CN103952463A - Specific primer based kit for identifying five common cryptoleste stored grain insects - Google Patents

Specific primer based kit for identifying five common cryptoleste stored grain insects Download PDF

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CN103952463A
CN103952463A CN201310298148.3A CN201310298148A CN103952463A CN 103952463 A CN103952463 A CN 103952463A CN 201310298148 A CN201310298148 A CN 201310298148A CN 103952463 A CN103952463 A CN 103952463A
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flat ostomatid
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CN103952463B (en
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李志红
王一椒
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China Agricultural University
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Abstract

The invention discloses a specific primer based kit for identifying five common cryptoleste stored grain insects. The kit provided by the invention comprises at least one pair of the following 5 pairs of primers: a primer pair 1 (sequence 1 and sequence 2), a primer pair 2 (sequence 3 and sequence 4), a primer pair 3 (sequence 5 and sequence 6), a primer pair 4 (sequence 7 and sequence 8) and a primer pair 5 (sequence 9 and sequence 10). Experiments show that compared with a traditional morphological identification method, the cryptoleste stored grain insect species identification method using the kit provided by the invention does not need morphological taxonomy base and realizes the identification of non-adult state; andcompared with other molecular biological identification methods, the method simplifies the molecular detection steps for identification of common cryptoleste stored grain insects, has the characteristics of simple operation, short time consumption, high sensitivity, stability and visual results. The invention lays a foundation for further research on identification of sibling species of cryptoleste.

Description

A kind of test kit of identifying 5 kinds of flat ostomatids of common grain storage 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 kits of 5 kinds of flat ostomatids of common grain storage of assistant identification.
Background technology
Flat ostomatid is under the jurisdiction of Coleoptera (Coleoptera), flat Ostomidae (Laemophloeidae), flat ostomatid genus (Cryptolestes), this genus is described altogether and is had more than 20 to plant both at home and abroad, the flat ostomatid that wherein endangers grain storage is about 10 kinds, and worldwide distributing is 5 kinds of rusty grain beetle, the flat ostomatid in long angle, the flat ostomatid of Turkey and the flat ostomatids of Kai Pu etc. comparatively widely.
Coleopteron quantity maximum, most species, quarantine meaning in grain storage pest is the most outstanding, flat ostomatid is a monoid in Coleoptera grain storage pest with Important Economic meaning, its larva and adult can endanger multiple agricultural-food and its converted productss such as cereal, beans, oil plant, during a large amount of generation, can cause comparatively serious financial loss, mainly by the trade of agricultural byproducts, carry out cross-border propagation.In recent years, China's flat ostomatid batch showed increased that inspection and quarantine department is intercepted and captured of passing in and out.Define the flat ostomatid kind of grain storage that intercept and capture and grain depot occur, quarantine decision-making and pest management tool are of great significance.The reasons such as formalness is very similar due to flat ostomatid individuality small (body long about 2mm) and between planting, traditional Morphological Identification technology only can be identified the adult with obvious characteristic form, the typoiogical classification of appraiser is learned a skill and had relatively high expectations, can not meet demand quick, the flat ostomatid kind of precise Identification.
The molecular biological variety identification method growing up in recent years mainly comprises isozyme electrophoretic techique, restriction fragment length polymorphism analysis (RFLP) technology, the analysis of random amplification DNA polymorphism (RAPD) technology, nucleic acid sequence analysis technology and specific primer PCR technology.Restriction fragment length polymorphism round pcr result is comparatively accurate, but it is larger to screen the workload of required restriction enzyme kind.So far, have no the report for the Molecular Identification technology of rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid, the flat ostomatid of Turkey and these 5 kinds of flat ostomatids of the common grain storage in the world of the flat ostomatid of Kai Pu.
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 the flat ostomatid of assistant identification grain storage.
Provided by the present invention for the identification of or the test kit of the flat ostomatid of assistant identification grain storage, can comprise at least one pair of in following 5 primer pairs:
(1) primer pair 1 that two single stranded DNAs shown in sequence 1 and sequence 2 form in sequence table;
(2) primer pair 2 that two single stranded DNAs shown in sequence 3 and sequence 4 form in sequence table;
(3) primer pair 3 that two single stranded DNAs shown in sequence 5 and sequence 6 form in sequence table;
(4) primer pair 4 that two single stranded DNAs shown in sequence 7 and sequence 8 form in sequence table;
(5) primer pair 5 that two single stranded DNAs shown in sequence 9 and sequence 10 form in sequence table.
Wherein, sequence 1 is comprised of 21 Nucleotide; Sequence 2 is comprised of 20 Nucleotide; Sequence 3 is comprised of 21 Nucleotide; Sequence 4 is comprised of 23 Nucleotide; Sequence 5 is comprised of 22 Nucleotide; Sequence 6 is comprised of 22 Nucleotide; Sequence 7 is comprised of 21 Nucleotide; Sequence 8 is comprised of 22 Nucleotide; Sequence 9 is comprised of 19 Nucleotide; Sequence 10 is comprised of 22 Nucleotide.
In described test kit, the mole number that forms two single stranded DNAs of each primer pair equates.
The flat ostomatid of described grain storage can be as lower at least one: rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid, the flat ostomatid of Turkey and the flat ostomatid of Kai Pu.
In described test kit, described primer pair 1 is specific to rusty grain beetle; Described primer pair 2 is specific to the flat ostomatid in long angle; Described primer pair 3 is specific to micro-flat ostomatid; Described primer pair 4 is specific to the flat ostomatid of Turkey; Described primer pair 5 is specific to out general flat ostomatid.
A further object of the present invention is to provide the preparation method of described test kit.
The preparation method of described test kit, each single stranded DNA that specifically can comprise the steps: to form each primer pair in described test kit is respectively separately after packing, is packaged in same reagent box: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP with at least one in following substances.
Also object of the present invention is to provide a kind of evaluation or the method for the flat ostomatid of assistant identification grain storage.
