CN103305616B

CN103305616B - Kit for verifying warehouse booklice based on specific primer

Info

Publication number: CN103305616B
Application number: CN201310250516.7A
Authority: CN
Inventors: 李志红; 崔冰艺
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2013-06-21
Filing date: 2013-06-21
Publication date: 2015-02-25
Anticipated expiration: 2033-06-21
Also published as: CN103305616A

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 based on special primer qualification storage booklice

Technical field

The invention belongs to biological technical field, relate to a kind of for the identification of or assistant identification storage booklice test kit.

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 pests in world's grain storage protection.This genus insect world widely distributes, current Known Species more than 120 is planted, China records booklice 27 kinds altogether, wherein relevant with grain storage booklice mainly comprises Leposcelis entomophia, l.bostrychophila, L. decolor, L. paeta, nibbles booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax, three look booklices.

Booklice nibbles the of paramount importance monoid of economic implications in order insect.Booklice individuality small (become polypide be about 1mm), occurrence scope is extensive, and mobility is strong, can with grain storage allocation and transportation and people for carrying long-distance communications, and very easily set up population, strong resistance is difficult to smoked kill, prevents and treats very difficult, serious harm stored grain safety.Day by day frequent along with international trade and grain storage allocation and transportation, grain storage pest Spreading and diffusion risk increases day by day.In recent years, inspection and quarantining for import/export department of China intercepts and captures the number of times showed increased of booklice, but is only difficult to be identified kind by traditional Morphological Identification method, and the method requires high to professional skill, length consuming time, brings difficulty to the formulation of quarantine and decision-making of removing the evil.

The molecular biological variety identification method grown 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 the workload of the required restriction enzyme kind of screening is larger.Have no the report of the molecular engineering of the 10 kinds of common storages in the qualification world of system at present.

Summary of the invention

An object of the present invention is to provide a kind of for the identification of or assistant identification storage booklice test kit.

Provided by the present invention for the identification of or assistant identification storage booklice test kit, at least one pair of in following 10 primer pairs can be comprised:

(1) primer pair 1 be made up of two single stranded DNAs shown in sequence in sequence table 1 and sequence 2;

(2) primer pair 2 be made up of two single stranded DNAs shown in sequence in sequence table 3 and sequence 4;

(3) primer pair 3 be made up of two single stranded DNAs shown in sequence in sequence table 5 and sequence 6;

(4) primer pair 4 be made up of two single stranded DNAs shown in sequence in sequence table 7 and sequence 8;

(5) primer pair 5 be made up of two single stranded DNAs shown in sequence in sequence table 9 and sequence 10;

(6) primer pair 6 be made up of two single stranded DNAs shown in sequence in sequence table 11 and sequence 12;

(7) primer pair 7 be made up of two single stranded DNAs shown in sequence in sequence table 13 and sequence 14;

(8) primer pair 8 be made up of two single stranded DNAs shown in sequence in sequence table 15 and sequence 16;

(9) primer pair 9 be made up of two single stranded DNAs shown in sequence in sequence table 17 and sequence 18;

(10) primer pair 10 be made up of two single stranded DNAs shown in sequence in sequence table 19 and sequence 20.

Wherein, sequence 1 is made up of 21 Nucleotide; Sequence 2 is made up of 21 Nucleotide; Sequence 3 is made up of 21 Nucleotide; Sequence 4 is made up of 21 Nucleotide; Sequence 5 is made up of 20 Nucleotide; Sequence 6 is made up of 20 Nucleotide; Sequence 7 is made up of 21 Nucleotide; Sequence 8 is made up of 22 Nucleotide; Sequence 9 is made up of 20 Nucleotide; Sequence 10 is made up of 21 Nucleotide; Sequence 11 is made up of 20 Nucleotide; Sequence 12 is made up of 21 Nucleotide; Sequence 13 is made up of 21 Nucleotide; Sequence 14 is made up of 21 Nucleotide; Sequence 15 is made up of 21 Nucleotide; Sequence 16 is made up of 21 Nucleotide; Sequence 17 is made up of 21 Nucleotide; Sequence 18 is made up of 21 Nucleotide; Sequence 19 is made up of 21 Nucleotide; Sequence 20 is made up of 21 Nucleotide.

In described test kit, the mole number forming two single stranded DNAs of each primer pair is equal.

Described storage booklice can be following at least one: l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, 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 Leposcelis entomophia; Described primer pair 3 is specific to L. decolor; Described primer pair 4 is specific to L. paeta; 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 Pi Shi booklice; Described primer pair 9 is specific to L. mendax; Described primer pair 10 is specific to three look booklices.

Another object of the present invention is to provide the preparation method of described test kit.

The preparation method of described test kit, specifically comprise the steps:, by after in the described test kit of composition, each single stranded DNA of each primer pair is individually packed, to be packaged in same reagent box with at least one in following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.

Also object of the present invention is to provide a kind of method of qualification or assistant identification storage booklice.

