CN102367482B - Specific primer pair for assisting booklice identification and application thereof - Google Patents

Specific primer pair for assisting booklice identification and application thereof Download PDF

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CN102367482B
CN102367482B CN 201110349328 CN201110349328A CN102367482B CN 102367482 B CN102367482 B CN 102367482B CN 201110349328 CN201110349328 CN 201110349328 CN 201110349328 A CN201110349328 A CN 201110349328A CN 102367482 B CN102367482 B CN 102367482B
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booklice
sample
pcr amplification
identification
primer pair
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CN102367482A (en
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李志红
杨倩倩
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China Agricultural University
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China Agricultural University
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Abstract

The invention discloses a specific primer pair for assisting booklice identification and application thereof. The specific primer pair provided by the invention is composed of DNA shown as a sequence 1 in a sequence table and DNA shown as a sequence 2 in the sequence table. The specific primer pair disclosed by the invention can be used for assisting the booklice identification from a molecular level, is free from influences of sample ontogenesis state and sample integrity, and has the advantages of high efficiency, rapidness and accuracy. According to the invention, the booklice variety identification problem which puzzles the stored grain protection and port quarantine departments for a long time is solved. The specific primer pair disclosed by the invention can be widely applied to the grain storage department and the quarantine department.

Description

The special primer that assistant identification is nibbled booklice to and use
Technical field
The present invention relates to special primer that a kind of assistant identification nibbles booklice to and use.
Background technology
Booklice (booklice) has another name called paper lice, rice lice, is under the jurisdiction of and nibbles order (Psocoptera), booklice section (Liposcelidiae), booklice genus (Liposcelis), is the common insect in world's grain storage protection.Booklice belongs to insect individuality small (become polypide be about 1mm), and mobility is strong, can very easily set up population with grain storage allocation and transportation and people for carrying long-distance communications, strong resistance and difficulty of prevention and cure is large, the serious harm stored grain safety.Day by day frequent along with international trade and grain storage allocation and transportation, grain storage pest is propagated spread risk and is day by day increased.Nibble booklice L.corrodens (Heymons) and extensively distribute abroad, report and there is not yet in China, have larger intrusion risk.
Booklice belongs to (Liposcelis) and comprises as sowing: nibble booklice (L.corrodens), colourless booklice (L.decolor), ommatidium booklice (L.paeta), l.bostrychophila (L.bostrychophila), have a liking for worm booklice (L.entomophila), dun booklice (L.brunnea), L. mendax (L.mendax), three look booklices (L.tricolor), red booklice (L.rufa) and Pi Shi booklice (L.pearmani) etc.
Traditional booklice Identification of Species is foundation mainly with the adult morphological specificity, because the booklice individuality is small, identification of morphology requires very high to professional technique, have the large defective of length consuming time and difficulty.Especially for the individuality (such as ovum, larva and pupa) in non-Adult Development stage, identification of morphology is difficult to accomplish.
Summary of the invention
The purpose of this invention is to provide special primer that a kind of assistant identification nibbles booklice to and use.
Assistant identification provided by the invention is nibbled the special primer pair of booklice (L.corrodens), is comprised of DNA (downstream primer) shown in the sequence 2 of DNA (upstream primer) shown in the sequence 1 of sequence table and sequence table.
Described special primer is nibbled booklice to can be used for assistant identification.
Described special primer is to can be used for preparing the test kit that assistant identification is nibbled booklice.
The present invention also protects a kind of assistant identification to nibble the test kit of booklice, comprises described special primer pair.
The present invention also protects a kind of assistant identification to nibble the method for booklice; comprise the steps: that the genomic dna (or diluent of described genomic dna) take booklice to be measured is template; with described special primer to carrying out pcr amplification; if obtain pcr amplification product booklice to be measured for the candidate nibble booklice, do not nibble booklice if obtain pcr amplification product booklice to be measured for candidate non-.
The size of described pcr amplification product specifically can be 243bp.
The reaction system of described pcr amplification specifically can be (26ul): 2 * Taq PCR Master Mix 13ul, described upstream primer (10uM) 0.5ul, described downstream primer (10uM) 0.5ul, described genomic dna 2ul, ddH 2O10ul.
