CN103305616A - Kit for verifying warehouse booklice based on specific primer - Google Patents

Kit for verifying warehouse booklice based on specific primer Download PDF

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CN103305616A
CN103305616A CN2013102505167A CN201310250516A CN103305616A CN 103305616 A CN103305616 A CN 103305616A CN 2013102505167 A CN2013102505167 A CN 2013102505167A CN 201310250516 A CN201310250516 A CN 201310250516A CN 103305616 A CN103305616 A CN 103305616A
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booklice
sequence
measured
primer pair
described storage
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CN103305616B (en
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李志红
崔冰艺
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China Agricultural University
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China Agricultural University
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Abstract

The invention discloses a kit for verification or assisted verification of a warehouse booklice. The kit provided by the invention comprises at least one pair of the following ten primer pairs: a primer pair 1 (sequences 1 and 2), a primer pair 2 (sequences 3 and 4), a primer pair 3 (sequence 5 and 6), a primer pair 4 (sequences 7 and 8), a primer pair 5 (sequence 9 and 10), a primer pair 6 (sequence 11 and 12), a primer pair 7 (sequence 13 and 14), a primer pair 8 (sequence 15 and 16), a primer pair 9 (sequence 17 and 18) and a primer pair 10 (sequence 19 and 20). Experiments prove that the kit for verifying the warehouse booklice based on specific primers provided by the invention can be used for verifying 10 types of booklices in the world, is strong in specificity, simple to operate, high in repeatability, high and stable in sensitivity, and is an ideal method for verifying the booklice.

Description

A kind of test kit of identifying the storage booklice based on special primer
Technical field
The invention belongs to biological technical field, relate to a kind of for the identification of or the test kit of assistant identification storage booklice.
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.This genus insect world widely distributes, Known Species more than 120 is planted at present, China puts down in writing 27 kinds of booklice altogether, and wherein the booklice relevant with grain storage mainly comprises and have a liking for worm booklice, l.bostrychophila, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax, three look booklices.
Booklice is to nibble the of paramount importance monoid of economic implications in the order insect.Booklice individuality small (become polypide be about 1mm), occurrence scope is extensive, mobility is strong, can very easily set up population with grain storage allocation and transportation and people for carrying long-distance communications, strong resistance is difficult to smoked kill, prevent and treat very difficult, 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.In recent years, China inspection and quarantining for import/export department intercepts and captures the number of times showed increased of booklice, but only is difficult to it is identified kind with traditional Morphological Identification method, and the method requires high to professional skill, length consuming time is given quarantine and the formulation of the decision-making of removing the evil brings difficulty.
The molecular biological variety identification method that grew up in recent years mainly comprises restriction fragment length polymorphism PCR(RFLP-PCR) technology and and nucleic acid sequence analysis authenticate technology.Restriction fragment length polymorphism round pcr result is comparatively accurate, but it is larger to screen the workload of required restriction enzyme kind.Have no at present the report of molecular engineering of the 10 kinds of common storages in the evaluation world of system.
Summary of the invention
An object of the present invention is to provide a kind of for the identification of or the test kit of assistant identification storage booklice.
Provided by the present invention for the identification of or the test kit of assistant identification storage booklice, can comprise at least one pair of in following 10 primer pairs:
(1) primer pair 1 that is formed by two single stranded DNAs shown in sequence in the sequence table 1 and the sequence 2;
(2) primer pair 2 that is formed by two single stranded DNAs shown in sequence in the sequence table 3 and the sequence 4;
(3) primer pair 3 that is formed by two single stranded DNAs shown in sequence in the sequence table 5 and the sequence 6;
(4) primer pair 4 that is formed by two single stranded DNAs shown in sequence in the sequence table 7 and the sequence 8;
(5) primer pair 5 that is formed by two single stranded DNAs shown in sequence in the sequence table 9 and the sequence 10;
(6) primer pair 6 that is formed by two single stranded DNAs shown in sequence in the sequence table 11 and the sequence 12;
(7) primer pair 7 that is formed by two single stranded DNAs shown in sequence in the sequence table 13 and the sequence 14;
(8) primer pair 8 that is formed by two single stranded DNAs shown in sequence in the sequence table 15 and the sequence 16;
(9) primer pair 9 that is formed by two single stranded DNAs shown in sequence in the sequence table 17 and the sequence 18;
(10) primer pair 10 that is formed by two single stranded DNAs shown in sequence in the sequence table 19 and the sequence 20.
Wherein, sequence 1 is comprised of 21 Nucleotide; Sequence 2 is comprised of 21 Nucleotide; Sequence 3 is comprised of 21 Nucleotide; Sequence 4 is comprised of 21 Nucleotide; Sequence 5 is comprised of 20 Nucleotide; Sequence 6 is comprised of 20 Nucleotide; Sequence 7 is comprised of 21 Nucleotide; Sequence 8 is comprised of 22 Nucleotide; Sequence 9 is comprised of 20 Nucleotide; Sequence 10 is comprised of 21 Nucleotide; Sequence 11 is comprised of 20 Nucleotide; Sequence 12 is comprised of 21 Nucleotide; Sequence 13 is comprised of 21 Nucleotide; Sequence 14 is comprised of 21 Nucleotide; Sequence 15 is comprised of 21 Nucleotide; Sequence 16 is comprised of 21 Nucleotide; Sequence 17 is comprised of 21 Nucleotide; Sequence 18 is comprised of 21 Nucleotide; Sequence 19 is comprised of 21 Nucleotide; Sequence 20 is comprised of 21 Nucleotide.
In described test kit, the mole number that forms two single stranded DNAs of each primer pair equates.
Described storage booklice can be as lower at least a: l.bostrychophila, have a liking for worm booklice, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax, three look booklices.
In described test kit, described primer pair 1 is specific to l.bostrychophila; Described primer pair 2 is specific to has a liking for the worm booklice; Described primer pair 3 is specific to colourless booklice; Described primer pair 4 is specific to the ommatidium booklice; Described primer pair 5 is specific to nibbles booklice; Described primer pair 6 is specific to dun booklice; Described primer pair 7 is specific to red booklice; Described primer pair 8 is specific to the Pi Shi booklice; Described primer pair 9 is specific to L. mendax; Described primer pair 10 is specific to three look booklices.
A further object of the present invention provides the preparation method of described test kit.
The preparation method of described test kit, each single stranded DNA that specifically comprises the steps: to form each primer pair in the described test kit is respectively separately after the packing, and at least a being packaged in the same reagent box in the following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
Also purpose of the present invention provides a kind of the evaluation or the method for assistant identification storage booklice.