The method of qualification provided by the present invention or assistant identification storage booklice, specifically can be any one in following (a)-(j):

A () identifies or whether assistant identification storage to be measured booklice is the method for l.bostrychophila, comprises the step of following (a1) and (a2):

(a1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 1 be made up of two single stranded DNAs shown in sequence in sequence table 1 and sequence 2;

(a2) size of PCR primer that obtains of detecting step (a1), determine whether described storage booklice to be measured is l.bostrychophila according to PCR primer as follows: if containing the DNA fragmentation of 177bp in described PCR primer, then described storage booklice to be measured is or candidate is l.bostrychophila; Otherwise, then described storage booklice to be measured be not or candidate for l.bostrychophila;

B () identifies or whether assistant identification storage to be measured booklice is the method for Leposcelis entomophia, comprises the step of following (b1) and (b2):

(b1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 2 be made up of two single stranded DNAs shown in sequence in sequence table 3 and sequence 4;

(b2) size of PCR primer that obtains of detecting step (b1), determine whether described storage booklice to be measured is Leposcelis entomophia according to PCR primer as follows: if containing the DNA fragmentation of 188bp in described PCR primer, then described storage booklice to be measured is or candidate is Leposcelis entomophia; Otherwise, then described storage booklice to be measured be not or candidate for Leposcelis entomophia;

C () identifies or whether assistant identification storage to be measured booklice is the method for L. decolor, comprises the step of following (c1) and (c2):

(c1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 3 be made up of two single stranded DNAs shown in sequence in sequence table 5 and sequence 6;

(c2) size of PCR primer that obtains of detecting step (c1), determine whether described storage booklice to be measured is L. decolor according to PCR primer as follows: if containing the DNA fragmentation of 175bp in described PCR primer, then described storage booklice to be measured is or candidate is L. decolor; Otherwise, then described storage booklice to be measured be not or candidate for L. decolor;

D () identifies or whether assistant identification storage to be measured booklice is the method for L. paeta, comprises the step of following (d1) and (d2):

(d1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 4 be made up of two single stranded DNAs shown in sequence in sequence table 7 and sequence 8;

(d2) size of PCR primer that obtains of detecting step (d1), determine whether described storage booklice to be measured is L. paeta according to PCR primer as follows: if containing the DNA fragmentation of 186bp in described PCR primer, then described storage booklice to be measured is or candidate is L. paeta; Otherwise, then described storage booklice to be measured be not or candidate for L. paeta;

E () identifies or whether assistant identification storage to be measured booklice is the method for nibbling booklice, comprises the step of following (e1) and (e2):

(e1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 5 be made up of two single stranded DNAs shown in sequence in sequence table 9 and sequence 10;

(e2) size of PCR primer that obtains of detecting step (e1), determine whether described storage booklice to be measured is nibble booklice according to PCR primer as follows: if containing the DNA fragmentation of 128bp in described PCR primer, then described storage booklice to be measured be or candidate for nibbling booklice; Otherwise, then described storage booklice to be measured not for or candidate not for nibbling booklice;

F () identifies or whether assistant identification storage to be measured booklice is the method for dun booklice, comprises the step of following (f1) and (f2):

(f1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 6 be made up of two single stranded DNAs shown in sequence in sequence table 11 and sequence 12;

(f2) size of PCR primer that obtains of detecting step (f1), determine whether described storage booklice to be measured is dun booklice according to PCR primer as follows: if containing the DNA fragmentation of 248bp in described PCR primer, then described storage booklice to be measured is or candidate is dun booklice; Otherwise, then described storage booklice to be measured be not or candidate for dun booklice;

G () identifies or whether assistant identification storage to be measured booklice is the method for red booklice, comprises the step of following (g1) and (g2):

(g1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 7 be made up of two single stranded DNAs shown in sequence in sequence table 13 and sequence 14;

(g2) size of PCR primer that obtains of detecting step (g1), determine whether described storage booklice to be measured is red booklice according to PCR primer as follows: if containing the DNA fragmentation of 199bp in described PCR primer, then described storage booklice to be measured is or candidate is red booklice; Otherwise, then described storage booklice to be measured be not or candidate for red booklice;

H () identifies or whether assistant identification storage to be measured booklice is the method for Pi Shi booklice, comprises the step of following (h1) and (h2):

(h1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 8 be made up of two single stranded DNAs shown in sequence in sequence table 15 and sequence 16;

(h2) size of PCR primer that obtains of detecting step (h1), determine whether described storage booklice to be measured is Pi Shi booklice according to PCR primer as follows: if containing the DNA fragmentation of 251bp in described PCR primer, then described storage booklice to be measured is or candidate is Pi Shi booklice; Otherwise, then described storage booklice to be measured be not or candidate for Pi Shi booklice;

(i) to identify or whether assistant identification storage to be measured booklice is the method for L. mendax, comprise the step of following (i1) and (i2):

(i1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 9 be made up of two single stranded DNAs shown in sequence in sequence table 17 and sequence 18;

(i2) size of PCR primer that obtains of detecting step (i1), determine whether described storage booklice to be measured is L. mendax according to PCR primer as follows: if containing the DNA fragmentation of 185bp in described PCR primer, then described storage booklice to be measured is or candidate is L. mendax; Otherwise, then described storage booklice to be measured be not or candidate for L. mendax;

J () identifies or whether assistant identification storage to be measured booklice is the method for three look booklices, comprises the step of following (j1) and (j2):

(j1) with described to be measured storage booklice genomic dna for template, carry out pcr amplification with the primer pair 10 be made up of two single stranded DNAs shown in sequence in sequence table 19 and sequence 20;

(j2) size of PCR primer that obtains of detecting step (j1), determine whether described storage booklice to be measured is three look booklices according to PCR primer as follows: if containing the DNA fragmentation of 250bp in described PCR primer, then described storage booklice to be measured is or candidate is three look booklices; Otherwise, then described storage booklice to be measured not for or candidate be not three look booklices.

In the above-mentioned methods, in step (a)-step (j), the annealing temperature of carrying out pcr amplification described in all is 53 DEG C.

In the above-mentioned methods, 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 respective PCR reaction system forming each primer pair is 1:1.In the present invention, the volumetric molar concentration of two single stranded DNAs in respective PCR reaction system of each primer pair is 0.2 μM.

