CN104232633A - Specific SS-COI primer pair for cryptorrhynchus lapathi(Linnaeus) and rapid molecular detection method - Google Patents
Specific SS-COI primer pair for cryptorrhynchus lapathi(Linnaeus) and rapid molecular detection method Download PDFInfo
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Abstract
The invention provides a specific SS-COI (species-specific cytochrome C oxidase I) primer pair for cryptorrhynchus lapathi(Linnaeus) and a rapid molecular detection method. A rapid molecular identification system for cryptorrhynchus lapathi(Linnaeus) is constructed. A rapid, stable, insect stage limitation-free, simple and easy cryptorrhynchus lapathi(Linnaeus) detection technology is provided, and can be widely applied to basic inspection and quarantine and the identification of a related department over cryptorrhynchus lapathi(Linnaeus). A molecular biological means is used for rapidly and accurately identifying the major forest quarantine pest cryptorrhynchus lapathi(Linnaeus) in China within a short time, and particularly identifying pests in other stages (eggs, larvae and pupae) except adults. The specific SS-COI primer pair and the rapid molecular detection method can be used for the rapid detection of cryptorrhynchus lapathi(Linnaeus) during the transportation of poplar seedlings at home and abroad, rough wood products, wooden packaging materials and the like.
Description
Technical field
The present invention relates to Molecular Detection field, particularly a kind of Osier weevil specificity SS-CO I primer pair and rapid molecular detection method.
Background technology
Osier weevil Cryptorrhynchus lapathi (Linnaeus), genus Coleoptera Coleoptera Culculionidae Curculionidae, hidden beak resemble subfamily Cruptorrhynchinae, Rhynchophorus Cryptorrhynchus.It is now national forestry quarantine harmful organisms.This worm is mainly with cadre's harm of the larva moth food Salicaceae such as willow and dry land willow trees, and the spray of imago feeding treelet and blade are as nutritional supplementation, and the tree vigor that often causes being injured is weak, and the position that is injured is withered, finally causes whole strain dead.Meanwhile, because larva eats into the burrow that food formed in wood, its use value (Ai Yunyan, 2007 have been had a strong impact on; Li Yajie etc., 1981).Along with the development of China's Poplar Introduction and afforestation cause, the distribution and damage of this worm has the trend grown with each passing day at present, and Poplar Plantation and three Norths Shelter-belt Ecosystem engineering and construction and consolidation in serious threat.Therefore, effectively control the generation of this worm and spread, the improvement of the ecological environment and Economic development ensureing China three northern areas of China is had very important significance.
According to 1999-2001 Forest Plant Quarantine object reconnaissance information result, Osier weevil is domestic is distributed in Heilungkiang, Jilin, Liaoning, Hebei, the Inner Mongol, Gansu, Xinjiang.Whole nation occurring area 14.18 ten thousand hm
2.This worm itself does not pass is with other harmful organism, and natural diffuseness is creeped by adult, and the nursery stock and clone strain or the new rough wood felled that carry this worm are are mainly allocated and transported in long-distance communications artificially.So Spreading and diffusion may be very large.Data shows, Osier weevil is classified as quarantine and dangerous venereal disease worm list by countries such as Korea, Germany, France, Canada, Czech, the U.S., Russia, Japan, Slovakia, Italy, Britain, and China's Plant Quarantine that enters the territory is classified as " host and quarantine or dangerous venereal disease worm list ".Potential economic damage is had in China.
2009, forest pest control master station of the State Administration of Forestry promulgated " Osier weevil Quarantine Techniques code ", specified, for willow international trade or domestic allocation and transportation, should carry out at Pest-or disease-free area as far as possible; Rough wood for seedlings of poplar and clone strain or new felling that area outward transport occurs from Osier weevil wants close inspection whether to be with worm; Identify according to external symptom, when confirming to occur without epidemic situation, sign and issue the allocation and transportation of plant quarantine certificate postscript.And the morphological specificity of Osier weevil is described with harm shape.
But, in order to sibling species weevil Osier weevil and China's same area occurred makes a distinction, need a large amount of morphology discriminating work, these work mostly time-consuming, effort and need specialty classification of insect gain knowledge.Meanwhile, be in ovum, larva, these three worm states of pupa weevil class insect morphologically closely similar, also there is no reliable identification mark at present.