The method of evaluation provided by the present invention or assistant identification storage booklice specifically can be in following (a)-(j) any:
(a) identify or whether assistant identification storage to be measured booklice is the method for l.bostrychophila, comprise following (a1) and step (a2):
(a1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 1 that is formed by two single stranded DNAs shown in sequence in the sequence table 1 and the sequence 2 to carry out pcr amplification;
(a2) size of the PCR product that obtains of detecting step (a1), determine according to the PCR product whether described storage booklice to be measured is l.bostrychophila as follows: if contain the dna fragmentation of 177bp in the described PCR product, then described storage booklice to be measured is or the candidate is l.bostrychophila; Otherwise, then described storage booklice to be measured be not or the candidate for l.bostrychophila;
(b) identify or whether assistant identification storage to be measured booklice is the method for having a liking for the worm booklice, comprise following (b1) and step (b2):
(b1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 2 that is formed by two single stranded DNAs shown in sequence in the sequence table 3 and the sequence 4 to carry out pcr amplification;
(b2) size of the PCR product that obtains of detecting step (b1), determine according to the PCR product whether described storage booklice to be measured is to have a liking for the worm booklice as follows: if contain the dna fragmentation of 188bp in the described PCR product, then described storage booklice to be measured for or the candidate for having a liking for the worm booklice; Otherwise, then described storage booklice to be measured not for or the candidate not for having a liking for the worm booklice;
(c) identify or whether assistant identification storage to be measured booklice is the method for colourless booklice, comprise following (c1) and step (c2):
(c1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 3 that is formed by two single stranded DNAs shown in sequence in the sequence table 5 and the sequence 6 to carry out pcr amplification;
(c2) size of the PCR product that obtains of detecting step (c1), determine according to the PCR product whether described storage booklice to be measured is colourless booklice as follows: if contain the dna fragmentation of 175bp in the described PCR product, then described storage booklice to be measured is or the candidate is colourless booklice; Otherwise, then described storage booklice to be measured be not or the candidate for colourless booklice;
(d) identify or whether assistant identification storage to be measured booklice is the method for ommatidium booklice, comprise following (d1) and step (d2):
(d1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 4 that is formed by two single stranded DNAs shown in sequence in the sequence table 7 and the sequence 8 to carry out pcr amplification;
(d2) size of the PCR product that obtains of detecting step (d1), determine according to the PCR product whether described storage booklice to be measured is the ommatidium booklice as follows: if contain the dna fragmentation of 186bp in the described PCR product, then described storage booklice to be measured is or the candidate is the ommatidium booklice; Otherwise, then described storage booklice to be measured be not or the candidate for the ommatidium booklice;
(e) identify or whether assistant identification storage to be measured booklice is the method for nibbling booklice, comprise following (e1) and step (e2):
(e1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 5 that is formed by two single stranded DNAs shown in sequence in the sequence table 9 and the sequence 10 to carry out pcr amplification;
(e2) size of the PCR product that obtains of detecting step (e1), determine according to the PCR product whether described storage booklice to be measured is to nibble booklice as follows: if contain the dna fragmentation of 128bp in the described PCR product, then described storage booklice to be measured for or the candidate for nibbling booklice; Otherwise, then described storage booklice to be measured not for or the candidate not for nibbling booklice;
(f) identify or whether assistant identification storage to be measured booklice is the method for dun booklice, comprise following (f1) and step (f2):
(f1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 6 that is formed by two single stranded DNAs shown in sequence in the sequence table 11 and the sequence 12 to carry out pcr amplification;
(f2) size of the PCR product that obtains of detecting step (f1), determine according to the PCR product whether described storage booklice to be measured is dun booklice as follows: if contain the dna fragmentation of 248bp in the described PCR product, then described storage booklice to be measured is or the candidate is dun booklice; Otherwise, then described storage booklice to be measured be not or the candidate for dun booklice;
(g) identify or whether assistant identification storage to be measured booklice is the method for red booklice, comprise following (g1) and step (g2):
(g1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 7 that is formed by two single stranded DNAs shown in sequence in the sequence table 13 and the sequence 14 to carry out pcr amplification;
(g2) size of the PCR product that obtains of detecting step (g1), determine according to the PCR product whether described storage booklice to be measured is red booklice as follows: if contain the dna fragmentation of 199bp in the described PCR product, then described storage booklice to be measured is or the candidate is red booklice; Otherwise, then described storage booklice to be measured be not or the candidate for red booklice;
(h) identify or whether assistant identification storage to be measured booklice is the method for Pi Shi booklice, comprise following (h1) and step (h2):
(h1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 8 that is formed by two single stranded DNAs shown in sequence in the sequence table 15 and the sequence 16 to carry out pcr amplification;
(h2) size of the PCR product that obtains of detecting step (h1), determine according to the PCR product whether described storage booklice to be measured is the Pi Shi booklice as follows: if contain the dna fragmentation of 251bp in the described PCR product, then described storage booklice to be measured is or the candidate is the Pi Shi booklice; Otherwise, then described storage booklice to be measured be not or the candidate for the Pi Shi booklice;
(i) identify or whether assistant identification storage to be measured booklice is the method for L. mendax, comprise following (i1) and step (i2):
(i1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 9 that is formed by two single stranded DNAs shown in sequence in the sequence table 17 and the sequence 18 to carry out pcr amplification;
(i2) size of the PCR product that obtains of detecting step (i1), determine according to the PCR product whether described storage booklice to be measured is L. mendax as follows: if contain the dna fragmentation of 185bp in the described PCR product, then described storage booklice to be measured is or the candidate is L. mendax; Otherwise, then described storage booklice to be measured be not or the candidate for L. mendax;
(j) identify or whether assistant identification storage to be measured booklice is the method for three look booklices, comprise following (j1) and step (j2):
(j1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 10 that is formed by two single stranded DNAs shown in sequence in the sequence table 19 and the sequence 20 to carry out pcr amplification;
(j2) size of the PCR product that obtains of detecting step (j1), determine according to the PCR product whether described storage booklice to be measured is three look booklices as follows: if contain the dna fragmentation of 250bp in the described PCR product, then described storage booklice to be measured for or the candidate be three look booklices; Otherwise, then described storage booklice to be measured for or the candidate be not three look booklices.
In aforesaid method, in step (a)-step (j), all described annealing temperatures of carrying out pcr amplification are 53 ℃.
In aforesaid method, described primer pair 1, described primer pair 2, described primer pair 3, described primer pair 4, described primer pair 5, described primer pair 6, described primer pair 7, described primer pair 8, described primer pair 9 and described primer pair 10, the mol ratio of two single stranded DNAs in PCR reaction system separately that forms each primer pair is 1:1.In the present invention, the volumetric molar concentration of two of each primer pair single stranded DNAs in PCR reaction system separately is 0.2 μ M.
In aforesaid method, in the step (a), the nucleotides sequence of the dna fragmentation of described 177bp is classified sequence 21 in the sequence table as; In the step (b), the nucleotides sequence of the dna fragmentation of described 188bp is classified sequence 22 in the sequence table as; In the step (c), the nucleotides sequence of the dna fragmentation of described 175bp is classified sequence 23 in the sequence table as; In the step (d), the nucleotides sequence of the dna fragmentation of described 186bp is classified sequence 24 in the sequence table as; In the step (e), the nucleotides sequence of the dna fragmentation of described 128bp is classified sequence 25 in the sequence table as; In the step (f), the nucleotides sequence of the dna fragmentation of described 248bp is classified sequence 26 in the sequence table as; In the step (g), the nucleotides sequence of the dna fragmentation of described 199bp is classified sequence 27 in the sequence table as; In the step (h), the nucleotides sequence of the dna fragmentation of described 251bp is classified sequence 28 in the sequence table as; In the step (i), the nucleotides sequence of the dna fragmentation of described 185bp is classified sequence 29 in the sequence table as; In the step (j), the nucleotides sequence of the dna fragmentation of described 250bp is classified sequence 30 in the sequence table as.
On the basis of brown booklice, red booklice, Pi Shi booklice, L. mendax, 10 kinds of common storage booklice ribosome-RNA(rRNA) the second transcribed spacer (ITS2rDNA) gene orders in this world of three look booklices, design also filters out 10 kinds of common storage booklice kinds and identifies special primers pair, formation has realized the quick and precisely evaluation of the common storage in world booklice based on the identification kit of special primer method.Utilize the 10 kinds of storages in test kit discriminating world booklice of identifying the storage booklice based on special primer provided by the present invention, high specificity, simple to operate and repeatable, strong highly sensitive and more stable is the Perfected process of differentiating booklice.