In the above-mentioned methods, in step (a), the nucleotides sequence of the DNA fragmentation of described 177bp is classified as sequence 21 in sequence table; In step (b), the nucleotides sequence of the DNA fragmentation of described 188bp is classified as sequence 22 in sequence table; In step (c), the nucleotides sequence of the DNA fragmentation of described 175bp is classified as sequence 23 in sequence table; In step (d), the nucleotides sequence of the DNA fragmentation of described 186bp is classified as sequence 24 in sequence table; In step (e), the nucleotides sequence of the DNA fragmentation of described 128bp is classified as sequence 25 in sequence table; In step (f), the nucleotides sequence of the DNA fragmentation of described 248bp is classified as sequence 26 in sequence table; In step (g), the nucleotides sequence of the DNA fragmentation of described 199bp is classified as sequence 27 in sequence table; In step (h), the nucleotides sequence of the DNA fragmentation of described 251bp is classified as sequence 28 in sequence table; Step (i) in, the nucleotides sequence of the DNA fragmentation of described 185bp is classified as sequence 29 in sequence table; In step (j), the nucleotides sequence of the DNA fragmentation of described 250bp is classified as sequence 30 in sequence table.

On the basis of brown booklice, red booklice, Pi Shi booklice, L. mendax, the common storage in 10 kinds, this world of three look booklices booklice ribosome-RNA(rRNA) second transcribed spacer (ITS2rDNA) gene order, design and filter out 10 kinds of common storage booklice category identification special primers pair, form the identification kit based on special primer method, achieve the quick and precisely qualification of the common storage in world booklice.The test kit based on special primer qualification storage booklice provided by the present invention is utilized to differentiate 10 kinds, world storage booklice, high specificity, simple to operate and repeatable, highly sensitive by force and more stable, be the Perfected process differentiating booklice.

Accompanying drawing explanation

Fig. 1 is the specific detection result of the primer pair LBF/LBR for the identification of l.bostrychophila.Wherein, swimming lane M is DNA Relative molecular weight markers (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: Leposcelis entomophia (Chongqing); 10: L. decolor (Chongqing); 11: L. paeta (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 of the primer pair 21LEnF/208LEnR-2 for the identification of Leposcelis entomophia.Wherein, swimming lane M is DNA Relative molecular weight markers (DNA Marker II); Other swimming lane information are 1: Leposcelis entomophia (Beijing); 2: Leposcelis entomophia (Guangxi); 3: Leposcelis entomophia (Czech); 4: Leposcelis entomophia (Chongqing); 5: l.bostrychophila (Guangxi); 6: L. decolor (Chongqing); 7: L. paeta (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 of the primer pair 164LDeF/319LDeR for the identification of L. decolor.Wherein, swimming lane M is DNA Relative molecular weight markers (DNA Marker II); Other swimming lane information are 1,2: L. decolor (Yunnan); 3,4: L. decolor (Chongqing); 5: Leposcelis entomophia (Chongqing); 6: l.bostrychophila (Guangxi); 7: L. paeta (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 of the primer pair LPa15F/LPa180R for the identification of L. paeta.Wherein, swimming lane M is DNA Relative molecular weight markers (DNA Marker II); Other swimming lane information are 1: L. paeta (Taian Shandong); 2: L. paeta (Cao County, Shandong) 3: L. paeta (Zhejiang); 4: L. paeta (Hubei); 5: L. paeta (U.S.); 6: Leposcelis entomophia (Chongqing); 7: l.bostrychophila (Guangxi); 8: L. decolor (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 nibbling booklice.Wherein, swimming lane M is DNA Relative molecular weight markers (D2000DNA Marker); Other swimming lane information are 1: nibble booklice (Denmark); 2: nibble booklice (Czech); 3: nibble booklice (U.S.); 4: Leposcelis entomophia (Chongqing); 5: l.bostrychophila (Guangxi); 6: L. decolor (Chongqing); 7: L. paeta (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 of the primer pair LBr350F/LBr577R for the identification of dun booklice.Wherein, swimming lane M is DNA Relative molecular weight markers (D2000DNA Marker); Other swimming lane information are 1: dun booklice (Czech); 2: dun booklice (U.S.); 3: Leposcelis entomophia (Chongqing); 4: l.bostrychophila (Guangxi); 5: L. decolor (U.S.); 6: L. paeta (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 of the primer pair 78LRuF-3/276LRuR-3 for the identification of red booklice.Wherein, swimming lane M is DNA Relative molecular weight markers (D2000DNA Marker); Other swimming lane information are 1-3: red booklice (U.S.); 4: Leposcelis entomophia (Chongqing); 5: l.bostrychophila (Guangxi); 6: L. decolor (Chongqing); 7: L. paeta (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 of the primer pair 186LPeF/436LPeR for the identification of Pi Shi booklice.Wherein, swimming lane M is DNA Relative molecular weight markers (D2000DNA Marker); Other swimming lane information are 1,2: Pi Shi booklice (U.S.); 3: Leposcelis entomophia (Chongqing); 4: l.bostrychophila (Guangxi); 5: L. decolor (Chongqing); 6: L. paeta (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 of the primer pair LM60F/LM224R for the identification of L. mendax.Wherein, swimming lane M is DNA Relative molecular weight markers (D2000DNA Marker); Other swimming lane information are 1-3: L. mendax (Jiangsu); 4: Leposcelis entomophia (Chongqing); 5: l.bostrychophila (Guangxi); 6: L. decolor (Chongqing); 7: L. paeta (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 of the primer pair LTri20F/LTri249R for the identification of three look booklices.Wherein, swimming lane M is DNA Relative molecular weight markers (D2000DNA Marker); Other swimming lane information are 1,2: three look booklices (Shandong); 3: Leposcelis entomophia (Chongqing); 4: l.bostrychophila (Guangxi); 5: L. decolor (Chongqing); 6: L. paeta (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 technique result of the primer pair LBF/LBR for the identification of l.bostrychophila.Wherein, swimming lane M is DNA Relative molecular weight markers (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 technique result of the primer pair 21LEnF/208LEnR-2 for the identification of Leposcelis entomophia.Wherein, swimming lane M is DNA Relative molecular weight markers (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 technique result of the primer pair 164LDeF/319LDeR for the identification of L. decolor.Wherein, swimming lane M is DNA Relative molecular weight markers (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 technique result of the primer pair LPa15F/LPa180R for the identification of L. paeta.Wherein, swimming lane M is DNA Relative molecular weight markers (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 technique result for the identification of the primer pair LC170F/LC277R nibbling booklice.Wherein, swimming lane M is DNA Relative molecular weight markers (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 technique result of the primer pair LBr350F/LBr577R for the identification of dun booklice.Wherein, swimming lane M is DNA Relative molecular weight markers (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 technique result of the primer pair 78LRuF-3/276LRuR-3 for the identification of red booklice.Wherein, swimming lane M is DNA Relative molecular weight markers (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 technique result of the primer pair 186LPeF/436LPeR for the identification of Pi Shi booklice.Wherein, swimming lane M is DNA Relative molecular weight markers (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 technique result of the primer pair LM60F/LM224R for the identification of L. mendax.Wherein, swimming lane M is DNA Relative molecular weight markers (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 technique result of the primer pair LTri20F/LTri249R for the identification of three look booklices.Wherein, swimming lane M is DNA Relative molecular weight markers (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