Prior art mainly relies on the morphological feature of Osier weevil adult to identify, these three worm states of ovum, larva and pupa are then difficult to carry out precise Identification.Therefore, in real work, need the Osier weevil of non-adult worm state to raise to adult just can identify, and Osier weevil raises the large and length consuming time of difficulty; Meanwhile, closely similar in the morphological specificity of ovum, larva, pupa and adult with equal other weevil kind, particularly sibling species of Osier weevil, be difficult to carry out taxonomic history by morphological method.In addition, in reality qualification, usually identified by the professional specializing in weevil classification, and the reviewer of basic unit does not mostly possess the ability of this respect, cannot meet actual quarantine needs.
In recent years, increasing research had proved that molecular biology method can provide strong foundation for insect qualification.The strict matrocliny of Mitochondrial DNA, Terminal oxidase I gene (COI) wherein has high conservative, Stability Analysis of Structures, the feature (Simon etc., 1994) of intronless.Therefore, research (Caterino etc., 2000 of species taxonomy, qualification and sibship are often used in; Herbert etc., 2003).
Summary of the invention
For solving above-mentioned prior art Problems existing, the object of the present invention is to provide a kind of Osier weevil specificity SS-CO I primer pair and rapid molecular detection method, based on the species-specific primer of COI gene design Osier weevil, build the rapid molecular identification system of Osier weevil.Be a kind of fast and stable, by the restriction of worm state, simple Osier weevil detection technique, basic unit's inspection and quarantine and relevant departments can be widely used in the qualification of Osier weevil.
For achieving the above object, technical scheme of the present invention is:
A kind of Osier weevil specificity SS-CO I primer 1 is to (CLSSF1/CLSSR1):
CLSSF1：5’-CTT?GGG?ATT?TGC?AGT?ATG?AGC-3’
CLSSR1：5’-TTT?GGA?GTT?TAG?GGT?GAG?AC?A-3’
The method that Osier weevil rapid molecular detects, comprises the steps:
The extraction of step one, insect genes group DNA;
The amplification of step 2, weevil CO I gene order;
Step 3, sibling species multiple alignment and species-specific primer design
PCR primer sent promise to match biological (Beijing) company and carry out two-way order-checking, obtain CO I sequence.Use DNAstar to splice sequence, remove redundant sequence, sequence results is carried out Blast sequence alignment in GeneBank.Be analyzed according to the base sequence of published 4 kinds of weevils in the sequencing result of 9 kinds of weevils and database, use software Primer Primer5.0 software design Osier weevil specificity SS-CO I primer 1 to (CLSSF1/CLSSR1):
CLSSF1：5’-CTT?GGG?ATT?TGC?AGT?ATG?AGC-3’
CLSSR1：5’-TTT?GGA?GTT?TAG?GGT?GAG?AC?A-3’
The foundation optimization of the species-specific primer amplification system of step 4, Osier weevil SS-CO I primer
Pcr amplification reaction uses
greenMaster Mix test kit (Promega), reaction system and CO I gene order amplification system identical (see table 1).The program of amplification is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 30s, 50 DEG C of renaturation 45s, and 72 DEG C extend 2min, 30 circulations; Last 72 DEG C extend 10min.Every secondary response sterile distilled water does a blank, in order to troubleshoot system errors.Getting 3 μ l products is loaded on 1.5%TAE sepharose; After 130V electrophoresis 20min, blob of viscose is soaked in 3min in EB dyestuff, detects under ultraviolet spectrophotometer and take pictures.
Relative to prior art, beneficial effect of the present invention is: authentication method fast and stable of the present invention, lower to the professional technique Capability Requirement of operator, does not limit by its worm state without the need to grasping the form diagnostic characteristics of Osier weevil, meanwhile.Inspection and quarantine and the relevant departments of basic unit can be widely used in.
Accompanying drawing explanation
Fig. 1 agarose gel electrophoresis detects detection figure: the a.CO I universal primer amplified production electrophorogram of nine kinds of weevil pcr amplification products; B. Osier weevil specificity SS-CO I primer extension product electrophorogram.
Caption: M:DL2000 DNA marker (from top to bottom 2000,1000,750,500,250,100bp); Swimming lane: 1-Osier weevil Cryptorrhynchus lapathi, 2-red plam weevil Rhynchophorus.ferrugineus, 3-ditch socket of the eye resembles Eucryptorrhynchus.chinensis, 4-Eucryptorrhynchus brandti Eucryptorrhynchus brandti, 5-mango fruit stone weevil Odoiporus. longicollis, 6-banana bulb weevil Cryptorrhynchus.lapathi, 7-Pinus massoniana Lamb angle shin resembles Shirahoshizo patruelis, 8-sitophilus zea-mais Sitophilus zeamais, 9-pine knurl resembles Hyposipalus gigas, 10-negative control.