Description of drawings
Fig. 1 is the specific detection result for the identification of the primer pair LBF/LBR of l.bostrychophila.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1: l.bostrychophila (Guangxi); 2: l.bostrychophila (Henan); 3: l.bostrychophila (Chongqing); 4: l.bostrychophila (Germany); 5: l.bostrychophila (U.S.); 6: l.bostrychophila (Prague, CZE); 7: l.bostrychophila (Beijing); 8: l.bostrychophila (Guangzhou); 9: have a liking for worm booklice (Chongqing); 10: colourless booklice (Chongqing); 11: ommatidium booklice (Zhejiang); 12: nibble booklice (U.S.); 13: dun booklice (U.S.); 14: red booklice (U.S.); 15: Pi Shi booklice (U.S.); 16: L. mendax (Jiangsu); 17: three look booklices (Shandong); CK: negative control.
Fig. 2 is the specific detection result for the identification of the primer pair 21LEnF/208LEnR-2 that has a liking for the worm booklice.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker II); Other swimming lane information are 1: have a liking for worm booklice (Beijing); 2: have a liking for worm booklice (Guangxi); 3: have a liking for worm booklice (Czech); 4: have a liking for worm booklice (Chongqing); 5: l.bostrychophila (Guangxi); 6: colourless booklice (Chongqing); 7: ommatidium booklice (Zhejiang); 8: nibble booklice (U.S.); 9: dun booklice (Czech); 10: red booklice (U.S.); 11: Pi Shi booklice (U.S.); 12: L. mendax (Jiangsu); 13: three look booklices (Shandong), CK: negative control.
Fig. 3 is the specific detection result for the identification of the primer pair 164LDeF/319LDeR of colourless booklice.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker II); Other swimming lane information are 1,2: colourless booklice (Yunnan); 3,4: colourless booklice (Chongqing); 5: have a liking for worm booklice (Chongqing); 6: l.bostrychophila (Guangxi); 7: ommatidium booklice (Zhejiang); 8: nibble booklice (U.S.); 9: dun booklice (Czech); 10: red booklice (U.S.); 11: L. mendax (Jiangsu); 12: Pi Shi booklice (U.S.); 13: three look booklices (Shandong); CK: negative control.
Fig. 4 is the specific detection result for the identification of the primer pair LPa15F/LPa180R of ommatidium booklice.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker II); Other swimming lane information are 1: ommatidium booklice (Taian Shandong); 2: ommatidium booklice (Cao County, Shandong) 3: ommatidium booklice (Zhejiang); 4: ommatidium booklice (Hubei); 5: ommatidium booklice (U.S.); 6: have a liking for worm booklice (Chongqing); 7: l.bostrychophila (Guangxi); 8: colourless booklice (Chongqing); 9: nibble booklice (Czech); 10: dun booklice (U.S.); 11: red booklice (U.S.); 12: Pi Shi booklice (U.S.); 13: L. mendax (Jiangsu); 14: three look booklices (Shandong); CK: negative control.
Fig. 5 is the specific detection result for the identification of the primer pair LC170F/LC277R that nibbles booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1: nibble booklice (Denmark); 2: nibble booklice (Czech); 3: nibble booklice (U.S.); 4: have a liking for worm booklice (Chongqing); 5: l.bostrychophila (Guangxi); 6: colourless booklice (Chongqing); 7: ommatidium booklice (Zhejiang); 8: dun booklice (U.S.); 9: red booklice (U.S.); 10: three look booklices (Shandong); 11: L. mendax (Jiangsu); 12: Pi Shi booklice (U.S.); CK: negative control.
Fig. 6 is the specific detection result for the identification of the primer pair LBr350F/LBr577R of dun booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1: dun booklice (Czech); 2: dun booklice (U.S.); 3: have a liking for worm booklice (Chongqing); 4: l.bostrychophila (Guangxi); 5: colourless booklice (U.S.); 6: ommatidium booklice (Zhejiang); 7: nibble booklice (U.S.); 8: red booklice (U.S.); 9: Pi Shi booklice (U.S.); 10: three look booklices (Shandong); 11: L. mendax (Jiangsu); CK: negative control.
Fig. 7 is the specific detection result for the identification of the primer pair 78LRuF-3/276LRuR-3 of red booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1-3: red booklice (U.S.); 4: have a liking for worm booklice (Chongqing); 5: l.bostrychophila (Guangxi); 6: colourless booklice (Chongqing); 7: ommatidium booklice (Czech); 8: nibble booklice (U.S.); 9: dun booklice (U.S.); 10: L. mendax (Jiangsu); 11: Pi Shi booklice (U.S.); 12: three look booklices (Shandong); CK: negative control.
Fig. 8 is the specific detection result for the identification of the primer pair 186LPeF/436LPeR of Pi Shi booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1,2: Pi Shi booklice (U.S.); 3: have a liking for worm booklice (Chongqing); 4: l.bostrychophila (Guangxi); 5: colourless booklice (Chongqing); 6: ommatidium booklice (Czech); 7: nibble booklice (U.S.); 8: dun booklice (U.S.); 9: red booklice (U.S.); 10: L. mendax (Jiangsu); 11: three look booklices (Shandong); CK: negative control.
Fig. 9 is the specific detection result for the identification of the primer pair LM60F/LM224R of L. mendax.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1-3: L. mendax (Jiangsu); 4: have a liking for worm booklice (Chongqing); 5: l.bostrychophila (Guangxi); 6: colourless booklice (Chongqing); 7: ommatidium booklice (Czech); 8: nibble booklice (U.S.); 9: dun booklice (U.S.); 10: red booklice (U.S.); 11: Pi Shi booklice (U.S.); 12: three look booklices (Shandong); CK: negative control.
Figure 10 is the specific detection result for the identification of the primer pair LTri20F/LTri249R of three look booklices.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1,2: three look booklices (Shandong); 3: have a liking for worm booklice (Chongqing); 4: l.bostrychophila (Guangxi); 5: colourless booklice (Chongqing); 6: ommatidium booklice (Czech); 7: nibble booklice (U.S.); 8: dun booklice (U.S.); 9: red booklice (U.S.); 10: L. mendax (Jiangsu); 11: Pi Shi booklice (U.S.); CK: negative control.
Figure 11 is the sensitivity detected result for the identification of the primer pair LBF/LBR of l.bostrychophila.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker II); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng; CK: negative control.
Figure 12 is the sensitivity detected result for the identification of the primer pair 21LEnF/208LEnR-2 that has a liking for the worm booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng.
Figure 13 is the sensitivity detected result for the identification of the primer pair 164LDeF/319LDeR of colourless booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng.
Figure 14 is the sensitivity detected result for the identification of the primer pair LPa15F/LPa180R of ommatidium booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng; CK: negative control.
Figure 15 is the sensitivity detected result for the identification of the primer pair LC170F/LC277R that nibbles booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng.
Figure 16 is the sensitivity detected result for the identification of the primer pair LBr350F/LBr577R of dun booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng; CK: negative control.
Figure 17 is the sensitivity detected result for the identification of the primer pair 78LRuF-3/276LRuR-3 of red booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng.
Figure 18 is the sensitivity detected result for the identification of the primer pair 186LPeF/436LPeR of Pi Shi booklice.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng; CK: negative control.
Figure 19 is the sensitivity detected result for the identification of the primer pair LM60F/LM224R of L. mendax.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng; CK: negative control.