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

10 kinds of storage booklices: l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, 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; Leposcelis entomophia relates to following geographical population: Beijing population, Chongqing population, Guangxi population, Czech population, population in Shandong; L. decolor relates to following geographical population: Yunnan population, Chongqing population, U.S. population; L. paeta 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; Pi Shi booklice relates to following geographical population: U.S. population; L. mendax relates to following geographical population: Jiangsu population; Three look booklices relate to following geographical population: population in Shandong.Above 10 kinds of storage booklices are documented in " Zhao Shuo. the common storage in the world based on 16S rDNA booklice Molecular Identification, China Agricultural University's master thesis, 2010 " in a literary composition.

Embodiment 1, the Design and synthesis of special primer for 10 kinds of storage booklices

One, the acquisition of 10 kinds of storage booklice ITS2rDNA sequences

Experiment material: relate to 10 kinds of storage booklices, be specially l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, 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; Leposcelis entomophia relates to following geographical population: Beijing population, Chongqing population, Guangxi population, Czech population, population in Shandong; L. decolor relates to following geographical population: Yunnan population, Chongqing population, U.S. population; L. paeta 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; Pi Shi booklice relates to following geographical population: U.S. population; L. mendax relates to following geographical population: 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 the STb gene of the whole polypide of single head booklice adult.Reference reagent box specification sheets carries out, specific as follows:

(1) get booklice adult sample, first kill with 75% alcohol, after dehydrated alcohol rinsing, be placed on filter paper and dry.

(2) the individuality after process is put into 1.5ml centrifuge tube, add 10 μ l GA liquid (subsidiary with test kit), with grinding rod, polypide is pulverized.

(3) 170 μ l GA liquid are joined in centrifuge tube, the centrifugal several seconds.

(4) join in centrifuge tube by unsettled for 10 μ l Proteinase K Solution (subsidiary with test kit), 56 DEG C of water-baths 1 hour, centrifugal several seconds.

By 200 μ l GB liquid (subsidiary with test kit) and 1 μ l Carrier RNA(subsidiary with test kit) join in centrifuge tube, 70 DEG C of water-baths 10 minutes, solution becomes clarification, centrifugal 30 seconds.

(6) add the dehydrated alcohol of 200 μ l precoolings, vibrate 15 seconds, centrifugal 1 minute, room temperature placed 5 minutes, all proceeded in adsorption column CR2, centrifugal 30 seconds.

(7) join in adsorption column CR2 by 500 μ l GD liquid (subsidiary with test kit), centrifugal 30 seconds of 12000rpm/s, outwells waste liquid.

(8) 700 μ l rinsing liquid PW(are subsidiary with test kit) join in adsorption column CR2, centrifugal 30 seconds of 12000rpm/s, outwells waste liquid.

(9) join in adsorption column CR2 by 500 μ l rinsing liquid PW, centrifugal 30 seconds of 12000rpm/s, outwells waste liquid.

(10), by adsorption column CR2 with 12000rpm/s centrifugal 2 minutes, open adsorption column CR2 lid, room temperature places 5 minutes.

(11) adsorption column CR2 is put in clean centrifuge tube, subsidiary with test kit to unsettled dropping 30 μ l elution buffer TB(in the middle of adsorption column CR2), place after 2-5 minute, with 12000rpm/s centrifugal 2 minutes.

(12) add in adsorption column CR2 again by the centrifugal solution obtained, room temperature places 2 minutes, centrifugal 2 minutes of 12000rpm/s.

(13) booklice genomic dna is eluted in centrifuge tube, and-20 DEG C save backup.

(2) ITS2rDNA sequence pcr amplification

Utilize the ITS2rDNA sequence of primer pair amplifies booklice be made up of 5 '-TGTGAACTGCAGGACACATG-3 ' and 5 '-GTCTTGTCTGATCTGAG-3 ', amplified production length not of the same race is not about 290bp ~ 650bp not etc.It is 50 μ L, wherein Premix EX Taq that reaction system: PCR reacts cumulative volume ^tMthe precious biotechnology Dalian company limited of 25 μ L(), 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 μMs.Amplification condition is: thermocycling program is 94 DEG C of 4min, then carries out 30 circulations (94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 1min), finally terminates after 72 DEG C of reaction 10min.