The method of evaluation provided by the present invention or the flat ostomatid of assistant identification grain storage, can be following (a)-any in (e):
(a) identify or whether the flat ostomatid of assistant identification grain storage to be measured is the method for rusty grain beetle, comprise following (a1) and step (a2):
(a1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 1 that two single stranded DNAs shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification;
(a2) size of the PCR product that detecting step (a1) obtains, according to PCR product, determine whether the flat ostomatid of described grain storage to be measured is rusty grain beetle as follows: if the DNA fragmentation that contains 158bp in described PCR product, the flat ostomatid of described grain storage to be measured is or candidate is rusty grain beetle; Otherwise, the flat ostomatid of described grain storage to be measured be not or candidate for rusty grain beetle;
(b) identify or whether the flat ostomatid of assistant identification grain storage to be measured is the method for the flat ostomatid in long angle, comprise following (b1) and step (b2):
(b1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 2 that two single stranded DNAs shown in sequence 3 and sequence 4 form in sequence table to carry out pcr amplification;
(b2) size of the PCR product that detecting step (b1) obtains, according to PCR product, determine that whether the flat ostomatid of described grain storage to be measured is the flat ostomatid in long angle as follows: if the DNA fragmentation that contains 371bp in described PCR product, the flat ostomatid of described grain storage to be measured be or candidate for growing the flat ostomatid in angle; Otherwise the flat ostomatid of described grain storage to be measured is not or candidate is not the flat ostomatid in long angle;
(c) identify or whether the flat ostomatid of assistant identification grain storage to be measured is the method for micro-flat ostomatid, comprise following (c1) and step (c2):
(c1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 3 that two single stranded DNAs shown in sequence 5 and sequence 6 form in sequence table to carry out pcr amplification;
(c2) size of the PCR product that detecting step (c1) obtains, according to PCR product, determine whether the flat ostomatid of described grain storage to be measured is micro-flat ostomatid as follows: if the DNA fragmentation that contains 585bp in described PCR product, the flat ostomatid of described grain storage to be measured is or candidate is micro-flat ostomatid; Otherwise, the flat ostomatid of described grain storage to be measured be not or candidate for micro-flat ostomatid;
(d) identify or whether the flat ostomatid of assistant identification grain storage to be measured is the method for the flat ostomatid of Turkey, comprise following (d1) and step (d2):
(d1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 4 that two single stranded DNAs shown in sequence 7 and sequence 8 form in sequence table to carry out pcr amplification;
(d2) size of the PCR product that detecting step (d1) obtains, according to PCR product, determine whether the flat ostomatid of described grain storage to be measured is the flat ostomatid of Turkey as follows: if the DNA fragmentation that contains 355bp in described PCR product, the flat ostomatid of described grain storage to be measured is or candidate is the flat ostomatid of Turkey; Otherwise the flat ostomatid of described grain storage to be measured is not or the flat ostomatid of candidate Bu Wei Turkey;
(e) identify or whether the flat ostomatid of assistant identification grain storage to be measured is out the method for general flat ostomatid, comprise following (e1) and step (e2):
(e1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 5 that two single stranded DNAs shown in sequence 9 and sequence 10 form in sequence table to carry out pcr amplification;
(e2) size of the PCR product that detecting step (e1) obtains, according to PCR product, determine whether the flat ostomatid of described grain storage to be measured is out general flat ostomatid as follows: if the DNA fragmentation that contains 289bp in described PCR product, the flat ostomatid of described grain storage to be measured for or candidate for opening general flat ostomatid; Otherwise, the flat ostomatid of described grain storage to be measured not for or candidate not for opening general flat ostomatid.
In aforesaid method, in step (a)-step (e), the annealing temperature of carrying out pcr amplification described in all is 50 ℃.
In aforesaid method, described primer pair 1, described primer pair 2, described primer pair 3, described primer pair 4 and described primer pair 5, the mol ratio of two single stranded DNAs that form each primer pair in PCR reaction system is separately 1:1.In the present invention, two of each primer pair single stranded DNAs volumetric molar concentration in PCR reaction system is separately 0.2 μ M.
In aforesaid method, in step (a), the nucleotide sequence of the DNA fragmentation of described 158bp is specially sequence 11 in sequence table; In step (b), the nucleotide sequence of the DNA fragmentation of described 371bp is specially sequence 12 in sequence table; In step (c), the nucleotide sequence of the DNA fragmentation of described 585bp is specially sequence 13 in sequence table; In step (d), the nucleotide sequence of the DNA fragmentation of described 355bp is specially sequence 14 in sequence table; In step (e), the nucleotide sequence of the DNA fragmentation of described 289bp is specially sequence 15 in sequence table.
In the method for above-mentioned arbitrary described evaluation or the flat ostomatid of assistant identification grain storage, the flat ostomatid that the flat ostomatid of described grain storage to be measured belongs to flat Ostomidae (Laemophloeidae) belongs to (Cryptolestes), is specially at least one in rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid, the flat ostomatid of Turkey and the flat ostomatid of Kai Pu.
The present invention is directed to rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid, the flat ostomatid of Turkey and these 5 kinds of flat ostomatids of the common grain storage in the world of the flat ostomatid of Kai Pu, based on mtDNA COI gene order, design respectively special primer, can directly differentiate above 5 kinds of flat ostomatids of grain storage.Compare with traditional form authentication method, the method does not need typoiogical classification basis and has realized the evaluation to non-adult form; And compare with other molecular biological variety identification method (as restriction fragment length polymorphism PCR, nucleic acid sequence analysis), the method has been simplified the Molecular Detection step of identifying the flat ostomatid of common grain storage, have easy and simple to handle, consuming time shorter, highly sensitive and more stable, the features such as visual result, the research that belongs to sibling species for the flat ostomatid of further evaluation is laid a good foundation.
Accompanying drawing explanation
Fig. 1 is the specific detection result for the identification of the primer pair CF84F21/CF222R20 of rusty grain beetle.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker II); Other swimming lane information are 1: open general flat ostomatid (Prague, CZE); 2: the flat ostomatid of Turkey (Prague, CZE); 3: the flat ostomatid of Turkey (Shangqiu, Henan); 4: rusty grain beetle (Prague, CZE); 5: rusty grain beetle (Kan.) 6: rusty grain beetle (Jining of Shandong Province); 7: rusty grain beetle (Zhanjiang); 8,9: micro-flat ostomatid (Luxi, Yunnan); 10: the long flat ostomatid in angle (Prague, CZE); 11: the long flat ostomatid in angle (Kan.); 12,13: the long flat ostomatid in angle (Wuhan, Hubei); 14,15: the long flat ostomatid in angle (Haikou); 16: negative control (ddH 2o).
Fig. 2 is the specific detection result for the identification of the primer pair CP53F21/CP401R23 of the flat ostomatid in long angle.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker II); Other swimming lane information are 1: open general flat ostomatid (Prague, CZE); 2: the flat ostomatid of Turkey (Prague, CZE); 3: the flat ostomatid of Turkey (Shangqiu, Henan); 4: rusty grain beetle (Prague, CZE); 5: rusty grain beetle (Kan.) 6: rusty grain beetle (Jining of Shandong Province); 7: rusty grain beetle (Zhanjiang); 8,9: micro-flat ostomatid (Luxi, Yunnan); 10: the long flat ostomatid in angle (Prague, CZE); 11: the long flat ostomatid in angle (Kan.); 12,13: the long flat ostomatid in angle (Wuhan, Hubei); 14,15: the long flat ostomatid in angle (Haikou); 16: negative control (ddH 2o).