Fig. 2 agarose gel electrophoresis detects the sensitivity of Osier weevil species-specific primer
Caption: M:DL2000 DNA marker (from top to bottom 2,000,1000,750,500,250,100bp); Swimming lane 1-6 represents respectively: the Osier weevil genomic dna of 20ng, 2ng, 200pg, 20pg, 2pg and 200fg, swimming lane 7-negative control.
Embodiment
Below in conjunction with the drawings and the specific embodiments, the present invention program is described in further detail:
Test example 1
1. the extraction of insect genes group DNA
Extracting genome DNA all adopts the EZgene of BIOMIGA company
tMinsect gDNA Kit (GD2413-02) test kit.
Concrete operation step:
(1), after being cleaned up by the sample ultrasonic instrument soaked in straight alcohol and drying, adult decaptitates and elytrum, and larva gets its abdominal tissues.Get being organized in mortar of < 50mg fully to grind, be placed in the centrifuge tube of 1.5ml.
(2) in centrifuge tube, add the Proteinase K (25mg/ml) of Buffer ITL and the 25ul of 350ul, fully mixing is placed on 60 DEG C of thermostat water baths two hours until organize completely digested.
(3) in centrifuge tube, the chloroform of 350 μ l is added: primary isoamyl alcohol (24:1) slowly mixes, in room temperature, centrifugal 2min under 10,000 rotating speed, and careful supernatant liquor being transferred in new clean 1.5ml centrifuge tube.
(4) add 1 times of volume BufferBL and the RNaseA of 5 μ l, slowly mix, 70 DEG C of water-bath half an hour.
(5) add the dehydrated alcohol (room temperature) of 1 times of volume, slowly mix.
(6) solution of back and flocks 700 μ are added (adsorption column is placed in collection tube) in adsorption column, under 10,000xg room temperature, centrifugal 1min, removes waste liquid, and adsorption column is placed in collection tube again.The 6th step is then repeated as now still having residual mixed liquor.
(7) be again placed in new collection tube by adsorption column, add the BufferKB of 500 μ l, under 10,000xg room temperature, centrifugal 30s, removes waste liquid, and adsorption column is placed in collection tube again.
(8) add the DNAWashBuffer of 600 μ l, the centrifugal 30s of 10,000xg, removes waste liquid, and adsorption column is placed in collection tube again.
(9) repeat the 8th step, be placed in by adsorption column in new collection tube, under the condition that the lid of adsorption column is opened, the centrifugal 2min of 13,000sg room temperature, to be placed under room temperature several minutes, alcohol is fully volatilized.
(10) adsorption column is placed in new 1.5ml centrifuge tube, unsettled the Elution Buffer adding 30 μ l, the Elution Buffer preheating under 60 DEG C of-70 DEG C of conditions to adsorption column middle part, room temperature places 2 minutes, centrifugal 2min under 13,000xg.
(11) the 10th step is repeated, to improve output.
2. the amplification of weevil CO I gene order
According to bibliographical information, synthesis weevil class insect CO I gene order amplification universal primer:
C1-J-2183:5’-CAA?CAT?TTA?TTT?TGA?TTT?TTT?GG-3’，
TL-2-N-3014：5’-TCC?ATT?GCA?CTA?TAC?TGC?CAT?ATT?A-3’
Pcr amplification reaction uses
greenMaster Mix test kit (Promega), be totally 25 μ l, each composition is as shown in table 1:
Table 1 PCR reaction system
Put into PCR instrument after mixing to increase, the program of amplification is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 30s, 49 DEG C of renaturation 45s, and 72 DEG C extend 2min, 30 circulations; Last 72 DEG C extend 10min.Every secondary response sterile distilled water does a blank, in order to troubleshoot system errors.Get 3 μ l products to be loaded on 1.5%TAE sepharose.After 130V electrophoresis 20min, blob of viscose is soaked in 3min in EB dyestuff, detects under ultraviolet spectrophotometer and take pictures.