(3) acquisition of ITS2rDNA sequence

Agarose gel electrophoresis is adopted to detect the quality of PCR primer; And adopt the gel of Beijing Tian Gen biochemical technology company limited to reclaim test kit recovery product; Be connected to by recovery product on pMD18-T carrier (precious biotechnology (Dalian) company limited), transformation of E. coli DH5 α, ammonia benzyl resistance LB culture medium flat plate carries out blue hickie screening; 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 DEG C of sex change 5min, 94 DEG C of sex change 1min, 58 DEG C of renaturation 1min, 72 DEG C extend 2min; 31 circulations; 72 DEG C fully extend 10min.Amplified production adopts the agarose gel electrophoresis of 2% to detect, and selects the clone of wherein clip size correct (inserting amplified fragments) to check order.Positive colony is sent company (Beijing AudioCodes prosperous biotechnology limited liability company) two-way order-checking, order-checking adopts carrier universal primer.Sequence will be recorded to compare splicing.

Two, for the Design and synthesis of the special primer of 10 kinds of storage booklices

(1) design of primers

After 10 kinds of storage booklice (each kind comprises multiple geographical population) ITS2rDNA sequences step one obtained carry out DNAMAN compare of analysis, employing Primer Premier5.0 software designs the special primer for 10 kinds of storage booklices respectively.

(2) primer assessment

Adopt Oligo7 software to assess primer pair indices, quantum evaluation and evaluation criteria as follows:

The Delta G value absolute value of upstream and downstream primer is no more than 9; What can form primer dimer or hairpin structure does not exceed 3 in conjunction with base pair; GC% content controls between 30%-70%, and does not differ too large between upstream and downstream; Mistake efficiency of initiation is no more than 100; Finally, provide different primers to the suitableeest annealing temperature in conjunction with Oligo7 software, be set as 53 DEG C by same for annealing temperature.

According to the above, design and synthesize the special primer following (table 1) obtained for 10 kinds of storage booklices:

The common 10 kinds of storage booklice special primers in table 1 world

Embodiment 2, specific detection for 10 kinds of storage booklice ITS2rDNA sequence specific primers

One, experiment material

Relate to 10 kinds of storage booklices, be specially l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, 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; Leposcelis entomophia relates to following geographical population: Beijing population, Chongqing population, Guangxi population, Czech population, population in Shandong; L. decolor relates to following geographical population: Yunnan population, Chongqing population, U.S. population; L. paeta 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; Pi Shi booklice relates to following geographical population: U.S. population; L. mendax relates to following geographical population: 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 the STb gene of the whole polypide of single head booklice to be measured adult.Reference reagent box specification sheets carries out, and concrete operations are with embodiment 1.With gained genomic dna for template, adopt and carry out substance pcr amplification respectively for each primer pairs in 10 kinds of storages booklice ITS2rDNA sequence specific primers (table 1) designed by embodiment 1.Thus the specificity for 10 kinds of storages booklice ITS2rDNA sequence specific primers (table 1) detected designed by embodiment 1.

1, it is 50 μ L, wherein Premix EX Taq that amplification system: PCR reacts cumulative volume ^tMthe precious biotechnology Dalian company limited of 25 μ L(), ddH ₂o20 μ L, template 3 μ L(30ng/ μ L), upstream primer 1 μ L(concentration is 10 μMs), downstream primer 1 μ L(concentration is 10 μMs).Upstream primer and the final concentration of downstream primer in PCR reaction system are 0.2 μM.

The negative control replacing template with water is set simultaneously.

2, amplification condition: 94 DEG C of denaturation 4min

3, after reaction terminates, get 5 μ L PCR reaction product, at the sepharose of 2%, electrophoresis detection in the TAE electrophoretic buffer of 1 times, after ethidium bromide (EB) dyeing, observes and imaging (Gel LogicalPro212) in gel imaging system.Meanwhile, amplified production directly send company (the prosperous bio tech ltd of AudioCodes) two-way order-checking, and sequencing result, by sequence alignment, determines that special primer institute amplified fragments belongs to designed kind further.

Three, experimental result

1, l.bostrychophila

The primer pair LBF/LBR(sequence 1 utilizing designed, designed to obtain and sequence 2) detect the specificity of this primer pair under conventional PCR method, in experiment material except the l.bostrychophila that qualification is distinguished, also by Leposcelis entomophia, L. decolor, L. paeta, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax carry out the reaction of special primer Standard PCR together with the common storage booklice of three look booklices these 9 kinds.Primer pair LBF/LBR standard PCR amplification product gel electrophoresis result as shown in Figure 1, except l.bostrychophila has specific amplification, a specific amplification fragment (in order-checking is as sequence table shown in sequence 21) is had outward at 177bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, the qualification l.bostrychophila that this primer can only be special is described, and invalid to other common storage booklices.

2, Leposcelis entomophia

The primer pair 21LEnF/208LEnR-2(sequence 3 utilizing designed, designed to obtain and sequence 4) detect the specificity of this primer pair under conventional PCR method, in experiment material except the Leposcelis entomophia that qualification is distinguished, also by l.bostrychophila, L. decolor, L. paeta, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax carry out the reaction of special primer Standard PCR together with the common storage booklice of three look booklices these 9 kinds.Primer pair 21LEnF/208LEnR-2 standard PCR amplification product gel electrophoresis result as shown in Figure 2, except Leposcelis entomophia has specific amplification, a specific amplification fragment (in order-checking is as sequence table shown in sequence 22) is had outward at 188bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, the qualification Leposcelis entomophia that this primer can only be special is described, and invalid to other common storage booklices.