Figure 20 is the sensitivity detected result for the identification of the primer pair LTri20F/LTri249R of three look booklices.Wherein, swimming lane M is DNA relative molecular weight standard (D2000DNA Marker); Other swimming lane information are 1:DNA template consumption 40ng; 2:DNA template consumption 20ng; 3:DNA template consumption 10ng; 4:DNA template consumption 1ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.01; 7:DNA template consumption 0.001ng; CK: negative control.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
10 kinds of storage booklices: l.bostrychophila, have a liking for worm booklice, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax, three look booklices.Wherein, l.bostrychophila relates to following geographical population: Guangxi population, Henan population, Chongqing population, U.S. population, Prague, CZE population, Beijing population, Guangzhou population, German population; Have a liking for the worm booklice and relate to following geographical population: Beijing population, Chongqing population, Guangxi population, Czech population, population in Shandong; Colourless booklice relates to following geographical population: Yunnan population, Chongqing population, U.S. population; The ommatidium booklice relates to following geographical population: Taian Shandong population, Cao County, Shandong population, Zhejiang population, Hubei population, U.S. population, Czech population; Nibble booklice and relate to following geographical population: Denmark population, Czech population, U.S. population; Dun booklice relates to following geographical population: Czech population, U.S. population; Red booklice relates to following geographical population: U.S. population; The Pi Shi booklice relates to following geographical population: U.S. population; L. mendax relates to following geographical population: the Jiangsu population; Three look booklices relate to following geographical population: population in Shandong.Above 10 kinds of storage booklices be documented in " Zhao Shuo. based on the common storage in the world of 16S rDNA booklice Molecular Identification, China Agricultural University's master thesis, 2010 " in the literary composition.
Embodiment 1, for the design of the special primers of 10 kinds of storage booklices with synthetic
One, the acquisition of 10 kinds of storage booklice ITS2rDNA sequences
Experiment material: relate to 10 kinds of storage booklices, be specially l.bostrychophila, have a liking for the worm booklice, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax and three look booklices.Wherein, l.bostrychophila relates to following geographical population: Guangxi population, Henan population, Chongqing population, U.S. population, Prague, CZE population, Beijing population, Guangzhou population, German population; Have a liking for the worm booklice and relate to following geographical population: Beijing population, Chongqing population, Guangxi population, Czech population, population in Shandong; Colourless booklice relates to following geographical population: Yunnan population, Chongqing population, U.S. population; The ommatidium booklice relates to following geographical population: Taian Shandong population, Cao County, Shandong population, Zhejiang population, Hubei population, U.S. population, Czech population; Nibble booklice and relate to following geographical population: Denmark population, Czech population, U.S. population; Dun booklice relates to following geographical population: Czech population, U.S. population; Red booklice relates to following geographical population: U.S. population; The Pi Shi booklice relates to following geographical population: U.S. population; L. mendax relates to following geographical population: the Jiangsu population; Three look booklices relate to following geographical population: population in Shandong.
(1) extraction of booklice genomic dna
Adopt the micro-example genome DNA extracting reagent kit of TIANGEN Biotech (Beijing) Co., Ltd., extract total DNA of the whole polypide of single head booklice adult.Reference reagent box specification sheets carries out, and is specific as follows:
⑴ get booklice adult sample, kills with 75% alcohol first, after the dehydrated alcohol rinsing, is placed on the filter paper and dries.
⑵ the individuality after will process is put into the 1.5ml centrifuge tube, adds 10 μ l GA liquid (subsidiary with test kit), with grinding rod polypide is pulverized.
⑶ join in the centrifuge tube the centrifugal several seconds with 170 μ l GA liquid.
⑷ with unsettled the joining in the centrifuge tube of 10 μ l Proteinase K solution (subsidiary with test kit), 56 ℃ of water-baths 1 hour, centrifugal several seconds.
⑸ subsidiary with test kit with 200 μ l GB liquid (subsidiary with test kit) and 1 μ l Carrier RNA() join in the centrifuge tube 70 ℃ of water-baths 10 minutes, solution becomes clarification, centrifugal 30 seconds.
⑹ add the dehydrated alcohol of 200 μ l precoolings, vibrated 15 seconds, and centrifugal 1 minute, room temperature was placed 5 minutes, all changed among the adsorption column CR2 centrifugal 30 seconds over to.
⑺ join 500 μ l GD liquid (subsidiary with test kit) among the adsorption column CR2, and centrifugal 30 seconds of 12000rpm/s outwells waste liquid.
⑻ subsidiary with test kit with 700 μ l rinsing liquid PW() join among the adsorption column CR2, centrifugal 30 seconds of 12000rpm/s outwells waste liquid.
⑼ join 500 μ l rinsing liquid PW among the adsorption column CR2, and centrifugal 30 seconds of 12000rpm/s outwells waste liquid.
⑽ open adsorption column CR2 lid with adsorption column CR2 with 12000rpm/s centrifugal 2 minutes, and room temperature was placed 5 minutes.
⑾ put into adsorption column CR2 in the clean centrifuge tube, and unsettled dropping 30 μ l elution buffer TB(are subsidiary with test kit in the middle of the adsorption column CR2), place after 2-5 minute with 12000rpm/s centrifugal 2 minutes.
⑿ add the centrifugal solution that obtains again among the adsorption column CR2, and room temperature was placed 2 minutes, centrifugal 2 minutes of 12000rpm/s.
⒀ booklice genomic dna is eluted in the centrifuge tube, and-20 ℃ save backup.
(2) ITS2rDNA sequence pcr amplification
The ITS2rDNA sequence of the primer pair amplification booklice that utilization is comprised of 5 '-TGTGAACTGCAGGACACATG-3 ' and 5 '-GTCTTGTCTGATCTGAG-3 ', amplified production length not of the same race is about 290bp～650bp and does not wait.Reaction system: PCR reaction cumulative volume is 50 μ L, wherein Premix EX Taq TMThe precious biotechnology of 25 μ L(Dalian company limited), ddH 2O20 μ L, template (genomic dna) 3 μ L(30ng/ μ L), upstream primer 1 μ L, downstream primer 1 μ L, primer concentration is 10 μ M.Amplification condition is: the thermal cycling program is 94 ℃ of 4min, then carries out 30 circulations (94 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min), finishes behind 72 ℃ of reaction 10min at last.
(3) acquisition of ITS2rDNA sequence
Adopt agarose gel electrophoresis to detect the quality of PCR product; And the gel that adopts sky, Beijing root biochemical technology company limited reclaims test kit recovery product; To reclaim product and be connected on the pMD18-T carrier (precious biotechnology (Dalian) company limited), transform bacillus coli DH 5 alpha, carry out blue hickie screening at ammonia benzyl resistance LB culture medium flat plate; Adopt carrier universal primer RV-M(5 '-GAGCGGATAACAATTTCACACAGG-3 ') and M13-47(5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ') carry out bacterium colony PCR detection positive colony, bacterium colony PCR reaction conditions is: 95 ℃ of sex change 5min, 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ are extended 2min; 31 circulations; 72 ℃ are fully extended 10min.Amplified production adopts 2% agarose gel electrophoresis detection, and selecting wherein, the clone of clip size correct (having inserted amplified fragments) checks order.Send company (the prosperous biotechnology of Beijing AudioCodes limited liability company) two-way order-checking positive colony, the carrier universal primer is adopted in order-checking.To record the sequence splicing of comparing.
Two, for the design of the special primers of 10 kinds of storage booklices with synthetic
(1) design of primers
After 10 kinds of storage booklice (each kind comprises a plurality of geographical population) ITS2rDNA sequences that step 1 is obtained are carried out the DNAMAN compare of analysis, adopt Primer Premier5.0 software to design respectively special primer for 10 kinds of storage booklices.