3. sibling species multiple alignment and species-specific primer design
PCR primer sent promise to match biological (Beijing) company and carry out two-way order-checking, obtain CO I sequence.Use DNAstar to splice sequence, remove redundant sequence, sequence results is carried out Blast sequence alignment in GeneBank.Be analyzed according to the base sequence of published 4 kinds of weevils in the sequencing result of 9 kinds of weevils and database, use software Primer Primer 5.0 software design Osier weevil specificity SS-CO I primer 1 to (CLSSF1/CLSSR1):
CLSSF1：5’-CTT?GGG?ATT?TGC?AGT?ATG?AGC-3’
CLSSR1：5’-TTT?GGA?GTT?TAG?GGT?GAG?AC?A-3’
4. the foundation optimization of the species-specific primer amplification system of Osier weevil SS-CO I primer
Pcr amplification reaction uses
greenMaster Mix test kit (Promega), reaction system and CO I gene order amplification system identical (see table 1).The program of amplification is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 30s, 50 DEG C of renaturation 45s, and 72 DEG C extend 2min, 30 circulations; Last 72 DEG C extend 10min.Every secondary response sterile distilled water does a blank, in order to troubleshoot system errors.Get 3 μ l products to be loaded on 1.5%TAE sepharose.After 130V electrophoresis 20min, blob of viscose is soaked in 3min in EB dyestuff, detects under ultraviolet spectrophotometer and take pictures.
Test example 2
1. the species specificity inspection of Osier weevil SS-CO I primer
Occur with other 8 kinds of common weevil class insect DNA for template with domestic with Osier weevil same area, with Osier weevil DNA for positive control (detecting the mother liquor that DNA is 10ng/ μ l concentration).The species specificity of inspection Osier weevil SS-CO I primer CLSSF1/CLSSR1.
Utilize CO I universal primer, 9 kinds of weevils all increase the product of about 830bp, and the Osier weevil specificity SS-CO I primer CLSSF1/CLSSR1 utilizing the present invention to design carries out PCR, only there is Osier weevil Successful amplification, product is 396bp, to other 8 kinds of weevils and negative control, all not there is amplification ability (see Fig. 1), show that this is the species-specific primer of Osier weevil to primer.
2. the sensitivity test of Osier weevil SS-CO I primer
Osier weevil DNA sample is diluted to successively the reference liquid of 10ng/ μ l, 1ng/ μ l, 100pg/ μ l, 10pg/ μ l, 1pg/ μ l, 100fg/ μ l according to the ratio of (1:10).The Osier weevil DNA standard substance of different concns gradient are utilized to carry out Auele Specific Primer sensitivity test (see Fig. 2).The minimum detection limit of PCR inspection is the genomic dna of 200pg.
Comprehensive above-mentioned research, Osier weevil species-specific primer (SSF1/SSR1) only has expanding effect to Osier weevil, does not have amplification ability to same area generation and other domestic common weevil class insects; Auele Specific Primer is highly sensitive, can detect the minimum genomic dna for 200pg; The Blast result of target sequence is shown, this fragment with report that Osier weevil sequence identity is 100%.Result shows that this invention can be used for the Rapid identification of Osier weevil in inspection and quarantining for import/export process.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any change of expecting without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, the protection domain that protection scope of the present invention should limit with claims is as the criterion.
Claims (6)
1. Osier weevil specificity SS-CO I primer pair, its sequence is as follows:
CLSSF1：5’-CTT?GGG?ATT?TGC?AGT?ATG?AGC-3’
CLSSR1：5’-TTT?GGA?GTT?TAG?GGT?GAG?AC?A-3’。
2. the method for an Osier weevil rapid molecular detection, it is characterized in that, the weevil class insect DNA utilizing the Osier weevil specificity SS-CO I primer pair Osier weevil described in claim 1 and other and Osier weevil same area to occur and to belong to nearly edge together carries out pcr amplification, with specific detection Osier weevil kind.
3. method according to claim 2, is characterized in that, comprises the steps:
The extraction of step one, insect genes group DNA;
The amplification of step 2, weevil CO I gene order;
Step 3, sibling species multiple alignment and species-specific primer design;
PCR primer is carried out two-way order-checking, obtain CO I sequence, DNAstar is used to splice sequence, remove redundant sequence, sequence results is carried out Blast sequence alignment in GeneBank, be analyzed according to the base sequence of published 4 kinds of weevils in the sequencing result of 9 kinds of weevils and database, design Osier weevil specificity SS-CO I primer 1 couple of CLSSF1/CLSSR1:
CLSSF1：5’-CTT?GGG?ATT?TGC?AGT?ATG?AGC-3’
CLSSR1：5’-TTT?GGA?GTT?TAG?GGT?GAG?AC?A-3’；
The foundation optimization of the species-specific primer amplification system of step 4, Osier weevil SS-CO I primer:
Pcr amplification reaction uses
greenMaster Mix test kit, reaction system is identical with CO I gene order amplification system, and the program of amplification is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 30s, 50 DEG C of renaturation 45s, and 72 DEG C extend 2min, 30 circulations; Last 72 DEG C extend 10min, and every secondary response sterile distilled water does a blank, in order to troubleshoot system errors, gets 3 μ l products and is loaded on 1.5%TAE sepharose; After 130V electrophoresis 20min, blob of viscose is soaked in 3min in EB dyestuff, detects under ultraviolet spectrophotometer and take pictures.