3, L. decolor

The primer pair 164LDeF/319LDeR(sequence 5 utilizing designed, designed to obtain and sequence 6) detect the specificity of this primer pair under conventional PCR method, in experiment material except the L. decolor that qualification is distinguished, also by l.bostrychophila, Leposcelis entomophia, L. paeta, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax carry out the reaction of special primer Standard PCR together with the common storage booklice of three look booklices these 9 kinds.Primer pair 164LDeF/319LDeR standard PCR amplification product gel electrophoresis result as shown in Figure 3, except L. decolor has specific amplification, a specific amplification fragment (in order-checking is as sequence table shown in sequence 23) is had outward at 175bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, the qualification L. decolor that this primer can only be special is described, and invalid to other common storage booklices.

4, L. paeta

The primer pair LPa15F/LPa180R(sequence 7 utilizing designed, designed to obtain and sequence 8) detect the specificity of this primer pair under conventional PCR method, in experiment material except the L. paeta that qualification is distinguished, also by l.bostrychophila, Leposcelis entomophia, L. decolor, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax carry out the reaction of special primer Standard PCR together with the common storage booklice of three look booklices these 9 kinds.Primer pair LPa15F/LPa180R standard PCR amplification product gel electrophoresis result as shown in Figure 4, except L. paeta has specific amplification, a specific amplification fragment (in order-checking is as sequence table shown in sequence 24) is had outward at 186bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, the qualification L. paeta that this primer can only be special is described, and invalid to other common storage booklices.

5, booklice is nibbled

The primer pair LC170F/LC277R(sequence 9 utilizing designed, designed to obtain and sequence 10) detect the specificity of this primer pair under conventional PCR method, that distinguishes except qualification in experiment material nibbles except booklice, also l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, dun booklice, red booklice, Pi Shi booklice, L. mendax is carried out the reaction of special primer Standard PCR together with the common storage booklice of three look booklices these 9 kinds.Primer pair LC170F/LC277R standard PCR amplification product gel electrophoresis result as shown in Figure 5, specific amplification is had except nibbling booklice, a specific amplification fragment (in order-checking is as sequence table shown in sequence 25) is had outward at 128bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, illustrate that this primer special qualification can only nibble booklice, and invalid to other common storage booklices.

6, dun booklice

The primer pair LBr350F/LBr577R(sequence 11 utilizing designed, designed to obtain and sequence 12) detect the specificity of this primer pair under conventional PCR method, in experiment material except the dun booklice that qualification is distinguished, also by l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, nibble booklice, red booklice, Pi Shi booklice, L. mendax carry out the reaction of special primer Standard PCR together with the common storage booklice of three look booklices these 9 kinds.Primer pair LBr350F/LBr577R standard PCR amplification product gel electrophoresis result as shown in Figure 6, except dun booklice has specific amplification, a specific amplification fragment (in order-checking is as sequence table shown in sequence 26) is had outward at 248bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, the dun booklice of qualification that this primer can only be special is described, and invalid to other common storage booklices.

7, red booklice

The primer pair 78LRuF-3/276LRuR-3(sequence 13 utilizing designed, designed to obtain and sequence 14) detect the specificity of this primer pair under conventional PCR method, in experiment material except the red booklice that qualification is distinguished, also by l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, nibble booklice, dun booklice, Pi Shi booklice, L. mendax carry out the reaction of special primer Standard PCR together with the common storage booklice of three look booklices these 9 kinds.Primer pair 78LRuF-3/276LRuR-3 standard PCR amplification product gel electrophoresis result as shown in Figure 7, except red booklice has specific amplification, a specific amplification fragment (in order-checking is as sequence table shown in sequence 27) is had outward at 199bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, the red booklice of qualification that this primer can only be special is described, and invalid to other common storage booklices.

8, Pi Shi booklice

The primer pair 186LPeF/436LPeR(sequence 15 utilizing designed, designed to obtain and sequence 16) detect the specificity of this primer pair under conventional PCR method, in experiment material except the Pi Shi booklice that qualification is distinguished, also by l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, nibble booklice, dun booklice, red booklice, L. mendax carry out the reaction of special primer Standard PCR together with the common storage booklice of three look booklices these 9 kinds.Primer pair 186LPeF/436LPeR standard PCR amplification product gel electrophoresis result as shown in Figure 8, except Pi Shi booklice has specific amplification, a specific amplification fragment (in order-checking is as sequence table shown in sequence 28) is had outward at 251bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, the qualification Pi Shi booklice that this primer can only be special is described, and invalid to other common storage booklices.

9, L. mendax

The primer pair LM60F/LM224R(sequence 17 utilizing designed, designed to obtain and sequence 18) detect the specificity of this primer pair under conventional PCR method, in experiment material except the L. mendax that qualification is distinguished, also by l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, nibble booklice, dun booklice, red booklice, Pi Shi booklice carry out the reaction of special primer Standard PCR together with the common storage booklice of three look booklices these 9 kinds.Primer pair LM60F/LM224R standard PCR amplification product gel electrophoresis result as shown in Figure 9, except L. mendax has specific amplification, a specific amplification fragment (in order-checking is as sequence table shown in sequence 29) is had outward at 185bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, the qualification L. mendax that this primer can only be special is described, and invalid to other common storage booklices.

10, three look booklices

The primer pair LTri20F/LTri249R(sequence 19 utilizing designed, designed to obtain and sequence 20) detect the specificity of this primer pair under conventional PCR method, in experiment material except the three look booklices that qualification is distinguished, also by l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, nibble together with booklice, dun booklice, red booklice, the common storage booklice of Pi Shi booklice and L. mendax these 9 kinds and carry out the reaction of special primer Standard PCR.Primer pair LTri20F/LTri249R standard PCR amplification product gel electrophoresis result as shown in Figure 10, except three look booklices have specific amplification, a specific amplification fragment (in order-checking is as sequence table shown in sequence 30) is had outward at 250bp place, other 9 kinds storage booklices and water negative control, all there is no amplified reaction, namely in product without specific target fragment, the qualification three look booklice that this primer can only be special is described, and invalid to other common storage booklices.