Fig. 3 is the specific detection result for the identification of the primer pair CO38F22/CO601R22 of micro-flat ostomatid.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker II); Other swimming lane information are 1: open general flat ostomatid (Prague, CZE); 2: the flat ostomatid of Turkey (Prague, CZE); 3: the flat ostomatid of Turkey (Shangqiu, Henan); 4: rusty grain beetle (Prague, CZE); 5: rusty grain beetle (Kan.) 6: rusty grain beetle (Jining of Shandong Province); 7: rusty grain beetle (Zhanjiang); 8,9: micro-flat ostomatid (Luxi, Yunnan); 10: the long flat ostomatid in angle (Prague, CZE); 11: the long flat ostomatid in angle (Kan.); 12,13: the long flat ostomatid in angle (Wuhan, Hubei); 14,15: the long flat ostomatid in angle (Haikou); 16: negative control (ddH 2o).
Fig. 4 is the specific detection result for the identification of the primer pair CT81F21/CT414R22 of the flat ostomatid of Turkey.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker II); Other swimming lane information are 1: open general flat ostomatid (Prague, CZE); 2: the flat ostomatid of Turkey (Prague, CZE); 3: the flat ostomatid of Turkey (Shangqiu, Henan); 4: rusty grain beetle (Prague, CZE); 5: rusty grain beetle (Kan.) 6: rusty grain beetle (Jining of Shandong Province); 7: rusty grain beetle (Zhanjiang); 8,9: micro-flat ostomatid (Luxi, Yunnan); 10: the long flat ostomatid in angle (Prague, CZE); 11: the long flat ostomatid in angle (Kan.); 12,13: the long flat ostomatid in angle (Wuhan, Hubei); 14,15: the long flat ostomatid in angle (Haikou); 16: negative control (ddH 2o).
Fig. 5 is for the identification of the specific detection result of opening the primer pair CC82F19/CC349R22 of general flat ostomatid.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker II); Other swimming lane information are 1: open general flat ostomatid (Prague, CZE); 2: the flat ostomatid of Turkey (Prague, CZE); 3: the flat ostomatid of Turkey (Shangqiu, Henan); 4: rusty grain beetle (Prague, CZE); 5: rusty grain beetle (Kan.) 6: rusty grain beetle (Jining of Shandong Province); 7: rusty grain beetle (Zhanjiang); 8,9: micro-flat ostomatid (Luxi, Yunnan); 10: the long flat ostomatid in angle (Prague, CZE); 11: the long flat ostomatid in angle (Kan.); 12,13: the long flat ostomatid in angle (Wuhan, Hubei); 14,15: the long flat ostomatid in angle (Haikou); 16: negative control (ddH 2o).
Fig. 6 is the sensitivity detected result for the identification of the primer pair CF84F21/CF222R20 of rusty grain beetle.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker I); Other swimming lane information are 1:DNA template consumption 10ng; 2:DNA template consumption 5ng; 3:DNA template consumption 1ng; 4:DNA template consumption 0.5ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.05ng; 7,8: negative control (ddH 2o).
Fig. 7 is the sensitivity detected result for the identification of the primer pair CP53F21/CP401R23 of the flat ostomatid in long angle.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker I); Other swimming lane information are 1:DNA template consumption 10ng; 2:DNA template consumption 5ng; 3:DNA template consumption 1ng; 4:DNA template consumption 0.5ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.05ng; 7,8: negative control (ddH 2o).
Fig. 8 is the sensitivity detected result for the identification of the primer pair CO38F22/CO601R22 of micro-flat ostomatid.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker I); Other swimming lane information are 1:DNA template consumption 10ng; 2:DNA template consumption 5ng; 3:DNA template consumption 1ng; 4:DNA template consumption 0.5ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.05ng; 7,8: negative control (ddH 2o).
Fig. 9 is the sensitivity detected result for the identification of the primer pair CT81F21/CT414R22 of the flat ostomatid of Turkey.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker I); Other swimming lane information are 1:DNA template consumption 10ng; 2:DNA template consumption 5ng; 3:DNA template consumption 1ng; 4:DNA template consumption 0.5ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.05ng; 7,8: negative control (ddH 2o).
Figure 10 is for the identification of the sensitivity detected result of opening the primer pair CC82F19/CC349R22 of general flat ostomatid.Wherein, swimming lane M is DNA relative molecular weight standard (DNA Marker I); Other swimming lane information are 1:DNA template consumption 10ng; 2:DNA template consumption 5ng; 3:DNA template consumption 1ng; 4:DNA template consumption 0.5ng; 5:DNA template consumption 0.1ng; 6:DNA template consumption 0.05ng; 7,8: negative control (ddH 2o).
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
5 kinds of flat ostomatids of grain storage: rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid, the flat ostomatid of Turkey and the flat ostomatid of Kai Pu.Wherein, rusty grain beetle relates to following geographical population: Prague, CZE population, Kan. population, Jining of Shandong Province population, Zhanjiang population; Flat ostomatid relates to following geographical population at long angle: Prague, CZE population, Kan. population, Wuhan, Hubei population, Haikou population; Micro-flat ostomatid relates to following geographical population: Luxi, Yunnan population; The flat ostomatid of Turkey relates to following geographical population: Prague, CZE population, Shangqiu, Henan population; Open general flat ostomatid and relate to following geographical population: Prague, CZE population.Above 5 kinds of flat ostomatids of grain storage are documented in " Xie Gengxiang, Tang Yi, open even medium. the flat ostomatid resistance in Hainan Region and real storehouse study on prevention. foodstuff preservation, 2013(1): 9-12 ", " wish Changqing etc. Henan entomography Coleoptera (). Henan science tech publishing house, 1999-12 publishes ", " Jiang Qingci, Huang Fuyuan, Jiang Wufeng etc. several Main Stored Grain Insects are taken a sample test report to the resistance of phosphuret-(t)ed hydrogen. the grain and oil scientific and technological communication of storing in a warehouse, 1992 (6): 46-52 ", " Zuzana Kucerova, Vaclav Stejskal.Comparative egg morphology of silvanid anda laemophloeid beetles (Coleptera) pccurring in stored products.Journal of Stored Products Research, 2002 (38): 219-227 ", " RENNIE ROESLI, BHADRIRAJU SUBRAMANYAM, et al.Stored-Product Insects Associated with a Retail Pet Store Chain in Kansas.JOURNAL OF ECONOMIC ENTOMOLOGY, 1958-1966 ", " David W.Hagstrum.Immigration of insects into bins storing newly harvested wheat on12Kansas farms.Journal of Stored Products Research, 2001 (37): 221-229 ", " Zhang Yongfu. the problem that merits attention of control rusty grain beetle. the grain and oil scientific and technological communication of storing in a warehouse, 2002(1): 33-40 ", " Liu Yongping, Zhang Shengfang. the discovery of the micro-flat ostomatid of China and preliminary investigation research. Plant Quarantine, 1984(3): 1-4 ", " Shang Yonghui, Du Minghua. explore booklice, the measure .137-143 of rusty grain beetle integrated control " in.