The reaction system of described pcr amplification specifically also can be (26ul): 2 * Taq PCR Master Mix 13ul, described upstream primer (10uM) 0.5ul, described downstream primer (10uM) 0.5ul, the diluent 1ul of described genomic dna, ddH 2O 11ul.
The reaction parameter of described pcr amplification specifically can be: 95 ℃ of denaturation 3min; 95 ℃ of sex change 30sec, 50 ℃ of annealing 40sec, 72 ℃ of extension 1min, 32 circulations; 72 ℃ of 8min.
Described booklice to be measured can be booklice and belongs to booklice, as nibbles booklice (L.corrodens), colourless booklice (L.decolor), ommatidium booklice (L.paeta), l.bostrychophila (L.bostrychophila), has a liking for worm booklice (L.entomophila), dun booklice (L.brunnea), L. mendax (L.mendax), three look booklices (L.tricolor), red booklice (L.rufa) or Pi Shi booklice (L.pearmani).
The present invention also protect a kind of from booklice the auxiliary method of nibbling booklice of differentiating, comprise the steps: genomic dna take booklice to be measured as template, to carrying out pcr amplification, the booklice to be measured that obtains pcr amplification product is candidate's the booklice that nibbles with described special primer.
The size of described pcr amplification product specifically can be 243bp.
The reaction system of described pcr amplification specifically can be (26ul): 2 * Taq PCR Master Mix 13ul, described upstream primer (10uM) 0.5ul, described downstream primer (10uM) 0.5ul, described genomic dna 2ul, ddH 2O10ul.
The reaction system of described pcr amplification specifically also can be (26ul): 2 * Taq PCR Master Mix 13ul, described upstream primer (10uM) 0.5ul, described downstream primer (10uM) 0.5ul, the diluent 1ul of described genomic dna, ddH 2O 11ul.
The reaction parameter of described pcr amplification specifically can be: 95 ℃ of denaturation 3min; 95 ℃ of sex change 30sec, 50 ℃ of annealing 40sec, 72 ℃ of extension 1min, 32 circulations; 72 ℃ of 8min.
Described booklice to be measured can be booklice and belongs to booklice, as nibbles booklice (L.corrodens), colourless booklice (L.decolor), ommatidium booklice (L.paeta), l.bostrychophila (L.bostrychophila), has a liking for worm booklice (L.entomophila), dun booklice (L.brunnea), L. mendax (L.mendax), three look booklices (L.tricolor), red booklice (L.rufa) or Pi Shi booklice (L.pearmani).
Using special primer provided by the invention nibbles booklice to assistant identification and has following advantage:
(1) fast, accurately and efficiently
Fragment in the special genomic dna for nibbling booklice of primer pair, detection sensitivity is high, consuming time short and can realize high throughput testing;
(2) easy and simple to handle, economy and facility
The traditional form authentication method generally needs slide sample to make technical ability, require high to sample integrity etc., but and the stdn of special primer molecular assay method, expense easy and simple to handle and required is lower, be convenient to without professional classification gain knowledge background staff operation with use.
Use special primer provided by the invention to nibbling booklice from the molecular level assistant identification, be not subjected to the impact of sample ontogeny state and sample integrity, have efficient, convenient, advantage accurately.The invention solves the booklice Identification of Species problem that perplexs for a long time grain storage protection and port quarantine department.The present invention has designed and has been used for differentiating the special primer pair of nibbling booklice, and provides a cover to differentiate the method for nibbling booklice, has significantly improved and has nibbled efficient and the accuracy that booklice is identified, have save time, efficiently, the characteristics such as directly perceived.This invention can be in foodstuff preservation department and sanitary authority widespread use.
Description of drawings
Fig. 1 is that special primer of the present invention belongs to the gel electrophoresis spectrum that various booklices carry out the PCR evaluation to booklice.
Fig. 2 nibbles the gel electrophoresis spectrum of the sensitivity of booklice for using special primer of the present invention to detection.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
Adopt following sample among the embodiment:
Sample 1: gather and to nibble booklice (L.corrodens) from Denmark;
Sample 2: gather and to nibble booklice (L.corrodens) from Czech;
Sample 3: gather and to nibble booklice (L.corrodens) from the U.S.;
Sample 4: gather from Portuguese and nibble booklice (L.corrodens);
Sample 5: gather the colourless booklice (L.decolor) from China;
Sample 6: gather the colourless booklice (L.decolor) from Czech;
Sample 7: gather the colourless booklice (L.decolor) from the U.S.;
Sample 8: gather the ommatidium booklice (L.paeta) from China;
Sample 9: gather the ommatidium booklice (L.paeta) from Czech;
Sample 10: gather the ommatidium booklice (L.paeta) from the U.S.;
Sample 11: gather the l.bostrychophila (L.bostrychophila) from China;
Sample 12: gather the l.bostrychophila (L.bostrychophila) from Czech;
Sample 13: gather the l.bostrychophila (L.bostrychophila) from the U.S.;
Sample 14: gather and to have a liking for worm booklice (L.entomophila) from China;