(2) primer assessment
Adopt Oligo7 software that the primer pair indices is assessed, assessment content and evaluation criteria are as follows:
The Delta G value absolute value of upstream and downstream primer is no more than 9; Can form primer dimer or hairpin structure in conjunction with base pair not above 3; GC% content is controlled between the 30%-70%, and does not differ too large between the upstream and downstream; The mistake efficiency of initiation is no more than 100; At last, provide different primers to the suitableeest annealing temperature in conjunction with Oligo7 software, be set as 53 ℃ with annealing temperature is same.
According to the above, design and synthesize the special primer following (table 1) that obtains for 10 kinds of storage booklices:
The common 10 kinds of storage booklice special primers in table 1 world
Figure BDA00003385164600121
Embodiment 2, for the specific detection of 10 kinds of storage booklice ITS2rDNA sequence specific primers
One, experiment material
Relate to 10 kinds of storage booklices, be specially l.bostrychophila, have a liking for the worm booklice, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax and three look booklices.Wherein, l.bostrychophila relates to following geographical population: Guangxi population, Henan population, Chongqing population, U.S. population, Prague, CZE population, Beijing population, Guangzhou population, German population; Have a liking for the worm booklice and relate to following geographical population: Beijing population, Chongqing population, Guangxi population, Czech population, population in Shandong; Colourless booklice relates to following geographical population: Yunnan population, Chongqing population, U.S. population; The ommatidium booklice relates to following geographical population: Taian Shandong population, Cao County, Shandong population, Zhejiang population, Hubei population, U.S. population, Czech population; Nibble booklice and relate to following geographical population: Denmark population, Czech population, U.S. population; Dun booklice relates to following geographical population: Czech population, U.S. population; Red booklice relates to following geographical population: U.S. population; The Pi Shi booklice relates to following geographical population: U.S. population; L. mendax relates to following geographical population: the Jiangsu population; Three look booklices relate to following geographical population: population in Shandong.
Two, experimental technique
Adopt the micro-example genome DNA extracting reagent kit of TIANGEN Biotech (Beijing) Co., Ltd., extract total DNA of the whole polypide of single head booklice adult to be measured.Reference reagent box specification sheets carries out, and concrete operations are with embodiment 1.Take the gained genomic dna as template, adopt embodiment 1 designed each primer pair in 10 kinds of storage booklice ITS2rDNA sequence specific primers (table 1) to carry out respectively the substance pcr amplification.Thereby detect the designed specificity for 10 kinds of storage booklice ITS2rDNA sequence specific primers (table 1) of embodiment 1.
1, amplification system: PCR reaction cumulative volume is 50 μ L, wherein Premix EX Taq TMThe precious biotechnology of 25 μ L(Dalian company limited), ddH 2O20 μ L, template 3 μ L(30ng/ μ L), upstream primer 1 μ L(concentration is 10 μ M) and, downstream primer 1 μ L(concentration is 10 μ M).Upstream primer and the downstream primer final concentration in the PCR reaction system is 0.2 μ M.
The negative control that replaces template with water is set simultaneously.
2, amplification condition: 94 ℃ of denaturation 4min
Figure BDA00003385164600122
3, after reaction finishes, get 5 μ L PCR reaction product, the sepharose 2%, electrophoresis detection in 1 times the TAE electrophoretic buffer after ethidium bromide (EB) dyeing, is observed and imaging (Gel Logical Pro212) in gel imaging system.Simultaneously, amplified production directly send company's (prosperous bio tech ltd of AudioCodes) two-way order-checking, and sequencing result determines further that by sequence alignment special primer institute amplified fragments belongs to designed kind.
Three, experimental result
1, l.bostrychophila
Primer pair LBF/LBR(sequence 1 and the sequence 2 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, except the l.bostrychophila of identify distinguishing, also will have a liking for worm booklice, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax and these 9 kinds common storage booklices of three look booklices will carry out together the conventional PCR of special primer and react in the experiment material.The conventional pcr amplification product gel electrophoresis of primer pair LBF/LBR result as shown in Figure 1, except l.bostrychophila has specific amplification, have outside the specific amplification fragment (through checking order shown in sequence in the sequence table 21) at the 177bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, the evaluation l.bostrychophila that this primer can only be special is described, and invalid to other common storage booklices.
2, have a liking for the worm booklice
Primer pair 21LEnF/208LEnR-2(sequence 3 and the sequence 4 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, in the experiment material except having a liking for the worm booklice of identify distinguishing, also with l.bostrychophila, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax and these 9 kinds common storage booklices of three look booklices and carry out together the conventional PCR of special primer and react.The conventional pcr amplification product gel electrophoresis of primer pair 21LEnF/208LEnR-2 result as shown in Figure 2, except having a liking for the worm booklice specific amplification is arranged, have outside the specific amplification fragment (through checking order shown in sequence in the sequence table 22) at the 188bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, illustrate that this primer can only special evaluation have a liking for the worm booklice, and invalid to other common storage booklices.
3, colourless booklice
Primer pair 164LDeF/319LDeR(sequence 5 and the sequence 6 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, in the experiment material except the colourless booklice of identify distinguishing, also with l.bostrychophila, have a liking for worm booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax and these 9 kinds common storage booklices of three look booklices carry out together the conventional PCR of special primer and react.The conventional pcr amplification product gel electrophoresis of primer pair 164LDeF/319LDeR result as shown in Figure 3, except colourless booklice has specific amplification, have outside the specific amplification fragment (through checking order shown in sequence in the sequence table 23) at the 175bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, the colourless booklice of evaluation that this primer can only be special is described, and invalid to other common storage booklices.
4, ommatidium booklice
Primer pair LPa15F/LPa180R(sequence 7 and the sequence 8 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, in the experiment material except the ommatidium booklice of identify distinguishing, also with l.bostrychophila, have a liking for worm booklice, colourless booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax and these 9 kinds common storage booklices of three look booklices carry out together the conventional PCR of special primer and react.The conventional pcr amplification product gel electrophoresis of primer pair LPa15F/LPa180R result as shown in Figure 4, except the ommatidium booklice has specific amplification, have outside the specific amplification fragment (through checking order shown in sequence 24 in the sequence table) at the 186bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, the evaluation ommatidium booklice that this primer can only be special is described, and invalid to other common storage booklices.
5, nibble booklice
Primer pair LC170F/LC277R(sequence 9 and the sequence 10 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, in the experiment material except nibbling the booklice of identify distinguishing, also with l.bostrychophila, have a liking for worm booklice, colourless booklice, ommatidium booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax and these 9 kinds common storage booklices of three look booklices and carry out together the conventional PCR of special primer and react.The conventional pcr amplification product gel electrophoresis of primer pair LC170F/LC277R result as shown in Figure 5, except nibbling booklice specific amplification is arranged, have outside the specific amplification fragment (through checking order shown in sequence in the sequence table 25) at the 128bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, illustrate that this primer can only special evaluation nibble booklice, and invalid to other common storage booklices.
6, dun booklice
Primer pair LBr350F/LBr577R(sequence 11 and the sequence 12 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, in the experiment material except the dun booklice of identify distinguishing, also with l.bostrychophila, have a liking for worm booklice, colourless booklice, ommatidium booklice, nibble booklice, red booklice, Pi Shi booklice, L. mendax and these 9 kinds common storage booklices of three look booklices carry out together the conventional PCR of special primer and react.The conventional pcr amplification product gel electrophoresis of primer pair LBr350F/LBr577R result as shown in Figure 6, except dun booklice has specific amplification, have outside the specific amplification fragment (through checking order shown in sequence in the sequence table 26) at the 248bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, the dun booklice of evaluation that this primer can only be special is described, and invalid to other common storage booklices.
