CN103409536B - DNA sequence, primers, identifying method and identifying kit for identifying specificity of cryptolaemus montrouzieri - Google Patents
DNA sequence, primers, identifying method and identifying kit for identifying specificity of cryptolaemus montrouzieri Download PDFInfo
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- CN103409536B CN103409536B CN201310360552.9A CN201310360552A CN103409536B CN 103409536 B CN103409536 B CN 103409536B CN 201310360552 A CN201310360552 A CN 201310360552A CN 103409536 B CN103409536 B CN 103409536B
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Abstract
The invention discloses a DNA sequence, primers, an identifying method and an identifying kit for identifying the specificity of cryptolaemus montrouzieri. The DNA sequence is shown in SEQ ID No.1, and the identifying primers are shown in SEQ ID No.2 and 3. The invention overcomes the problems that the traditional ladybird classification has high requirement on specimen completion, needs special classification experts and is high in identification cost, the provided PCR primers and the identifying method not only can improve the efficiency and the accuracy of identification for the cryptolaemus montrouzieri, can be operated simply and quickly, are suitable for detecting whether predatory cryptolaemus montrouzieri is mixed in mealybug pest population in fields, and have important theoretical significance and application value on the aspects of quality monitoring to the process of indoor culture and accurate evaluation on the biocontrol effect of the cryptolaemus montrouzieri.
Description
Technical field
The invention belongs to Applied Biotechnology field, be specifically related to the specific DNA sequences of qualification Cryptolaemus montrouzieri, primer, authentication method and identification kit.
Background technology
Cryptolaemus montrouzieri (Cryptolaemus montrouzieri Mulsant) belongs to Coleoptera, Coccinellidae, originates in Australia, is the important predator of mealybug class pest.Cryptolaemus montrouzieri can prey on more than 20 kind of coccid, and all has distribution in China's southeastern coast, South East Asia and most of tropical and subtropical region.On China, Egypt, India and other places, researchist carried out repeatedly the behavior of Cryptolaemus montrouzieri and was applied to the test of biological control, all proved that this ladybug is the wide natural enemy insect of kind of DEVELOPMENT PROSPECT.Due to its have feeding habits single-minded, heavy, be easy to many good characteristics such as indoor feeding, be more and more widely used in the biological control of mealybug.But the polypide of Cryptolaemus montrouzieri larva is covered by white wax completely, and mealybug class pest is closely similar, in the place that mealybug insect assembles, whether very difficult resolution exists predacious Cryptolaemus montrouzieri; In addition; the species of ladybirds that the whole world has recorded is more than 5000 kinds; phytophagy, bacterium feeding habits, predatism three major types can be divided into again according to its feeding habits; wherein; only have the Predaceous Coccinellids as pest natural enemy to be the object utilizing and protect, and in ladybug, some nearly source kinds are difficult to distinguish, and just must dissect sexual organ by identification of morphology; this just has high requirements to the integrity of collect specimen, and qualification takes time and effort.In recent years, utilize Protocols in Molecular Biology to carry out species identification and obtain application treasuring in species and inspection and quarantine, but also do not report in the qualification of natural enemy ladybug.Therefore, to set up a kind of efficient, quick, sensitive and Cryptolaemus montrouzieri authentication method that accuracy is high just very necessary in research.
Summary of the invention
First object of the present invention is to provide clones from Cryptolaemus montrouzieri gene order, can for the identification of the specific DNA sequences of Cryptolaemus montrouzieri.
The present invention to go forward side by side performing PCR amplification according to the Auele Specific Primer of Cryptolaemus montrouzieri est sequence design COI gene, obtain the DNA specific band of 449bp, tetraploid rice is carried out to after its recovery, clone, order-checking, determine that this specific DNA sequences can be used as the specific DNA sequences of qualification Cryptolaemus montrouzieri, the insect imago not easily differentiated on the natural enemy ladybug of distinguishing these species and nearly edge and form and larva, thus achieve object of the present invention.
The specific DNA sequences of qualification Cryptolaemus montrouzieri of the present invention, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.1.
Second object of the present invention provides the Specific PCR primers identifying Cryptolaemus montrouzieri for a pair, it is characterized in that, comprise forward primer: TAATGTTATTGTAACTGCCCATG(is as shown in SEQ ID NO.2) and reverse primer: CAGCTAAAACAGGTAAGGAAAG(as shown in SEQ ID NO.3).
3rd object of the present invention is to provide a kind of test kit of energy specificity identification Cryptolaemus montrouzieri, comprise DNA extraction reagent, PCR reaction reagent and primer, it is characterized in that, described primer is forward primer: TAATGTTATTGTAACTGCCCATG and reverse primer: CAGCTAAAACAGGTAAGGAAAG.
The genomic dna sample of the insect gathered with this primer amplification field, if any 449bp amplified band, then for being mixed with the sample of Cryptolaemus montrouzieri species.