Sample 15: gather and to have a liking for worm booklice (L.entomophila) from Czech;
Sample 16: gather and to have a liking for worm booklice (L.entomophila) from the U.S.;
Sample 17: gather the dun booklice (L.brunnea) from Czech;
Sample 18: gather the dun booklice (L.brunnea) from the U.S.;
Sample 19: gather the L. mendax (L.mendax) from China;
Sample 20: gather three look booklices (L.tricolor) from China;
Sample 21: gather the red booklice (L.rufa) from the U.S.;
Sample 22: gather from the Pi Shi of U.S. booklice (L.pearmani).
Nibble booklice, colourless booklice, ommatidium booklice, l.bostrychophila, have a liking for worm booklice, dun booklice and L. mendax, the public all can obtain from China Agricultural University; The reference of mentioning above 7 kinds of booklices is: Zhao Shuo, Li Zhihong, Qin Meng. booklice and Progress on Molecular Biology thereof. and plant protection, 2009,35 (6): 17-21..
Three look booklices, the public can obtain from China Agricultural University; The reference of mentioning three look booklices is: Dong Peng, Wang Jinjun. the Molecular Detection of the wsp gene of three look booklice parachorium microorganism Wolbachia. and zoological research, 2004,25 (5): 456-459..
Red booklice and the Pi Shi booklice public all can obtain from China Agricultural University; The reference of mentioning red booklice and Pi Shi booklice is: Li Fasheng. order will is nibbled by China. and Beijing: Science Press, 2002:91-94..
Embodiment 1, the right design of special primer
On the basis of ten kinds of common psocids mitochondrial cytochrome oxidase subunit I barcodes (mtDNA COI barcode) section that booklice belongs to, design is synthetic can identify the special primer pair of nibbling booklice:
Upstream primer (sequence 1 of sequence table): 5 '-GAACAATAATCGGGAGAGGA-3 '; Tm=58.0 ℃, GC%=45.0;
Downstream primer (sequence 2 of sequence table): 5 '-GTAGAAGAATTGCTGTAATAAG-3 '; Tm=52.8 ℃, GC%=31.8.
Embodiment 2, application special primer are nibbled booklice to evaluation
Respectively 22 samples are carried out following experiment:
1, extracts the genomic dna of single head booklice.
2, take the genomic dna of step 1 as template (using water as negative contrast), to carrying out pcr amplification, obtain pcr amplification product with the special primer of embodiment 1 design.
PCR reaction system (26ul): 2 * Taq PCR Master Mix 13ul, upstream primer (10uM) 0.5ul, downstream primer (10uM) 0.5ul, genomic dna 2ul, ddH 2O 10ul.
PCR reaction parameter: 95 ℃ of denaturation 3min; 95 ℃ of sex change 30sec, 50 ℃ of annealing 40sec, 72 ℃ of extension 1min, 32 circulations; 72 ℃ of 8min; 4 ℃ of preservations.
3, the pcr amplification product with 4ul step 2 carries out 1.5% agarose gel electrophoresis, and ethidium bromide (EB) dyeing is observed and imaging analysis in gel systems.
The electrophoresis result of each sample is seen Fig. 1.Among Fig. 1, M:DNA relative molecular weight standard (D2000); Swimming lane 1 to swimming lane 22 is followed successively by sample 1 to sample 22, swimming lane 23 negative contrasts.Collection all shows a specific band about 250bp from the booklice (sample 1 is to sample 4) that nibbles of country variant, and other sample standard deviations do not show any band.
4, reclaim respectively pcr amplification product and checking order, sample 1 to the pcr amplification product of sample 4 is 243bp.The sequencing result of sample 1 is seen the sequence 3 of sequence table, and the sequencing result of sample 2 is seen the sequence 4 of sequence table, and the sequencing result of sample 3 is seen the sequence 5 of sequence table, and the sequencing result of sample 4 is seen the sequence 6 of sequence table.
Embodiment 3, the right sensitivity of special primer detect
Sample 1 is carried out following experiment:
1, extracts the genomic dna of single head booklice.
2, detect the concentration of genomic dna, it is carried out serial dilution with the TE damping fluid, obtain 8 kinds of diluents; Genomic dna concentration in 8 kinds of diluents is respectively: 0.1ng/ul, 1ng/ul, 5ng/ul, 10ng/ul, 25ng/ul, 50ng/ul, 75ng/ul and 100ng/ul are followed successively by diluent 1 to diluent 8.
3, respectively take every kind of diluent as template, to carrying out pcr amplification, obtain pcr amplification product with the special primer of embodiment 1 design.
PCR reaction system (26ul): 2 * Taq PCR Master Mix 13ul, upstream primer (10uM) 0.5ul, downstream primer (10uM) 0.5ul, diluent (containing genomic dna) 1ul, ddH 2O 11ul.
PCR reaction parameter: 95 ℃ of denaturation 3min; 95 ℃ of sex change 30sec, 50 ℃ of annealing 40sec, 72 ℃ of extension 1min, 32 circulations; 72 ℃ of 8min; 4 ℃ of preservations.
4, the pcr amplification product with 4ul step 3 carries out 1.5% agarose gel electrophoresis, and ethidium bromide (EB) dyeing is observed and imaging analysis in gel systems.
The electrophoresis result of the pcr amplification product of each diluent is seen Fig. 2.Among Fig. 2, M:DNA relative molecular weight standard (D2000); Swimming lane 1 to swimming lane 8 is followed successively by diluent 1 to diluent 8.The result shows, the concentration of genomic dna is all can amplify the purpose band more than the 1ng/ul in the reaction system, and purpose band increasing progressively and strengthen with genomic dna concentration on the electrophorogram.
Figure IDA0000106026720000021