7, red booklice
Primer pair 78LRuF-3/276LRuR-3(sequence 13 and the sequence 14 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, in the experiment material except the red booklice of identify distinguishing, also with l.bostrychophila, have a liking for worm booklice, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, Pi Shi booklice, L. mendax and these 9 kinds common storage booklices of three look booklices carry out together the conventional PCR of special primer and react.The conventional pcr amplification product gel electrophoresis of primer pair 78LRuF-3/276LRuR-3 result as shown in Figure 7, except red booklice has specific amplification, have outside the specific amplification fragment (through checking order shown in sequence in the sequence table 27) at the 199bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, the red booklice of evaluation that this primer can only be special is described, and invalid to other common storage booklices.
8, Pi Shi booklice
Primer pair 186LPeF/436LPeR(sequence 15 and the sequence 16 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, in the experiment material except the Pi Shi booklice of identify distinguishing, also with l.bostrychophila, have a liking for worm booklice, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, L. mendax and these 9 kinds common storage booklices of three look booklices carry out together the conventional PCR of special primer and react.The conventional pcr amplification product gel electrophoresis of primer pair 186LPeF/436LPeR result as shown in Figure 8, except the Pi Shi booklice has specific amplification, have outside the specific amplification fragment (through checking order shown in sequence in the sequence table 28) at the 251bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, the evaluation Pi Shi booklice that this primer can only be special is described, and invalid to other common storage booklices.
9, L. mendax
Primer pair LM60F/LM224R(sequence 17 and the sequence 18 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, in the experiment material except the L. mendax of identify distinguishing, also with l.bostrychophila, have a liking for worm booklice, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice and these 9 kinds common storage booklices of three look booklices carry out together the conventional PCR of special primer and react.The conventional pcr amplification product gel electrophoresis of primer pair LM60F/LM224R result as shown in Figure 9, except L. mendax has specific amplification, have outside the specific amplification fragment (through checking order shown in sequence in the sequence table 29) at the 185bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, the evaluation L. mendax that this primer can only be special is described, and invalid to other common storage booklices.
10, three look booklices
Primer pair LTri20F/LTri249R(sequence 19 and the sequence 20 of utilizing designed, designed to obtain) detect the specificity of this primer pair under the conventional PCR method, in the experiment material except the three look booklices of identify distinguishing, also with l.bostrychophila, have a liking for worm booklice, colourless booklice, ommatidium booklice, nibble these 9 kinds common storage booklices of booklice, dun booklice, red booklice, Pi Shi booklice and L. mendax and carry out together the conventional PCR of special primer and react.The conventional pcr amplification product gel electrophoresis of primer pair LTri20F/LTri249R result as shown in figure 10, except three look booklices have specific amplification, have outside the specific amplification fragment (through checking order shown in sequence in the sequence table 30) at the 250bp place, other 9 kinds store in a warehouse booklice and water negative controls, all there is not amplified reaction, be in the product without specific target fragment, the evaluation three look booklices that this primer can only be special are described, and invalid to other common storage booklices.
Comprehensive above experimental result, what visible embodiment 1 was designed has higher specificity for 10 kinds of booklice ITS2rDNA sequence specific primers (table 1) of storing in a warehouse.
Embodiment 3, detect for the sensitivity of 10 kinds of storage booklice ITS2rDNA sequence specific primers
One, experiment material
Relate to 10 kinds of storage booklices, be specially l.bostrychophila, have a liking for the worm booklice, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax and three look booklices.Wherein, the geographical population of l.bostrychophila are specially the Guangxi population; The geographical population of having a liking for the worm booklice are specially the Chongqing population; The geographical population of colourless booklice are specially the Chongqing population; The geographical population of ommatidium booklice are specially Zhejiang population; The geographical population of nibbling booklice are specially the Czech population; The geographical population of dun booklice are specially U.S. population; The geographical population of red booklice are specially U.S. population; The geographical population of Pi Shi booklice are specially U.S. population; The geographical population of L. mendax are specially the Jiangsu population; The geographical population of three look booklices are specially population in Shandong.
Two, experimental technique
Adopt the micro-example genome DNA extracting reagent kit of TIANGEN Biotech (Beijing) Co., Ltd., extract total DNA of the whole polypide of single head booklice adult to be measured.Concrete operations are referring to the test kit specification sheets.Take the gained genomic dna as template, adopt embodiment 1 designed each primer pair in 10 kinds of storage booklice ITS2rDNA sequence specific primers (table 1) to carry out respectively the substance pcr amplification.Thereby detect the designed sensitivity for 10 kinds of storage booklice ITS2rDNA sequence specific primers (table 1) of embodiment 1.
1, amplification system: PCR reaction cumulative volume is 50 μ L, wherein Premix EX Taq TMThe precious biotechnology of 25 μ L(Dalian company limited), ddH 2O20 μ L, template 3 μ L, upstream primer 1 μ L(concentration is 10 μ M), downstream primer 1 μ L(concentration is 10 μ M).Upstream primer and the downstream primer final concentration in the PCR reaction system is 0.2 μ M.Wherein, as the consumption of booklice genomic dna to be measured in reaction system of template gradient is set and is: 40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01 and 0.001ng.
The negative control that replaces template with water is set simultaneously.
2, amplification condition: 94 ℃ of denaturation 4min
Figure BDA00003385164600161
3, after reaction finishes, get 5 μ L PCR reaction product, the sepharose 2%, electrophoresis detection in 1 times the TAE electrophoretic buffer after ethidium bromide (EB) dyeing, is observed and imaging (Gel Logical Pro212) in gel imaging system.Simultaneously, amplified production directly send company's (prosperous bio tech ltd of AudioCodes) two-way order-checking, and sequencing result determines further that by sequence alignment special primer institute amplified fragments belongs to designed kind.
Three, experimental result
1, l.bostrychophila
Primer pair LBF/LBR(sequence 1 and the sequence 2 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.The conventional PCR product gel of the l.bostrychophila of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) electrophoresis result as shown in figure 11.When template concentrations is 01ng, there is not the amplified band of specificity product, when template concentrations was 1ng, amplified band was fainter, only had the target fragment of trace to occur, and when template concentrations during greater than 10ng, all specific amplified can occur.
2, have a liking for the worm booklice
Primer pair 21LEnF/208LEnR-2(sequence 3 and the sequence 4 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.Different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) have a liking for the conventional PCR product gel of worm booklice electrophoresis result as shown in figure 12.When template concentrations was 0.1ng, amplified band was fainter, only had the target fragment of trace to occur, and when template concentrations during greater than 1ng, all specific amplified can occur.
3, colourless booklice
Primer pair 164LDeF/319LDeR(sequence 5 and the sequence 6 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.The conventional PCR product gel of the colourless booklice electrophoresis result of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) as shown in figure 13.When template concentrations is 0.1ng, a little less than amplified band is atomic, and when template concentrations during greater than 1ng, all specific amplified can occur.
4, ommatidium booklice
Primer pair LPa15F/LPa180R(sequence 7 and the sequence 8 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.The conventional PCR product gel of the ommatidium booklice of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) electrophoresis result as shown in figure 14.When template concentrations is 0.1ng, a little less than amplified band is atomic, and when template concentrations during greater than 1ng, all specific amplified can occur.
5, nibble booklice
Primer pair LC170F/LC277R(sequence 9 and the sequence 10 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.Different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) nibble the conventional PCR product gel of booklice electrophoresis result as shown in figure 15.When template concentrations was 1ng, amplified band was fainter, only had the target fragment of trace to occur, and when template concentrations during greater than 10ng, all specific amplified can occur.
6, dun booklice
Primer pair LBr350F/LBr577R(sequence 11 and the sequence 12 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.The conventional PCR product gel of the dun booklice electrophoresis result of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) as shown in figure 16.When template concentrations is 0.01ng, a little less than amplified band is atomic, and when template concentrations during greater than 0.1ng, all specific amplified can occur.