Embodiment 1, for the design of the special primers of 5 kinds of flat ostomatids of the common grain storage in the world with synthetic
One, the acquisition of the 5 kinds of flat ostomatid mtDNA of the common grain storage in world COI sequences
Experiment material: relate to 5 kinds of flat ostomatids of grain storage, be specially rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid, the flat ostomatid of Turkey and the flat ostomatid of Kai Pu.Wherein, rusty grain beetle relates to following geographical population: Prague, CZE population, Kan. population, Jining of Shandong Province population, Zhanjiang population; Flat ostomatid relates to following geographical population at long angle: Prague, CZE population, Kan. population, Wuhan, Hubei population, Haikou population; Micro-flat ostomatid relates to following geographical population: Luxi, Yunnan population; The flat ostomatid of Turkey relates to following geographical population: Prague, CZE population, Shangqiu, Henan population; Open general flat ostomatid and relate to following geographical population: Prague, CZE population.
(1) extraction of flat ostomatid genomic dna
Adopt blood/cell/tissue genome DNA extracting reagent kit of TIANGEN Biotech (Beijing) Co., Ltd., according to test kit specification sheets, operate, total DNA of polypide or the whole polypide of the non-adult form of single head after the flat ostomatid adult removal of extraction single head belly, according to extraction effect, do suitable adjustment, guarantee to extract DNA quality good, can be used for next step experiment.Concrete extraction procedure is as follows:
1. get adult sample to be measured and under binocular anatomical lens, remove its belly, the flat ostomatid adult sample without belly or whole non-adult sample are cleaned 1-3 time with distilled water, be then placed in seasoning on filter paper.
2. get the flat ostomatid of above-mentioned dried single head in 1.5ml volume centrifuge tube, add liquid nitrogen, with the rifle head burning after molten sealing, sample is thoroughly ground broken rapidly.
3. add 200 μ L damping fluid GA, concussion is to thoroughly suspending.
4. add 10 μ L Proteinase Ks to mix, under 56 ℃ of water bath condition, place at least 3 hours, until histolysis, per hour putting upside down mixes sample 2-3 time.The centrifugal 30 seconds globules with removal cap wall, then carry out next step.
5. add 200 μ L damping fluid GB, fully put upside down and mix, place 10 minutes for 70 ℃, solution strain is limpid, and centrifugal 30 seconds to remove the globule of cap wall.
6. add 200 μ L dehydrated alcohols, fully vibration mixes 15 seconds, now may occur flocks, brief centrifugal to remove the globule of cap wall.
7. previous step gained solution is added in an adsorption column CB3 (adsorption column is put into collection tube), centrifugal 30 seconds of 12000rpm, outwells waste liquid, and CB3 adsorption column is put back in collection tube.
8. in adsorption column CB3, add 500 μ L damping fluid GD, centrifugal 30 seconds of 12000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
9. in adsorption column CB3, add 700 μ L damping fluid PW, centrifugal 30 seconds of 12000rpm, outwells waste liquid, and adsorption column CB3 is put into collection tube.
10. in adsorption column CB3, add 500ul damping fluid PW, centrifugal 30 seconds of 12000rpm, outwells waste liquid.
(11) adsorption column CB3 is put back in collection tube, centrifugal 2 minutes of 12000rpm, outwells waste liquid.Adsorption column CB3 is placed in to room temperature and places 15 minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
(12) adsorption column CB3 is proceeded in a clean centrifuge tube, to the unsettled dropping in the middle part 20ul elution buffer TE of adsorption film, room temperature is placed 2-5 minute, and centrifugal 2 minutes of 12000rpm, collects solution in centrifuge tube.
(13) the genomic dna of flat ostomatid is eluted in centrifuge tube, and-20 ℃ save backup.
(2) mtDNA COI sequence pcr amplification
It is template that the step (1) of take is extracted the flat ostomatid genomic dna obtain, utilizes universal primer pair: the increase mtDNA COI sequence of flat ostomatid of LCO1490 and HCO2198, amplified production length not of the same race is about 700bp.Reaction system: PCR reaction cumulative volume is 50 μ L, wherein 2 * Taq PCR MasterMix25 μ L(Shanghai Sheng Gong biotechnology company limited), ddH 2o21 μ L, template 2 μ L, upstream primer 1 μ L, downstream primer 1 μ L, primer concentration is 10 μ M.Thermal cycling program is: 94 ℃ of denaturation 3min, then carry out 35 circulations (94 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 1min), and finally after 72 ℃ of reaction 10min, finish.
LCO1490：5’-GGTCAACAAATCATAAAGATATTGG-3’；
HCO2198：5’-TAAACTTCAGGGTGACCAAAAAATCA-3’。
(3) acquisition of mtDNA COI sequence
Adopt agarose gel electrophoresis to detect the quality of PCR product, sample amplified band is bright and clean background entrusts Beijing AudioCodes biotech company to carry out purifying two-way order-checking, and order-checking adopts above universal primer to (LCO1490/HCO2198).Sequencing reaction all carries out on ABI-3730 sequenator.To record the DNAMAN6.0 software splicing of comparing for sequence, excision primer section obtains the sequence fragment that final length is 658bp afterwards.
Two, for the design of 5 kinds of flat ostomatid special primers of grain storage
(1) design of primers
The mtDNA COI sequence of 5 kinds of flat ostomatids (every kind comprises 1 to a plurality of geographical population) that step 1 is obtained is carried out after DNAMAN compare of analysis, adopts Primer Premier5.0 software to design respectively the special primer for 5 kinds of flat ostomatids.
(2) primer assessment
Adopt Oligo7 software to assess primer pair indices, 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 the combination base pair of primer dimer or hairpin structure not over 3; GC% content is controlled between 30%-60%, and between upstream and downstream, does not differ too large; Mistake efficiency of initiation is no more than 100; Finally, in conjunction with Oligo7 software, provide different primers to the suitableeest annealing temperature, annealing temperature unification is set as to 50 ℃.