Comprehensive above experimental result, has higher specificity for 10 kinds of booklices ITS2rDNA sequence specific primers (table 1) of storing in a warehouse designed by visible embodiment 1.

Embodiment 3, sensitivity technique for 10 kinds of storage booklice ITS2rDNA sequence specific primers

One, experiment material

Relate to 10 kinds of storage booklices, be specially l.bostrychophila, Leposcelis entomophia, L. decolor, L. paeta, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax and three look booklices.Wherein, the geographical population of l.bostrychophila are specially Guangxi population; The geographical population of Leposcelis entomophia are specially Chongqing population; The geographical population of L. decolor are specially Chongqing population; The geographical population of L. paeta are specially Zhejiang population; The geographical population of nibbling booklice are specially 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 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 the STb gene of the whole polypide of single head booklice to be measured adult.Concrete operations are see test kit specification sheets.With gained genomic dna for template, adopt and carry out substance pcr amplification respectively for each primer pairs in 10 kinds of storages booklice ITS2rDNA sequence specific primers (table 1) designed by embodiment 1.Thus the sensitivity for 10 kinds of storages booklice ITS2rDNA sequence specific primers (table 1) detected designed by embodiment 1.

1, it is 50 μ L, wherein Premix EX Taq that amplification system: PCR reacts cumulative volume ^tMthe precious biotechnology Dalian company limited of 25 μ L(), ddH ₂o20 μ L, template 3 μ L, upstream primer 1 μ L(concentration is 10 μMs), downstream primer 1 μ L(concentration is 10 μMs).Upstream primer and the final concentration of downstream primer in PCR reaction system are 0.2 μM.Wherein, the consumption of booklice genomic dna to be measured in reaction system as template arranges gradient and is: 40ng, 20ng, 10ng, 1ng, 0.1ng, and 0.01 and 0.001ng.

The negative control replacing template with water is set simultaneously.

2, amplification condition: 94 DEG C of denaturation 4min

Three, experimental result

1, l.bostrychophila

The primer pair LBF/LBR(sequence 1 utilizing designed, designed to obtain and sequence 2) detect the sensitivity of this primer pair under conventional PCR method.The l.bostrychophila Standard PCR product gel electrophoresis result of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) as shown in figure 11.When template concentrations is 01ng, do not have the amplified band of specific product, when template concentrations is 1ng, amplified band is fainter, only has the target fragment of trace to occur, and when template concentrations is greater than 10ng, all specific amplified can occur.

2, Leposcelis entomophia

The primer pair 21LEnF/208LEnR-2(sequence 3 utilizing designed, designed to obtain and sequence 4) detect the sensitivity of this primer pair under conventional PCR method.The Leposcelis entomophia Standard PCR product gel electrophoresis result of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) as shown in figure 12.When template concentrations is 0.1ng, amplified band is fainter, only has the target fragment of trace to occur, and when template concentrations is greater than 1ng, all specific amplified can occur.

3, L. decolor

The primer pair 164LDeF/319LDeR(sequence 5 utilizing designed, designed to obtain and sequence 6) detect the sensitivity of this primer pair under conventional PCR method.The L. decolor Standard PCR product gel 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, amplified band is atomic weak, and when template concentrations is greater than 1ng, all specific amplified can occur.

4, L. paeta

The primer pair LPa15F/LPa180R(sequence 7 utilizing designed, designed to obtain and sequence 8) detect the sensitivity of this primer pair under conventional PCR method.The L. paeta Standard PCR product gel electrophoresis result of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) as shown in figure 14.When template concentrations is 0.1ng, amplified band is atomic weak, and when template concentrations is greater than 1ng, all specific amplified can occur.

5, booklice is nibbled

The primer pair LC170F/LC277R(sequence 9 utilizing designed, designed to obtain and sequence 10) detect the sensitivity of this primer pair under conventional PCR method.Different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) nibble booklice Standard PCR product gel electrophoresis result as shown in figure 15.When template concentrations is 1ng, amplified band is fainter, only has the target fragment of trace to occur, and when template concentrations is greater than 10ng, all specific amplified can occur.

6, dun booklice

The primer pair LBr350F/LBr577R(sequence 11 utilizing designed, designed to obtain and sequence 12) detect the sensitivity of this primer pair under conventional PCR method.The dun booklice Standard PCR product gel 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, amplified band is atomic weak, and when template concentrations is greater than 0.1ng, all specific amplified can occur.

7, red booklice

The primer pair 78LRuF-3/276LRuR-3(sequence 13 utilizing designed, designed to obtain and sequence 14) detect the sensitivity of this primer pair under conventional PCR method.The red booklice Standard PCR product gel 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, amplified band is atomic weak, and when template concentrations is greater than 1ng, all specific amplified can occur.

8, Pi Shi booklice

The primer pair 186LPeF/436LPeR(sequence 15 utilizing designed, designed to obtain and sequence 16) detect the sensitivity of this primer pair under conventional PCR method.The Pi Shi booklice Standard PCR product gel electrophoresis result of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) as shown in figure 18.When template concentrations is 0.1ng, amplified band is fainter, and when template concentrations is greater than 1ng, all specific amplified can occur.

9, L. mendax

The primer pair LM60F/LM224R(sequence 17 utilizing designed, designed to obtain and sequence 18) detect the sensitivity of this primer pair under conventional PCR method.The L. mendax Standard PCR product gel electrophoresis result of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) as shown in figure 19.When template concentrations is 0.1ng, amplified band is fainter, and when template concentrations is greater than 1ng, all specific amplified can occur.