7, red booklice
Primer pair 78LRuF-3/276LRuR-3(sequence 13 and the sequence 14 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.The conventional PCR product gel of the red booklice electrophoresis result of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) as shown in figure 17.When template concentrations is 0.1ng, a little less than amplified band is atomic, and when template concentrations during greater than 1ng, all specific amplified can occur.
8, Pi Shi booklice
Primer pair 186LPeF/436LPeR(sequence 15 and the sequence 16 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.The conventional PCR product gel of the Pi Shi booklice of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) electrophoresis result as shown in figure 18.When template concentrations was 0.1ng, amplified band was fainter, and when template concentrations during greater than 1ng, all specific amplified can occur.
9, L. mendax
Primer pair LM60F/LM224R(sequence 17 and the sequence 18 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.The conventional PCR product gel of the L. mendax of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) electrophoresis result as shown in figure 19.When template concentrations was 0.1ng, amplified band was fainter, and when template concentrations during greater than 1ng, all specific amplified can occur.
10, three look booklices
Primer pair LTri20F/LTri249R(sequence 19 and the sequence 20 of utilizing designed, designed to obtain) detect the sensitivity of this primer pair under the conventional PCR method.The conventional PCR product gel of three look booklices of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) electrophoresis result as shown in figure 20.When template concentrations is 0.1ng, a little less than amplified band is atomic, and when template concentrations during greater than 1ng, all specific amplified can occur.
Comprehensive above experimental result, what visible embodiment 1 was designed has higher sensitivity for 10 kinds of booklice ITS2rDNA sequence specific primers (table 1) of storing in a warehouse.
Embodiment 4, use for the evaluation of 10 kinds of storage booklice ITS2rDNA sequence specific primers
Adopt detecting the inventor for 10 kinds of storage booklice ITS2rDNA sequence specific primers and adopting 5 unknown species booklice samples in Yantai, Shandong grain depot in 2011 of embodiment 1 design.Adopt the micro-example genome DNA extracting reagent kit of TIANGEN Biotech (Beijing) Co., Ltd., extract total DNA of whole polypide; Take the booklice genomic dna as template, use and carry out respectively the substance pcr amplification for special primer not of the same race (seeing the table 1 of embodiment 1); The employing agarose gel electrophoresis detects.Relating operation detects embodiment 1-3.
The result shows, wherein 4 unknown species booklice samples use be specific to occur in the product that the primer pair 21LEnF/208LEnR-2 that has a liking for the worm booklice carries out pcr amplification 188bp(through order-checking shown in sequence in the sequence table 22) bright band, 1 sample in the primer pair 164LDeF/319LDeR that use is specific to colourless booklice carries out the product of pcr amplification, occur 175bp(through order-checking shown in sequence in the sequence table 23) bright band.Except foregoing description, these 5 unknown species booklice samples all do not obtain corresponding purpose band when other special primers carry out pcr amplification in the table 1 that uses embodiment 1.Above result shows, detects by Auele Specific Primer shown in the table 1 that utilizes embodiment 1, and in 5 unknown species booklice samples, 4 for having a liking for the worm booklice, and 1 is colourless booklice.
Simultaneously, the present inventor has adopted existing Morphological Identification method.China Agricultural University's Plant Quarantine and the biological invasion laboratory Li Zhi Red Sect of Lamaism award and were engaged in the booklice morphological research more than 20 years, possesses abundant Morphological Identification experience, through the Li Zhi Red Sect of Lamaism award to 5 unknown species booklice sample Morphological Identifications (authentication method referring to " Li Zhihong. Chinese lice nibbles the sort research of genus; Beijing Agricultural University's master thesis; 1994 " literary composition), the result confirms, in 5 unknown species booklice samples, through Auele Specific Primer shown in the table 1 of the above-mentioned embodiment of utilization 1 detect be accredited as have a liking for the worm booklice 4 booklices really for having a liking for the worm booklice, detecting 1 booklice that is accredited as colourless booklice through Auele Specific Primer shown in the table 1 of the above-mentioned embodiment of utilization 1 is colourless booklice really.This shows the evaluation that the present invention utilizes Auele Specific Primer shown in the table 1 of embodiment 1 that booklice to be measured is carried out kind, reliable results.
Figure IDA00003385165500011
Figure IDA00003385165500021
Figure IDA00003385165500031
Figure IDA00003385165500041
Figure IDA00003385165500051
Figure IDA00003385165500061

Claims (8)

  1. One kind for the identification of or the test kit of assistant identification storage booklice, comprise at least one pair of in following 10 primer pairs:
    (1) primer pair 1 that is formed by two single stranded DNAs shown in sequence in the sequence table 1 and the sequence 2;
    (2) primer pair 2 that is formed by two single stranded DNAs shown in sequence in the sequence table 3 and the sequence 4;
    (3) primer pair 3 that is formed by two single stranded DNAs shown in sequence in the sequence table 5 and the sequence 6;
    (4) primer pair 4 that is formed by two single stranded DNAs shown in sequence in the sequence table 7 and the sequence 8;
    (5) primer pair 5 that is formed by two single stranded DNAs shown in sequence in the sequence table 9 and the sequence 10;
    (6) primer pair 6 that is formed by two single stranded DNAs shown in sequence in the sequence table 11 and the sequence 12;
    (7) primer pair 7 that is formed by two single stranded DNAs shown in sequence in the sequence table 13 and the sequence 14;
    (8) primer pair 8 that is formed by two single stranded DNAs shown in sequence in the sequence table 15 and the sequence 16;
    (9) primer pair 9 that is formed by two single stranded DNAs shown in sequence in the sequence table 17 and the sequence 18;
    (10) primer pair 10 that is formed by two single stranded DNAs shown in sequence in the sequence table 19 and the sequence 20.
  2. 2. test kit according to claim 1 is characterized in that: the mole number that forms two single stranded DNAs of each primer pair in the described test kit equates.
  3. 3. test kit according to claim 1 and 2 is characterized in that: described storage booklice for as lower at least a: have a liking for worm booklice, l.bostrychophila, colourless booklice, ommatidium booklice, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax, three look booklices.