According to the above, design and synthesize the special primer following (table 1) obtaining for 5 kinds of flat ostomatids of grain storage:
The special primer table look-up of the common 5 kinds of flat ostomatids of grain storage in table 1 world
Embodiment 2, for the specific detection of the 5 kinds of flat ostomatid mtDNA of grain storage COI sequence specific primers
One, experiment material
Experiment material: relate to 5 kinds of flat ostomatids of grain storage, be specially rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid, the flat ostomatid of Turkey and the flat ostomatid of Kai Pu.Wherein, rusty grain beetle relates to following geographical population: Prague, CZE population, Kan. population, Jining of Shandong Province population, Zhanjiang population; Flat ostomatid relates to following geographical population at long angle: Prague, CZE population, Kan. population, Wuhan, Hubei population, Haikou population; Micro-flat ostomatid relates to following geographical population: Luxi, Yunnan population; The flat ostomatid of Turkey relates to following geographical population: Prague, CZE population, Shangqiu, Henan population; Open general flat ostomatid and relate to following geographical population: Prague, CZE population.
Two, experimental technique
Adopt blood/cell/tissue genome DNA extracting reagent kit of TIANGEN Biotech (Beijing) Co., Ltd., total DNA of polypide or the whole polypide of single head non-adult form to be measured after extraction single head flat ostomatid adult removal belly to be measured.Take gained genomic dna as template, and what employing embodiment 1 was designed carries out respectively substance pcr amplification for each primer pair in 5 kinds of grain storage flat ostomatid mtDNA COI sequence specific primers (table 1).Thereby detect the designed specificity for 5 kinds of grain storage flat ostomatid mtDNA COI sequence specific primers (table 1) of embodiment 1.
1, amplification system: PCR reaction cumulative volume is 50 μ L, wherein 2 * Taq PCR MasterMix25 μ L(Shanghai Sheng Gong biotechnology company limited), ddH 2o21 μ L, template 2 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 PCR reaction system is 0.2 μ M.
Arrange with ddH simultaneously 2o replaces the negative control of template.
2, amplification condition: 94 ℃ of denaturation 3min, then carry out 35 circulations (72 ℃ are extended 1min for 94 ℃ of sex change 1min, 50 ℃ of annealing 1min), finally after 72 ℃ of reaction 10min, finish.
3, after reaction finishes, get 5 μ L PCR reaction product, the sepharose 1%, electrophoresis detection in the TAE electrophoretic buffer of 1 times after ethidium bromide (EB) dyeing, is observed and imaging (Gel Logical Pro212) 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, further determines that special primer institute amplified fragments belongs to designed kind.
Three, experimental result
1, rusty grain beetle
Primer pair CF84F21/CF222R20(sequence 1 and the sequence 2 of utilizing designed, designed to obtain) check the specificity of this primer pair under conventional PCR method, the rusty grain beetle of differentiating except need in experiment material, also together with these 4 kinds of flat ostomatids of common grain storage of the flat ostomatid in length angle, micro-flat ostomatid, the flat ostomatid of Turkey and the flat ostomatid of Kai Pu, carry out the conventional PCR reaction of special primer.The special primer the result of rusty grain beetle as shown in Figure 1, except rusty grain beetle has specific amplification, at 158bp place, have outside a specific amplification fragment (through checking order as shown in sequence in sequence table 11), other 4 kinds of flat ostomatids of grain storage and water negative controls, all there is no amplified reaction, be in product without specific target fragment, the evaluation rusty grain beetle that primer pair CF84F21/CF222R20 can only be special is described, and invalid to the flat ostomatid of other common grain storages.
2, grow the flat ostomatid in angle
Primer pair CP53F21/CP401R23(sequence 3 and the sequence 4 of utilizing designed, designed to obtain) check the specificity of this primer pair under conventional PCR method, the flat ostomatid in long angle of differentiating except need in experiment material, also together with these 4 kinds of flat ostomatids of common grain storage of rusty grain beetle, micro-flat ostomatid, the flat ostomatid of Turkey and the flat ostomatid of Kai Pu, carry out the conventional PCR reaction of special primer.The special primer the result of the flat ostomatid in long angle as shown in Figure 2, except the flat ostomatid in long angle has specific amplification, at 371bp place, have outside a specific amplification fragment (through checking order as shown in sequence in sequence table 12), other 4 kinds of flat ostomatids of grain storage and water negative controls, all there is no amplified reaction, be in product without specific target fragment, the flat ostomatid in the long angle of evaluation that primer pair CP53F21/CP401R23 can only be special is described, and invalid to the flat ostomatid of other common grain storages.
3, micro-flat ostomatid
Primer pair CO38F22/CO601R22(sequence 5 and the sequence 6 of utilizing designed, designed to obtain) check the specificity of this primer pair under conventional PCR method, the micro-flat ostomatid of differentiating except need in experiment material, also together with rusty grain beetle, the flat ostomatid in long angle, these 4 kinds of flat ostomatids of common grain storage of the flat ostomatid of Turkey and the flat ostomatid of Kai Pu, carry out the conventional PCR reaction of special primer.The special primer the result of micro-flat ostomatid as shown in Figure 3, except micro-flat ostomatid has specific amplification, at 585bp place, have outside a specific amplification fragment (through checking order as shown in sequence in sequence table 13), other 4 kinds of flat ostomatids of grain storage and water negative controls, all there is no amplified reaction, be in product without specific target fragment, the micro-flat ostomatid of evaluation that primer pair CO38F22/CO601R22 can only be special is described, and invalid to the flat ostomatid of other common grain storages.
4, the flat ostomatid of Turkey
Primer pair CT81F21/CT414R22(sequence 7 and the sequence 8 of utilizing designed, designed to obtain) check the specificity of this primer pair under conventional PCR method, the flat ostomatid of Turkey of differentiating except need in experiment material, also together with rusty grain beetle, the flat ostomatid in long angle, these 4 kinds of flat ostomatids of common grain storage of micro-flat ostomatid and the flat ostomatid of Kai Pu, carry out the conventional PCR reaction of special primer.The special primer the result of the flat ostomatid of Turkey as shown in Figure 4, except the flat ostomatid of Turkey has specific amplification, at 355bp place, have outside a specific amplification fragment (through checking order as shown in sequence in sequence table 14), other 4 kinds of flat ostomatids of grain storage and water negative controls, all there is no amplified reaction, be in product without specific target fragment, the flat ostomatid of evaluation Turkey that primer pair CT81F21/CT414R22 can only be special is described, and invalid to the flat ostomatid of other common grain storages.
5, open general flat ostomatid
Primer pair CC82F19/CC349R22(sequence 9 and the sequence 10 of utilizing designed, designed to obtain) check the specificity of this primer pair under conventional PCR method, in experiment material, except opening general flat ostomatid that need are differentiated, also rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid are carried out to the conventional PCR reaction of special primer with together with Turkey these 4 kinds of flat ostomatids of common grain storage of flat ostomatid.Open the special primer the result of general flat ostomatid as shown in Figure 5, except opening general flat ostomatid, there is specific amplification, at 289bp place, have outside a specific amplification fragment (through checking order as shown in sequence in sequence table 15), other 4 kinds of flat ostomatids of grain storage and water negative controls, all there is no amplified reaction, be in product without specific target fragment, illustrate that primer pair CC82F19/CC349R22 can only special evaluation open general flat ostomatid, and invalid to the flat ostomatid of other common grain storages.