10, three look booklices

The primer pair LTri20F/LTri249R(sequence 19 utilizing designed, designed to obtain and sequence 20) detect the sensitivity of this primer pair under conventional PCR method.Three look booklice Standard PCR product gel electrophoresis result of different concns (40ng, 20ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng) as shown in figure 20.When template concentrations is 0.1ng, amplified band is atomic weak, and when template concentrations is greater than 1ng, all specific amplified can occur.

Comprehensive above experimental result, has higher sensitivity for 10 kinds of booklices ITS2rDNA sequence specific primers (table 1) of storing in a warehouse designed by visible embodiment 1.

Embodiment 4, qualification application for 10 kinds of storage booklice ITS2rDNA sequence specific primers

What adopt embodiment 1 to design detects the present inventor for 10 kinds of storage booklice ITS2rDNA sequence specific primers and to adopt 5 unknown species booklice samples in Yantai, Shandong grain depot in 2011.Adopt the micro-example genome DNA extracting reagent kit of TIANGEN Biotech (Beijing) Co., Ltd., extract the STb gene of whole polypide; With booklice genomic dna for template, use and carry out substance pcr amplification respectively for special primer (table 1 see embodiment 1) not of the same race; Employing agarose gel electrophoresis detects.Relating operation detects embodiment 1-3.

Result shows, wherein 4 unknown species booklice samples to use the primer pair 21LEnF/208LEnR-2 being specific to Leposcelis entomophia to carry out to occur 188bp(in the product of pcr amplification in order-checking is as sequence table shown in sequence 22) bright band, 1 sample to use the primer pair 164LDeF/319LDeR being specific to L. decolor to carry out to occur 175bp(in the product of pcr amplification in order-checking is as sequence table shown in sequence 23) bright band.Apart from the above description, these 5 unknown species booklice samples all do not obtain corresponding object band when other special primers carry out pcr amplification in the table 1 using embodiment 1.Above result shows, by utilizing Auele Specific Primer shown in the table 1 of embodiment 1 to detect, in 5 unknown species booklice samples, 4 is Leposcelis entomophia, and 1 is L. decolor.

Meanwhile, the present inventor have employed existing Morphological Identification method.China Agricultural University's Plant Quarantine and the biological invasion laboratory Li Zhi Red Sect of Lamaism award and are engaged in more than 20 years of booklice morphological research, possesses abundant Morphological Identification experience, through the Li Zhi Red Sect of Lamaism award to the qualification of 5 unknown species booklice sample morphology (authentication method see " Li Zhihong. Chinese lice nibbles the sort research of genus, Beijing Agricultural University's master thesis, 1994 " literary composition), result confirms, in 5 unknown species booklice samples, carrying out through Auele Specific Primer shown in the above-mentioned table 1 utilizing embodiment 14 booklices that Testing and appraisal is Leposcelis entomophia is Leposcelis entomophia really, carrying out through Auele Specific Primer shown in the above-mentioned table 1 utilizing embodiment 11 booklice that Testing and appraisal is L. decolor is L. decolor really.As can be seen here, the present invention utilizes Auele Specific Primer shown in the table 1 of embodiment 1 booklice to be measured to be carried out to the qualification of planting, reliable results.

Claims

1. for the identification of or assistant identification storage booklice a test kit, comprise following 10 primer pairs:

(10) primer pair 10 be made up of two single stranded DNAs shown in sequence in sequence table 19 and sequence 20;

Described storage booklice be following at least one: Leposcelis entomophia, l.bostrychophila, L. decolor, L. paeta, nibble booklice, dun booklice, red booklice, Pi Shi booklice, L. mendax, three look booklices.

2. test kit according to claim 1, is characterized in that: the mole number forming two single stranded DNAs of each primer pair in described test kit is equal.

3. the preparation method of test kit described in claim 1 or 2, comprise the steps:, by after in the described test kit of composition, each single stranded DNA of each primer pair is individually packed, to be packaged in same reagent box with at least one in following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.

4. the method for qualification or assistant identification storage booklice is any one in following (a)-(j):

I () identifies or whether assistant identification storage to be measured booklice is the method for L. mendax, comprises the step of following (i1) and (i2):

5. method according to claim 4, is characterized in that: in step (a)-step (j), and the annealing temperature of carrying out pcr amplification described in all is 53 DEG C.

6. the method according to claim 4 or 5, 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 respective PCR reaction system forming each primer pair is 1:1.

7. method according to claim 4, is characterized in that: in step (a), and the nucleotides sequence of the DNA fragmentation of described 177bp is classified as sequence 21 in sequence table; In step (b), the nucleotides sequence of the DNA fragmentation of described 188bp is classified as sequence 22 in sequence table; In step (c), the nucleotides sequence of the DNA fragmentation of described 175bp is classified as sequence 23 in sequence table; In step (d), the nucleotides sequence of the DNA fragmentation of described 186bp is classified as sequence 24 in sequence table; In step (e), the nucleotides sequence of the DNA fragmentation of described 128bp is classified as sequence 25 in sequence table; In step (f), the nucleotides sequence of the DNA fragmentation of described 248bp is classified as sequence 26 in sequence table; In step (g), the nucleotides sequence of the DNA fragmentation of described 199bp is classified as sequence 27 in sequence table; In step (h), the nucleotides sequence of the DNA fragmentation of described 251bp is classified as sequence 28 in sequence table; In step (i), the nucleotides sequence of the DNA fragmentation of described 185bp is classified as sequence 29 in sequence table; In step (j), the nucleotides sequence of the DNA fragmentation of described 250bp is classified as sequence 30 in sequence table.