  4. 4. the preparation method of arbitrary described test kit among the claim 1-3, each single stranded DNA that comprises the steps: to form each primer pair in the described test kit is respectively separately after the packing, and at least a being packaged in the same reagent box in the following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
  5. 5. identify or the method for assistant identification storage booklice, be in following (a)-(j) any:
    (a) identify or whether assistant identification storage to be measured booklice is the method for l.bostrychophila, comprise following (a1) and step (a2):
    (a1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 1 that is formed by two single stranded DNAs shown in sequence in the sequence table 1 and the sequence 2 to carry out pcr amplification;
    (a2) size of the PCR product that obtains of detecting step (a1), determine according to the PCR product whether described storage booklice to be measured is l.bostrychophila as follows: if contain the dna fragmentation of 177bp in the described PCR product, then described storage booklice to be measured is or the candidate is l.bostrychophila; Otherwise, then described storage booklice to be measured be not or the candidate for l.bostrychophila;
    (b) identify or whether assistant identification storage to be measured booklice is the method for having a liking for the worm booklice, comprise following (b1) and step (b2):
    (b1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 2 that is formed by two single stranded DNAs shown in sequence in the sequence table 3 and the sequence 4 to carry out pcr amplification;
    (b2) size of the PCR product that obtains of detecting step (b1), determine according to the PCR product whether described storage booklice to be measured is to have a liking for the worm booklice as follows: if contain the dna fragmentation of 188bp in the described PCR product, then described storage booklice to be measured for or the candidate for having a liking for the worm booklice; Otherwise, then described storage booklice to be measured not for or the candidate not for having a liking for the worm booklice;
    (c) identify or whether assistant identification storage to be measured booklice is the method for colourless booklice, comprise following (c1) and step (c2):
    (c1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 3 that is formed by two single stranded DNAs shown in sequence in the sequence table 5 and the sequence 6 to carry out pcr amplification;
    (c2) size of the PCR product that obtains of detecting step (c1), determine according to the PCR product whether described storage booklice to be measured is colourless booklice as follows: if contain the dna fragmentation of 175bp in the described PCR product, then described storage booklice to be measured is or the candidate is colourless booklice; Otherwise, then described storage booklice to be measured be not or the candidate for colourless booklice;
    (d) identify or whether assistant identification storage to be measured booklice is the method for ommatidium booklice, comprise following (d1) and step (d2):
    (d1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 4 that is formed by two single stranded DNAs shown in sequence in the sequence table 7 and the sequence 8 to carry out pcr amplification;
    (d2) size of the PCR product that obtains of detecting step (d1), determine according to the PCR product whether described storage booklice to be measured is the ommatidium booklice as follows: if contain the dna fragmentation of 186bp in the described PCR product, then described storage booklice to be measured is or the candidate is the ommatidium booklice; Otherwise, then described storage booklice to be measured be not or the candidate for the ommatidium booklice;
    (e) identify or whether assistant identification storage to be measured booklice is the method for nibbling booklice, comprise following (e1) and step (e2):
    (e1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 5 that is formed by two single stranded DNAs shown in sequence in the sequence table 9 and the sequence 10 to carry out pcr amplification;
    (e2) size of the PCR product that obtains of detecting step (e1), determine according to the PCR product whether described storage booklice to be measured is to nibble booklice as follows: if contain the dna fragmentation of 128bp in the described PCR product, then described storage booklice to be measured for or the candidate for nibbling booklice; Otherwise, then described storage booklice to be measured not for or the candidate not for nibbling booklice;
    (f) identify or whether assistant identification storage to be measured booklice is the method for dun booklice, comprise following (f1) and step (f2):
    (f1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 6 that is formed by two single stranded DNAs shown in sequence in the sequence table 11 and the sequence 12 to carry out pcr amplification;
    (f2) size of the PCR product that obtains of detecting step (f1), determine according to the PCR product whether described storage booklice to be measured is dun booklice as follows: if contain the dna fragmentation of 248bp in the described PCR product, then described storage booklice to be measured is or the candidate is dun booklice; Otherwise, then described storage booklice to be measured be not or the candidate for dun booklice;
    (g) identify or whether assistant identification storage to be measured booklice is the method for red booklice, comprise following (g1) and step (g2):
    (g1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 7 that is formed by two single stranded DNAs shown in sequence in the sequence table 13 and the sequence 14 to carry out pcr amplification;
    (g2) size of the PCR product that obtains of detecting step (g1), determine according to the PCR product whether described storage booklice to be measured is red booklice as follows: if contain the dna fragmentation of 199bp in the described PCR product, then described storage booklice to be measured is or the candidate is red booklice; Otherwise, then described storage booklice to be measured be not or the candidate for red booklice;
    (h) identify or whether assistant identification storage to be measured booklice is the method for Pi Shi booklice, comprise following (h1) and step (h2):
    (h1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 8 that is formed by two single stranded DNAs shown in sequence in the sequence table 15 and the sequence 16 to carry out pcr amplification;
    (h2) size of the PCR product that obtains of detecting step (h1), determine according to the PCR product whether described storage booklice to be measured is the Pi Shi booklice as follows: if contain the dna fragmentation of 251bp in the described PCR product, then described storage booklice to be measured is or the candidate is the Pi Shi booklice; Otherwise, then described storage booklice to be measured be not or the candidate for the Pi Shi booklice;
    (i) identify or whether assistant identification storage to be measured booklice is the method for L. mendax, comprise following (i1) and step (i2):
    (i1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 9 that is formed by two single stranded DNAs shown in sequence in the sequence table 17 and the sequence 18 to carry out pcr amplification;
    (i2) size of the PCR product that obtains of detecting step (i1), determine according to the PCR product whether described storage booklice to be measured is L. mendax as follows: if contain the dna fragmentation of 185bp in the described PCR product, then described storage booklice to be measured is or the candidate is L. mendax; Otherwise, then described storage booklice to be measured be not or the candidate for L. mendax;
    (j) identify or whether assistant identification storage to be measured booklice is the method for three look booklices, comprise following (j1) and step (j2):
    (j1) take the genomic dna of described storage to be measured booklice as template, use the primer pair 10 that is formed by two single stranded DNAs shown in sequence in the sequence table 19 and the sequence 20 to carry out pcr amplification;
    (j2) size of the PCR product that obtains of detecting step (j1), determine according to the PCR product whether described storage booklice to be measured is three look booklices as follows: if contain the dna fragmentation of 250bp in the described PCR product, then described storage booklice to be measured for or the candidate be three look booklices; Otherwise, then described storage booklice to be measured for or the candidate be not three look booklices.
  6. 6. method according to claim 5, it is characterized in that: in step (a)-step (j), all described annealing temperatures of carrying out pcr amplification are 53 ℃.
  7. 7. according to claim 5 or 6 described methods, it is characterized in that: described primer pair 1, described primer pair 2, described primer pair 3, described primer pair 4, described primer pair 5, described primer pair 6, described primer pair 7, described primer pair 8, described primer pair 9 and described primer pair 10, the mol ratio of two single stranded DNAs in PCR reaction system separately that forms each primer pair is 1:1.
  8. 8. arbitrary described method according to claim 5-7 is characterized in that: in the step (a), the nucleotides sequence of the dna fragmentation of described 177bp is classified sequence 21 in the sequence table as; In the step (b), the nucleotides sequence of the dna fragmentation of described 188bp is classified sequence 22 in the sequence table as; In the step (c), the nucleotides sequence of the dna fragmentation of described 175bp is classified sequence 23 in the sequence table as; In the step (d), the nucleotides sequence of the dna fragmentation of described 186bp is classified sequence 24 in the sequence table as; In the step (e), the nucleotides sequence of the dna fragmentation of described 128bp is classified sequence 25 in the sequence table as; In the step (f), the nucleotides sequence of the dna fragmentation of described 248bp is classified sequence 26 in the sequence table as; In the step (g), the nucleotides sequence of the dna fragmentation of described 199bp is classified sequence 27 in the sequence table as; In the step (h), the nucleotides sequence of the dna fragmentation of described 251bp is classified sequence 28 in the sequence table as; In the step (i), the nucleotides sequence of the dna fragmentation of described 185bp is classified sequence 29 in the sequence table as; In the step (j), the nucleotides sequence of the dna fragmentation of described 250bp is classified sequence 30 in the sequence table as.
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CN114295696A (en) * 2021-12-29 2022-04-08 广西大学 Electrochemical DNA sensor for rapidly detecting environmental DNA of starfish

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CN105567840A (en) * 2016-02-03 2016-05-11 中国农业大学 Method for identifying warehousing booklice or assisting in warehousing booklice identification and special complete set of reagent thereof
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WO2021164396A1 (en) * 2020-02-18 2021-08-26 南京农业大学 Viral nucleic acid extraction method based on glass fiber filter paper
CN114295696A (en) * 2021-12-29 2022-04-08 广西大学 Electrochemical DNA sensor for rapidly detecting environmental DNA of starfish
CN114295696B (en) * 2021-12-29 2024-02-20 广西大学 Electrochemical DNA sensor for rapidly detecting long-thorn starfish environmental DNA

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