Comprehensive above experimental result, what visible embodiment 1 was designed has higher specificity for 5 kinds of grain storage flat ostomatid mtDNA COI sequence specific primers (table 1).
Embodiment 3, for the sensitivity of the 5 kinds of flat ostomatid mtDNA of grain storage COI sequence specific primers, detect
One, experiment material
Experiment material: relate to 5 kinds of flat ostomatids of grain storage, be specially rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid, the flat ostomatid of Turkey and the flat ostomatid of Kai Pu.Wherein, rusty grain beetle relates to following geographical population: Jining of Shandong Province population; Flat ostomatid relates to following geographical population at long angle: Prague, CZE population; Micro-flat ostomatid relates to following geographical population: Luxi, Yunnan population; The flat ostomatid of Turkey relates to following geographical population: Prague, CZE population; Open general flat ostomatid and relate to following geographical population: Prague, CZE population.
Two, experimental technique
Adopt blood/cell/tissue genome DNA extracting reagent kit of TIANGEN Biotech (Beijing) Co., Ltd., total DNA of polypide or the whole polypide of single head non-adult form to be measured after extraction single head flat ostomatid adult removal belly to be measured.The gained genomic dna of series concentration of take is template, adopt embodiment 1 designed for each primer pair in 5 kinds of grain storage flat ostomatid mtDNA COI sequence specific primers (table 1), carry out respectively substance pcr amplification.Thereby detect the designed sensitivity for 5 kinds of grain storage flat ostomatid mtDNA COI sequence specific primers (table 1) of embodiment 1.
1, amplification system: PCR reaction cumulative volume is 50 μ L, wherein 2 * Taq PCR MasterMix25 μ L(Shanghai Sheng Gong biotechnology company limited), ddH 2o21 μ L, template 2 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 PCR reaction system is 0.2 μ M.Wherein, as the to be measured flat ostomatid genomic dna of template, the consumption in reaction system arranges gradient and is: 10ng, 5ng, 1ng, 0.5ng, 0.1ng and 0.05ng.
Arrange with ddH simultaneously 2o replaces the negative control of template.
2, amplification condition: 94 ℃ of denaturation 3min, then carry out 35 circulations (72 ℃ are extended 1min for 94 ℃ of sex change 1min, 50 ℃ of annealing 1min), finally after 72 ℃ of reaction 10min, finish.
3, after reaction finishes, get 5 μ L PCR reaction product, the sepharose 1%, electrophoresis detection in the TAE electrophoretic buffer of 1 times after ethidium bromide (EB) dyeing, is observed and imaging (Gel Logical Pro212) 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, further determines that special primer institute amplified fragments belongs to designed kind.
Three, experimental result
1, rusty grain beetle
Primer pair CF84F21/CF222R20(sequence 1 and the sequence 2 of utilizing designed, designed to obtain) check the sensitivity of this primer pair under conventional PCR method.The conventional PCR product gel of the rusty grain beetle electrophoresis result of different amounts (10ng, 5ng, 1ng, 0.5ng, 0.1ng and 0.05ng) as shown in Figure 6.When template consumption is 0.1ng, the amplified band that there is no specificity product, when template consumption is 0.5ng, amplified band is fainter, only there is micro-target fragment to occur, and when template consumption is greater than 1ng, all can there is specific amplified, this suitable template amount that conventional PCR method evaluation rusty grain beetle is described should be not less than 1ng.
2, grow the flat ostomatid in angle
Primer pair CP53F21/CP401R23(sequence 3 and the sequence 4 of utilizing designed, designed to obtain) check the sensitivity of this primer pair under conventional PCR method.The conventional PCR product gel of the flat ostomatid in the long angle electrophoresis result of different amounts (10ng, 5ng, 1ng, 0.5ng, 0.1ng and 0.05ng) as shown in Figure 7.When template consumption is 0.05ng and 0.1ng, amplified band is fainter, only has micro-target fragment to occur, and when template consumption is greater than 0.5ng, all specific amplified can occur, this suitable template amount that flat ostomatid in long angle of conventional PCR method evaluation is described should be not less than 0.5ng.
3, micro-flat ostomatid
Primer pair CO38F22/CO601R22(sequence 5 and the sequence 6 of utilizing designed, designed to obtain) check the sensitivity of this primer pair under conventional PCR method.The conventional PCR product gel of the micro-flat ostomatid electrophoresis result of different amounts (10ng, 5ng, 1ng, 0.5ng, 0.1ng and 0.05ng) as shown in Figure 8.When template consumption is 0.05ng, the amplified band that there is no specificity product, when template consumption is 0.1ng, amplified band is fainter, only there is micro-target fragment to occur, and when template consumption is greater than 0.5ng, all can there is specific amplified, this suitable template amount that micro-flat ostomatid of conventional PCR method evaluation is described should be not less than 0.5ng.
4, the flat ostomatid of Turkey
Primer pair CT81F21/CT414R22(sequence 7 and the sequence 8 of utilizing designed, designed to obtain) check the sensitivity of this primer pair under conventional PCR method.The conventional PCR product gel of the flat ostomatid of the Turkey electrophoresis result of different amounts (10ng, 5ng, 1ng, 0.5ng, 0.1ng and 0.05ng) as shown in Figure 9.When template consumption is 0.1ng, the amplified band that there is no specificity product, when template consumption is 0.5ng, amplified band is fainter, only there is micro-target fragment to occur, and when template consumption is greater than 1ng, all can there is specific amplified, this suitable template amount that flat ostomatid of conventional PCR method evaluation Turkey is described should be not less than 1ng.
5, open general flat ostomatid
Primer pair CC82F19/CC349R22(sequence 9 and the sequence 10 of utilizing designed, designed to obtain) check the sensitivity of this primer pair under conventional PCR method.Different amounts (10ng, 5ng, 1ng, 0.5ng, 0.1ng and 0.05ng) open the conventional PCR product gel of general flat ostomatid electrophoresis result as shown in figure 10.When template consumption is 0.1ng, the amplified band that there is no specificity product, when template consumption is 0.5ng, amplified band is fainter, only there is micro-target fragment to occur, and when template consumption is greater than 1ng, all can there is specific amplified, this suitable template amount that illustrates that general flat ostomatid is opened in conventional PCR method evaluation should be not less than 1ng.
Comprehensive above experimental result, what visible embodiment 1 was designed has higher sensitivity for 5 kinds of grain storage flat ostomatid mtDNA COI sequence specific primers (table 1).