4. method according to claim 3, is characterized in that, the concrete operations of described step one are:
(1), after being cleaned up by the sample ultrasonic instrument soaked in straight alcohol and drying, adult decaptitates and elytrum, and larva gets its abdominal tissues, gets being organized in mortar of < 50mg and fully grinds, be placed in the centrifuge tube of 1.5ml;
(2) in centrifuge tube, add 350ul Buffer ITL and 25ul concentration are the Proteinase K of 25mg/ml, and fully mixing is placed on 60 DEG C of thermostat water baths two hours until organize completely digested;
(3) in centrifuge tube, the chloroform of 350 μ l is added: primary isoamyl alcohol is that 24:1 mixture slowly mixes, in room temperature, centrifugal 2min under 10,000 rotating speed, and careful supernatant liquor is transferred in new clean 1.5ml centrifuge tube;
(4) add 1 times of volume BufferBL and the RNaseA of 5 μ l, slowly mix, 70 DEG C of water-bath half an hour;
(5) add the dehydrated alcohol of 1 times of volume under room temperature, slowly mix;
(6) add in adsorption column by the solution of back and flocks 700 μ, adsorption column is placed in collection tube, and 10, centrifugal 1min under 000xg room temperature, remove waste liquid, adsorption column is placed in collection tube again, then repeats the 6th step as now still having residual mixed liquor;
(7) be again placed in new collection tube by adsorption column, add the BufferKB of 500 μ l, under 10,000xg room temperature, centrifugal 30s, removes waste liquid, and adsorption column is placed in collection tube again;
(8) add the DNAWashBuffer of 600 μ l, the centrifugal 30s of 10,000xg, removes waste liquid, and adsorption column is placed in collection tube again;
(9) repeat the 8th step, be placed in by adsorption column in new collection tube, under the condition that the lid of adsorption column is opened, the centrifugal 2min of 13,000sg room temperature, to be placed under room temperature several minutes, alcohol is fully volatilized;
(10) adsorption column is placed in new 1.5ml centrifuge tube, unsettled the Elution Buffer adding 30 μ l, the Elution Buffer preheating under 60 DEG C of-70 DEG C of conditions to adsorption column middle part, room temperature places 2 minutes, centrifugal 2min under 13,000xg.
5. method according to claim 3, is characterized in that, in described step 2 and step 3, pcr amplification reaction system is: use
greenMaster Mix test kit, is totally 25 μ l, comprises: 2 × GO Taq GreenMaster Mix 12.5 μ l, two kinds of each 1 μ l of primer, DNA profiling 1.5 μ l, ddH
2o 9 μ l.
6. method according to claim 3, is characterized in that, in described step 4, the minimum detection limit of PCR inspection is the genomic dna of 200pg.
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| CN107904315A (en) * | 2017-11-22 | 2018-04-13 | 北京林业大学 | Rust palm weevil specific primer and rapid molecular detection method |
| CN111269968A (en) * | 2020-03-13 | 2020-06-12 | 华南师范大学 | PCR-RFLP-based method for rapidly identifying Spodoptera frugiperda |
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| CN102409097A (en) * | 2011-11-25 | 2012-04-11 | 中国热带农业科学院环境与植物保护研究所 | Specific primers for quick identification of three kinds of sternochetus spp. and detection method thereof |
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| CN107904315A (en) * | 2017-11-22 | 2018-04-13 | 北京林业大学 | Rust palm weevil specific primer and rapid molecular detection method |
| CN107904315B (en) * | 2017-11-22 | 2021-01-15 | 北京林业大学 | Rusty palm weevil specific primer and rapid molecular detection method |
| CN111269968A (en) * | 2020-03-13 | 2020-06-12 | 华南师范大学 | PCR-RFLP-based method for rapidly identifying Spodoptera frugiperda |
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