atagctttcc cacgaataaa taatataaga ttttgattat tacccccatc 300
tattacttta ttaatttcaa gaagaattgt agaaaatgga gctggaacag gatgaactgt 360
ttacccccct ttatcatcca atattgctca tggaggtaga tctgttgatt tagctatttt 420
ttctttacat ttagctggaa tttcttcaat tttaggggct attaatttta ttacaactat 480
tattaatata aaattaaatg gtttatcttt tgatcaaata cctttatttg tttgagctgt 540
aggaattaca gctttattac tgttattatc attacctgta ttagctggag ctattactat 600
attactaaca gatcgaaatc ttaatacatc tttttttgac cctgctggag gtggagaccc 660
tattttatat caacatttat tttgattttt tggtcaccct gaagttta 708
<210> 9
<211> 708
<212> DNA
<213>small heart-eating peach worm (Carposina sasakii)
<400> 9
ggtcaacaat cataaagata ttggaacatt atattttatc tttggaattt gagcaggaat 60
agtaggaaca tcattaagat tattaattcg agctgaatta ggaaatccag gatcattaat 120
tggagatgat caaatttata attcaattgt tacagctcat gcatttatta taattttctt 180
tatagtaata cctattataa ttggtggatt tggaaattga cttgttccat taatattagg 240
agcaccagat atagcattcc ctcgtataaa taatataaga ttttgattgc ttcccccttc 300
aattttatta ctaatttcaa gaagaattgt agaaaatgga gcaggaaccg gatgaacagt 360
gtacccccca ctttcatcta atattgctca tggaggaaga tcagtagatt tagctatttt 420
ctcacttcat ttagcaggga tttcctcaat tttaggagca attaatttta ttacaactat 480
tattaacata cgaattaata atttatcatt tgaccaaata cctttatttg tatgagctgt 540
aggaattact gctctattat tacttttatc tcttccagtt cttgctggag ctattactat 600
attattaact gatcgaaatt taaatacatc attttttgac ccggctggag gaggtgatcc 660
tattttatac caacatttat tttgattttt tggtcaccct gaagttta 708
<210> 10
<211> 202
<212> DNA
<213>artificial sequence
<400> 10
cctctcctcc aatattgccc atagaggaag atcagttgac ttagcaattt tttctctgca 60
cttagctggt atttcatcaa ttttaggagc tgttaatttt attacaacta tcattaatat 120
acgaccaaat aatatatcac tagatcaaat accattattt gtatgagctg ttggtattac 180
agccttatta cttctacttt ca 202
Claims (9)
1. a kind of PCR detection primer of Lee's Grapholita spp, it is characterised in that: the sequence of the detection primer are as follows:
Upstream primer Gf-346-F:5 '-CCTCTCCTCCAATATTGCCCA-3 '；
Downstream primer Gf-528-R:5 '-TGAAAGTAGAAGTAATAAGG-3 '.
2. the method for carrying out PCR detection using primer pair Lee Grapholita spp described in claim 1, it is characterised in that: including following
Step:
A, the genomic DNA of insect to be detected is extracted；
B, using the genomic DNA in step a as template, PCR detection is carried out with detection primer；
C, detected through gel electrophoresis is carried out to the pcr amplification product in step b.
3. the method for carrying out PCR detection to Lee's Grapholita spp as claimed in claim 2, it is characterised in that: adopted in the step a
DNA extraction is carried out with larva abdominal tissues.
4. the method for carrying out PCR detection such as described in any item pairs of Lee's Grapholita spps of claim 2-3, it is characterised in that: described
The extraction of genomic DNA is extracted using CTAB method or genome extraction kit is extracted.
5. the method for carrying out PCR detection to Lee's Grapholita spp as claimed in claim 4, it is characterised in that: the genome of extraction
DNA carries out PCR detection with COI universal primer, according to the accuracy for the genomic DNA that amplified production judgement is extracted.
6. the method for carrying out PCR detection to Lee's Grapholita spp as claimed in claim 5, it is characterised in that: the COI is general to be drawn
Object are as follows:
COI-F:5 '-GGTCAACAATCATAAAGATATTGG-3 '；
COI-R:5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 '.
7. the method for carrying out PCR detection to Lee's Grapholita spp as claimed in claim 2, it is characterised in that: in the step b
The amplification system of PCR detection is 25 μ l, comprising: the Gf- of 2 × Premix Ex Taq of 12.5 μ l, the Gf-346-F of 1 μ l, 1 μ l
The deionized water of 528-R, the DNA profiling of 1 μ l and 9.5 μ l.
8. the method for carrying out PCR detection to Lee's Grapholita spp as claimed in claim 7, it is characterised in that: the Gf-346-F
Concentration with Gf-528-R is 10 μM.
9. the method for carrying out PCR detection to Lee's Grapholita spp as claimed in claim 7, it is characterised in that: the PCR amplification item
Part are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 51 DEG C are moved back 30s, and 72 DEG C of extension 30s run 30 circulations；72 DEG C of extensions
10min。
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