Embodiment 4, for the evaluation application of the 5 kinds of flat ostomatid mtDNA of grain storage COI sequence specific primers
Adopt adopting in 2011 in Jining of Shandong Province grain depot and each 5 flat ostomatid samples of unknown species that pick up from Luxi, Yunnan grain depot for the 5 kinds of flat ostomatid mtDNA of grain storage COI sequence specific primers detection inventor of embodiment 1 design.Adopt blood/cell/tissue genome DNA extracting reagent kit of TIANGEN Biotech (Beijing) Co., Ltd., total DNA of polypide after extraction single head removal belly; The flat ostomatid genomic dna extracting of take is template, uses and carries out pcr amplification for special primer not of the same race (seeing the table 1 of embodiment 1); Employing agarose gel electrophoresis detects.Relating operation is referring to embodiment 1-3.
Result shows, the flat ostomatid sample of 5 unknown species that wherein picks up from Jining of Shandong Province grain depot is specific to primer pair CF84F21/CF222R20(sequence 1 and the sequence 2 of rusty grain beetle in use) all occur in the product of amplification size for 158bp(through order-checking as shown in sequence in sequence table 11) bright band; And the flat ostomatid sample of 5 unknown species that picks up from Luxi, Yunnan grain depot is specific to primer pair CO38F22/CO601R22(sequence 5 and the sequence 6 of micro-flat ostomatid in use) occur in the product of amplification size for 585bp(through order-checking as shown in sequence in sequence table 13) bright band.Except foregoing description, the flat ostomatid sample of 5 unknown species that picks up from Jining of Shandong Province grain depot does not all obtain corresponding object band when other special primers carry out pcr amplification in the table 1 that uses embodiment 1, and the flat ostomatid sample of 5 unknown species that picks up from Luxi, Yunnan grain depot does not all obtain corresponding object band yet when other special primers carry out pcr amplification in the table 1 that uses embodiment 1.Above result shows, by utilizing Auele Specific Primer shown in the table 1 of embodiment 1 to detect, the flat ostomatid sample of 5 unknown species that picks up from Jining of Shandong Province grain depot is rusty grain beetle, and the flat ostomatid sample of 5 unknown species that picks up from Luxi, Yunnan grain depot is micro-flat ostomatid.
Meanwhile, the present inventor has adopted existing Morphological Identification method.The Zhang Shengfang researcher of Institute of quarantine of animals and plants, Chinese Academy of Inspection and is engaged in flat ostomatid morphological research more than 30 years, possesses abundant Morphological Identification experience, through Zhang Shengfang researcher, to adopting in Jining of Shandong Province grain depot and each 5 flat ostomatid samples of unknown species that pick up from Luxi, Yunnan grain depot, carry out Morphological Identification (authentication method is referring to " identification of three kinds of flat ostomatids and tabletting technology a thereof " literary composition), result confirms, through Auele Specific Primer shown in the table 1 of the above-mentioned embodiment of utilization 1, detecting 5 the flat ostomatids (picking up from Jining of Shandong Province grain depot) that are accredited as rusty grain beetle is rusty grain beetle really, through Auele Specific Primer shown in the table 1 of the above-mentioned embodiment of utilization 1, detecting 5 the flat ostomatids (picking up from Luxi, Yunnan grain depot) that are accredited as micro-flat ostomatid is micro-flat ostomatid really.As can be seen here, the evaluation that the present invention utilizes Auele Specific Primer shown in the table 1 of embodiment 1 to carry out kind flat ostomatid to be measured, reliable results.

Claims (8)

  1. For the identification of or the test kit of the flat ostomatid of assistant identification grain storage, comprise at least one pair of in following 5 primer pairs:
    (1) primer pair 1 that two single stranded DNAs shown in sequence 1 and sequence 2 form in sequence table;
    (2) primer pair 2 that two single stranded DNAs shown in sequence 3 and sequence 4 form in sequence table;
    (3) primer pair 3 that two single stranded DNAs shown in sequence 5 and sequence 6 form in sequence table;
    (4) primer pair 4 that two single stranded DNAs shown in sequence 7 and sequence 8 form in sequence table;
    (5) primer pair 5 that two single stranded DNAs shown in sequence 9 and sequence 10 form in sequence table.
  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 described test kit equates.
  3. 3. test kit according to claim 1 and 2, is characterized in that: the flat ostomatid of described grain storage for as lower at least one: rusty grain beetle, the flat ostomatid in long angle, micro-flat ostomatid, the flat ostomatid of Turkey and the flat ostomatid of Kai Pu.
  4. 4. the preparation method of arbitrary described test kit in claim 1-3, each single stranded DNA that comprises the steps: to form each primer pair in described test kit is respectively separately after packing, is packaged in same reagent box: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP with at least one in following substances.
  5. 5. identifying or the method for the flat ostomatid of assistant identification grain storage, is any in following (a)-(e):
    (a) identify or whether the flat ostomatid of assistant identification grain storage to be measured is the method for rusty grain beetle, comprise following (a1) and step (a2):
    (a1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 1 that two single stranded DNAs shown in sequence 1 and sequence 2 form in sequence table to carry out pcr amplification;
    (a2) size of the PCR product that detecting step (a1) obtains, according to PCR product, determine whether the flat ostomatid of described grain storage to be measured is rusty grain beetle as follows: if the DNA fragmentation that contains 158bp in described PCR product, the flat ostomatid of described grain storage to be measured is or candidate is rusty grain beetle; Otherwise, the flat ostomatid of described grain storage to be measured be not or candidate for rusty grain beetle;
    (b) identify or whether the flat ostomatid of assistant identification grain storage to be measured is the method for the flat ostomatid in long angle, comprise following (b1) and step (b2):
    (b1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 2 that two single stranded DNAs shown in sequence 3 and sequence 4 form in sequence table to carry out pcr amplification;
    (b2) size of the PCR product that detecting step (b1) obtains, according to PCR product, determine that whether the flat ostomatid of described grain storage to be measured is the flat ostomatid in long angle as follows: if the DNA fragmentation that contains 371bp in described PCR product, the flat ostomatid of described grain storage to be measured be or candidate for growing the flat ostomatid in angle; Otherwise the flat ostomatid of described grain storage to be measured is not or candidate is not the flat ostomatid in long angle;
    (c) identify or whether the flat ostomatid of assistant identification grain storage to be measured is the method for micro-flat ostomatid, comprise following (c1) and step (c2):
    (c1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 3 that two single stranded DNAs shown in sequence 5 and sequence 6 form in sequence table to carry out pcr amplification;
    (c2) size of the PCR product that detecting step (c1) obtains, according to PCR product, determine whether the flat ostomatid of described grain storage to be measured is micro-flat ostomatid as follows: if the DNA fragmentation that contains 585bp in described PCR product, the flat ostomatid of described grain storage to be measured is or candidate is micro-flat ostomatid; Otherwise, the flat ostomatid of described grain storage to be measured be not or candidate for micro-flat ostomatid;
    (d) identify or whether the flat ostomatid of assistant identification grain storage to be measured is the method for the flat ostomatid of Turkey, comprise following (d1) and step (d2):
    (d1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 4 that two single stranded DNAs shown in sequence 7 and sequence 8 form in sequence table to carry out pcr amplification;
    (d2) size of the PCR product that detecting step (d1) obtains, according to PCR product, determine whether the flat ostomatid of described grain storage to be measured is the flat ostomatid of Turkey as follows: if the DNA fragmentation that contains 355bp in described PCR product, the flat ostomatid of described grain storage to be measured is or candidate is the flat ostomatid of Turkey; Otherwise the flat ostomatid of described grain storage to be measured is not or the flat ostomatid of candidate Bu Wei Turkey;
    (e) identify or whether the flat ostomatid of assistant identification grain storage to be measured is out the method for general flat ostomatid, comprise following (e1) and step (e2):
    (e1) take the genomic dna of the flat ostomatid of described grain storage to be measured is template, uses the primer pair 5 that two single stranded DNAs shown in sequence 9 and sequence 10 form in sequence table to carry out pcr amplification;
    (e2) size of the PCR product that detecting step (e1) obtains, according to PCR product, determine whether the flat ostomatid of described grain storage to be measured is out general flat ostomatid as follows: if the DNA fragmentation that contains 289bp in described PCR product, the flat ostomatid of described grain storage to be measured for or candidate for opening general flat ostomatid; Otherwise, the flat ostomatid of described grain storage to be measured not for or candidate not for opening general flat ostomatid.