4th object of the present invention is to provide a kind of method identifying Cryptolaemus montrouzieri, it is characterized in that, using forward primer: TAATGTTATTGTAACTGCCCATG and reverse primer: CAGCTAAAACAGGTAAGGAAAG is increased to genomic dna sample as PCR primer by PCR method, if any 449bp amplified band, then for being mixed with the sample of Cryptolaemus montrouzieri species.
Instant invention overcomes that the classification of traditional ladybug is high to sample integrity demands, the problem such as need professional classification expert, appraisal cost high, the PCR primer provided and authentication method can not only improve efficiency and the accuracy of Cryptolaemus montrouzieri qualification, and it is simple and efficient to handle, be suitable for whether being mixed with predacious Cryptolaemus montrouzieri in Fields detection mealybug class pest population, indoor culture process is carried out to quality monitoring, carried out the aspects such as accurate evaluation to the biocontrol effect of Cryptolaemus montrouzieri, there is important theory significance and using value.
Accompanying drawing explanation
Fig. 1 is the 1% agarose gel electrophoresis figure carrying out PCR detection with PCR primer pair 18 kinds of insect DNA samples of design;
M:D2000DNA Marker; 1: negative control; 2: Cryptolaemus montrouzieri Cryptolaemus montrouzieri; 3: Propylaea japonica Propylea japonica; 4: Menochilus sexmaculata Menochilus sexmaculata; 5: little black ladybug Cryptolaemus sp.; 6: beautiful Chrysopa Chrysopa sinica; 7: citrus fruit fly Bactrocera dorsalis; 8: red flour beetle Tribolium castaneum; 9: diaphorina citri Diaphorina citri; 10: citrus mealy bug Planococcus citri; 11: two bar strokes mealybug Ferrisia virgata; 12: Egyptian icerya purchasi Icerya aegyptiaca; 13: Japan continuous mealybug Phenacoccus solenopsis; 14: aphid Aphidoidea sp.; 15: Tessaratoma Papillosa Drury Tessaratoma papillosa; 16: leaf roller Archips sp.; 17: aleyrodid Aleurodicus sp.; 18: plant hopper section Fulgoridae(sp.); 19: small cabbage moth Plutella xyllostella;
Fig. 2 is the electrophorogram using test kit of the present invention to detect single head ladybug, ladybug and its prey citrus mealy bug mixing sample pcr amplification result; M:DNA molecular weight standard (D2000DNA Marker); 1: Cryptolaemus montrouzieri larva; 2: the citrus mealy bug population biased sample being mixed with Cryptolaemus montrouzieri; 3: the sample only having citrus mealy bug population.
Embodiment
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: the acquisition of Cryptolaemus montrouzieri specific DNA sequences and qualification
1, the design of the Specific PCR primers of Cryptolaemus montrouzieri is identified
Based on the fragment being noted as COI gene in Cryptolaemus montrouzieri est sequence library, design and synthesis can identify the Specific PCR primers of Cryptolaemus montrouzieri:
Forward primer sequence: TAATGTTATTGTAACTGCCCATG(is as shown in SEQ ID NO.2);
Reverse primer sequences: CAGCTAAAACAGGTAAGGAAAG(is as shown in SEQ ID NO.3).
Primer is synthesized by Hua Da gene (Shenzhen) company, and for all amplifications for examination species DNA of this experiment.
2, sample preparation and DNA extraction
By 18 kinds of insects (Cryptolaemus montrouzieri Cryptolaemus montrouzieri; Propylaea japonica Propylea japonica; Menochilus sexmaculata Menochilus sexmaculata; Little black ladybug Cryptolaemus sp.; Beautiful Chrysopa Chrysopa sinica; Citrus fruit fly Bactrocera dorsalis; Red flour beetle Tribolium castaneum; Diaphorina citri Diaphorina citri; Citrus mealy bug Planococcus citri; Two bar strokes mealybug Ferrisia virgata; Egyptian icerya purchasi Icerya aegyptiaca; Japan continuous mealybug Phenacoccus solenopsis; Aphid Aphidoidea sp.; Tessaratoma Papillosa Drury Tessaratoma papillosa; Leaf roller Archips sp.; Aleyrodid Aleurodicus sp.; Plant hopper section Fulgoridae(sp.); Small cabbage moth Plutella xyllostella) get respectively single head insect specimen distilled water clean, be placed in 1.5ml centrifuge tube, add 200 μ l lysis buffer (10mmol/L Tris-HCl pH8.0; 50mmol/L NaCl; 0.45%Tween20; 0.45%NP-40), with grinding rod, sample is ground, vortex mixes, add 10 μ l Proteinase Ks (100 μ g/ml) again, 68 DEG C of water-baths 30 minutes, then 95 DEG C of process 5 minutes, centrifugal 5 minutes of 12000rpm, transfer supernatant is in new centrifuge tube, and namely obtain genomic dna, the DNA sample obtained is for pcr amplification.