Claims (8)

1. assistant identification is nibbled the special primer pair of booklice, is comprised of DNA shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table.
2. the described special primer of claim 1 is to nibbling the application in the booklice in assistant identification.
3. the described special primer of claim 1 is to the application in the test kit of nibbling booklice in the preparation assistant identification.
4. an assistant identification is nibbled the test kit of booklice, comprises the described special primer of claim 1 pair.
5. an assistant identification is nibbled the method for booklice, comprise the steps: that genomic dna take booklice to be measured is as template, with the described special primer of claim 1 to carrying out pcr amplification, if obtain pcr amplification product booklice to be measured for the candidate nibble booklice, do not nibble booklice if obtain pcr amplification product booklice to be measured for candidate non-.
6. method as claimed in claim 5, it is characterized in that: the size of described pcr amplification product is 243bp.
7. auxiliary method of nibbling booklice of differentiating from booklice comprises the steps: genomic dna take booklice to be measured as template, and to carrying out pcr amplification, the booklice to be measured that obtains pcr amplification product is candidate's the booklice that nibbles with the described special primer of claim 1.
8. method as claimed in claim 7, it is characterized in that: the size of described pcr amplification product is 243bp.
CN 201110349328 2011-11-07 2011-11-07 Specific primer pair for assisting booklice identification and application thereof Active CN102367482B (en)

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CN103305616B (en) * 2013-06-21 2015-02-25 中国农业大学 Kit for verifying warehouse booklice based on specific primer
CN105483121B (en) * 2014-09-18 2019-01-01 中国农业大学 One group of primer and its application for identifying storage booklice

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CN101608238B (en) * 2009-07-29 2011-09-14 中国农业大学 Primer pair for identifying booklice in stored booklices and application thereof
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