  6. 6. method according to claim 5, is characterized in that: in step (a)-step (e), the annealing temperature of carrying out pcr amplification described in all is 50 ℃.
  7. 7. according to the method described in claim 5 or 6, it is characterized in that: described primer pair 1, described primer pair 2, described primer pair 3, described primer pair 4 and described primer pair 5, the mol ratio of two single stranded DNAs that form each primer pair in PCR reaction system is separately 1:1.
  8. 8. according to arbitrary described method in claim 5-7, it is characterized in that: in step (a), the nucleotides sequence of the DNA fragmentation of described 158bp is classified sequence 11 in sequence table as; In step (b), the nucleotides sequence of the DNA fragmentation of described 371bp is classified sequence 12 in sequence table as; In step (c), the nucleotides sequence of the DNA fragmentation of described 585bp is classified sequence 13 in sequence table as; In step (d), the nucleotides sequence of the DNA fragmentation of described 355bp is classified sequence 14 in sequence table as; In step (e), the nucleotides sequence of the DNA fragmentation of described 289bp is classified sequence 15 in sequence table as.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104164431A (en) * 2014-08-26 2014-11-26 中国农业大学 Primer group for identifying tribolium stored grain pests and application thereof
CN104894117A (en) * 2015-05-28 2015-09-09 中国农业大学 Real-time fluorescent PCR (polymerase chain reaction) detection kit for identifying six types of flour beetle type stored-grain pests
CN104946789A (en) * 2015-07-24 2015-09-30 扬州大学 Set of specific primers for quickly identifying eight important bruchidae species, kit comprising set of specific primers, and detecting method
CN105420365A (en) * 2015-12-17 2016-03-23 中国农业科学院棉花研究所 Molecular method capable of identifying gynaikothrips ficorum and gynairothrips uzeli zimmerman
CN104164431B (en) * 2014-08-26 2017-01-04 中国农业大学 One group for the primer and application thereof identifying to intend ostomatid class grain storage pest
CN106701971A (en) * 2017-01-21 2017-05-24 河南工业大学 Primer pair, kit and method for identifying storage flour beetles
CN110592228A (en) * 2018-06-13 2019-12-20 中国农业大学 Real-time fluorescent PCR (polymerase chain reaction) primer and probe for identifying Eupatorium odoratum
CN110592229A (en) * 2018-06-13 2019-12-20 中国农业大学 Real-time fluorescent PCR (polymerase chain reaction) primer and probe for identifying Thelephora scabrosus
CN110592199A (en) * 2018-06-13 2019-12-20 中国农业大学 Real-time fluorescent PCR (polymerase chain reaction) primer and probe for identifying thefts of dalbergia odorifera

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102605093A (en) * 2012-04-10 2012-07-25 董志平 Method for identifying molecule of athetis lepigone
CN103004979A (en) * 2012-12-11 2013-04-03 甘肃农业大学 Preparation method for natural insect repellent

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102605093A (en) * 2012-04-10 2012-07-25 董志平 Method for identifying molecule of athetis lepigone
CN103004979A (en) * 2012-12-11 2013-04-03 甘肃农业大学 Preparation method for natural insect repellent

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
D.H.LUNT等: "The insect cytochrome oxidase Ⅰgene: evolutionary patterns and conserved primers for phylogenetic studies", 《INSECT MOLECULAR BIOLOGY》 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104164431A (en) * 2014-08-26 2014-11-26 中国农业大学 Primer group for identifying tribolium stored grain pests and application thereof
CN104164431B (en) * 2014-08-26 2017-01-04 中国农业大学 One group for the primer and application thereof identifying to intend ostomatid class grain storage pest
CN104894117A (en) * 2015-05-28 2015-09-09 中国农业大学 Real-time fluorescent PCR (polymerase chain reaction) detection kit for identifying six types of flour beetle type stored-grain pests
CN104946789A (en) * 2015-07-24 2015-09-30 扬州大学 Set of specific primers for quickly identifying eight important bruchidae species, kit comprising set of specific primers, and detecting method
CN105420365A (en) * 2015-12-17 2016-03-23 中国农业科学院棉花研究所 Molecular method capable of identifying gynaikothrips ficorum and gynairothrips uzeli zimmerman
CN106701971A (en) * 2017-01-21 2017-05-24 河南工业大学 Primer pair, kit and method for identifying storage flour beetles
CN110592228A (en) * 2018-06-13 2019-12-20 中国农业大学 Real-time fluorescent PCR (polymerase chain reaction) primer and probe for identifying Eupatorium odoratum
CN110592229A (en) * 2018-06-13 2019-12-20 中国农业大学 Real-time fluorescent PCR (polymerase chain reaction) primer and probe for identifying Thelephora scabrosus
CN110592199A (en) * 2018-06-13 2019-12-20 中国农业大学 Real-time fluorescent PCR (polymerase chain reaction) primer and probe for identifying thefts of dalbergia odorifera

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