3, specific PCR amplification
Reaction system is 25 μ l, adds 10 × PCR buffer2.5 μ l, each 1 μ l, the dNTPs(10mM of PCR primer pair (10mM)) 1 μ l, Taq enzyme (50U) 0.5 μ l and 5-10ng DNA profiling, add sterilizing distilled water to 25 μ l.With sterilizing distilled water for negative control.PCR response procedures: pre-amplification 94 DEG C of 2min; 94 DEG C of 30s, 50 DEG C ~ 55 DEG C 30s, 72 DEG C of 1min, totally 35 circulations; Last extends 72 DEG C, 7min, 4 DEG C of preservations.Get 5 μ l PCR primer and carry out 1% agarose gel electrophoresis, result as shown in Figure 1, negative control is sterilizing distilled water, there is the band of clear and unique 449bp size in the electrophoresis result of Cryptolaemus montrouzieri sample, otherwise, other occur without this band or occur the non-specific amplifications such as hangover, primer dimer, be then other insect samples.
Embodiment 2: the application of test kit
1, the formation of test kit
Lysis buffer (10mmol/L Tris-HCl pH8.0; 50mmol/L NaCl; 0.45%Tween20; 0.45%NP-40); Proteinase K (100 μ g/ml); Forward primer (TAATGTTATTGTAACTGCCCATG) (10mmol/L); Anyway primer (CAGCTAAAACAGGTAAGGAAAG) (10mmol/L); 2 × PCR premixed liquid (0.1U/ μ l Taq enzyme; 0.2 μ l/ μ l10 × PCR buffer; 0.6mmol/L dNTPs); Cryptolaemus montrouzieri qualification process specifications.
2, concrete operation step:
(1) the doubtful Cryptolaemus montrouzieri larva sample 1, by field collected and the doubtful citrus mealy bug sample 2 and 3 being mixed with Cryptolaemus montrouzieri, often kind of sample is got 0.1g and is placed in 1.5ml centrifuge tube, adds 500 μ l lysis buffer grinding rods and is ground by sample; Add 500 μ l lysis buffers, 10 μ l Proteinase Ks again, vortex mixes; 68 DEG C of water-baths 30 minutes; Then 95 DEG C process 5 minutes; Centrifugal 5 minutes of 12000rpm; By for subsequent use in the DNA sample of supernatant to new centrifuge tube.
(2), PCR reaction system is configured:
(3), PCR response procedures is arranged:
Pre-amplification 94 DEG C of 2min; 94 DEG C of 30s, 50 DEG C ~ 55 DEG C 30s, 72 DEG C of 1min, totally 35 circulations; Last extends 72 DEG C, 7min, 4 DEG C of preservations.
(4), 1.0 ~ 1.5% agarose gel electrophoresis detect, PCR primer and DNA marker respectively get 3 μ l loadings, 120V electrophoresis 25min.
(5), uv photography result as shown in Figure 2, the corresponding swimming lane 1,2 of sample 1,2() have the object band of 449bp to occur, sample 3(correspondence swimming lane 3) do not have the object band of 449bp to occur, therefore the insect larvae of sample 1 is Cryptolaemus montrouzieri larva; Sample 2 is for being mixed with the citrus mealy bug population of Cryptolaemus montrouzieri; Sample 3 is not for be mixed with Cryptolaemus montrouzieri citrus mealy bug population.
Claims (4)
1. identify a specific DNA sequences for Cryptolaemus montrouzieri, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.1.
2. the PCR primer of a pair energy specificity identification Cryptolaemus montrouzieri, is characterized in that, comprise forward primer: TAATGTTATTGTAACTGCCCATG and reverse primer: CAGCTAAAACAGGTAAGGAAAG.
3. the test kit of an energy specificity identification Cryptolaemus montrouzieri, comprise DNA extraction reagent, PCR reaction reagent and primer, it is characterized in that, described primer is forward primer: TAATGTTATTGTAACTGCCCATG and reverse primer: CAGCTAAAACAGGTAAGGAAAG.
4. identify the method for Cryptolaemus montrouzieri for one kind, it is characterized in that, using forward primer: TAATGTTATTGTAACTGCCCATG and reverse primer: CAGCTAAAACAGGTAAGGAAAG is increased to genomic dna sample as PCR primer by PCR method, if any 449bp amplified band, then for being mixed with the sample of Cryptolaemus montrouzieri species.
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Non-Patent Citations (4)
| Title |
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| Pasquer F等.GenBank登录号:FM210139.《NCBI》.2010, * |
| 付景等.27种瓢虫mtDNA-COI基因序列分析及系统发育研究.《昆虫分类学报》.2006,第28卷(第3期), * |
| 基于细胞色素氧化酶I亚基基因的动物分子系统学概述;郭晓华等;《生物学教学》;20091231;第34卷(第10期);摘要、第8页以及第9页左栏第一段 * |
| 红色蚋细胞色素氧化酶COI基因克隆及鉴定;罗洪斌等;《中国公共卫生》;20101130;第26卷(第11期);第1383页右栏倒数第1-7行 * |
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