CN106367506A - Quarantine fruit fly multi-target rapid identification method - Google Patents
Quarantine fruit fly multi-target rapid identification method Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention relates to a fruit fly quarantine technology, in particular to a quarantine fruit fly multi-target rapid identification method. The quarantine fruit fly multi-target rapid identification method comprises the following steps: extracting genome DNA of a to-be-detected sample; performing PCR by taking the genome DNA as a template and by using specific primers as shown in SEQ ID NO.1 and SEQ ID NO.2; and detecting the PCR product by gel electrophoresis, wherein the sample belongs to quarantine fruit flies if a 603bp specific band occurs, and the sample does not belong to the quarantine fruit flies if the 603bp specific band does not occur. The quarantine fruit flies relate to anastrepha schiner, bactrocera macquart, carpomya, ceratitis, dacus fabricius and/or rhagoletis; and the specific primers can distinguish the quarantine fruit flies from other tephritidae and diptera close species, have high universality and provide powerful technological support for effectively preventing and controlling introduction and diffusion of the quarantine fruit flies and protecting the ecological safety in China.
Description
Technical field
The present invention relates to trypetid Quarantine Techniques are and in particular to a kind of quarantine fruit fly multiple target rapid identification method.
Background technology
Trypetid fruit flies, belongs to Diptera diptera Tephritidae tephritidae insecticide, and the whole world is widely distributed,
Mostly occur in subtropical and tropical zones.4500 kinds of 500 genus is there are about, wherein about 1500 kinds are relevant with various fruits known to the world,
The trypetid with economic implications reaches more than 250 kinds.Trypetid host range is wide, is laid eggs shape in the fruit surface of water fruits and vegetables with adult
Become hole, larva takes food inside fruit causes harm, and causes fruit rot or shedding, its economy that can cause up to 100% that causes harm
Fruit and vegerable are not only produced and constitute serious harm by loss, and are fed the export trade and bring have a strong impact on.
Extremely the quarantine trypetid of concern mainly has 5 genus in the whole world, that is, press Anastrepha anastrepha, Bactrocera
Bactrocera, little bar Anastrepha ceratitis, few hair on the neck Anastrepha dacus and around Anastrepha rhagoletis.In China, real
Fly invasion prevention and control are highly valued.In 2007, issue is combined by the Ministry of Agriculture and State Administration for Quality Supervision and Inspection and Quarantine
" People's Republic of China (PRC) enter the territory plant quarantine harmful organism register " in, quarantine fruit fly comprises by Anastrepha, fruit fly
Genus, little bar Anastrepha, few hair on the neck trypetid (non-China seed), around 10 kinds such as trypetids (non-China seed)；2013 2 months, the Ministry of Agriculture
Issue " state key management alien species register (first) " (No. 1897 bulletin), this register includes 3 kinds of trypetid classes
Insect, respectively citrus fruit fly b.dorsalis, melonfly b.cucurbitae and jujube fly carpomya vesuviana.With
When, the acquisition of information of China's port last decade shows, often has trypetid to intercept and capture, and species is numerous in passenger's belongings and goods quarantine
Many.
The trypetid of intercept and capture mostly is non-adult form, need to raise through 1-2 week as greatly could being dropped by precise Identification after adult
Low quarantine ageing.With developing rapidly of international trade, tourism and traffic, the danger of trypetid invasion increasingly increases
Plus, after invasion, easily outburst is caused disaster and is led to huge economic loss, and its whole world invasion attracts great attention, its quarantine identification
The demand of particularly Rapid identification is more urgent.
Nearly ten years, different kinds of molecules biology techniques have been applied to the Rapid identification of trypetid, for example: dna bar codes technique
(dna barcoding), pcr technology, restriction fragment length polymorphism technology (restriction fragment length
Polymorphism, rflp), DNArandom amplified polymorphic DNA dna technology (random amplified polymorphic dna,
Rapd), amplified fragment length polymorphism technology (amplified fragment length polymorphism, aflp) and
Biochip technology etc., but from the point of view of currently reported, all researchs establish quick detection just for indivedual trypetid species
Method, still fails to fully meet China and enters the territory in plant quarantine harmful organism register in units of trypetid superior classification rank unit
Detection demand.
It is desirable to set up the multiple target Rapid Identification system of Tephritidae Important Economic trypetid, thus improving China
Border Quarantine Techniques level, more fully and effectively takes precautions against the invasion of quarantine fruit fly, suppresses it to colonize and diffusion spreads, and ensures
China's agricultural and forestry production and ecological safety.
Content of the invention
The limitation of indivedual trypetids can only be detected to make up existing trypetid rapid identification method, the present invention provides a kind of
Trypetid multiple target rapid identification method and its specific primer, have broad applicability, have reformed quarantine means and measure, for having
The incoming and diffusion of effect prevention and control quarantine fruit fly, crosses over external plant quarantine technology barriers, protection China's agricultural production and ecology
Safely provide strong technical support.
The technical scheme that the present invention is claimed is as follows:
Specific primer for quarantine fruit fly multiple target Rapid identification is it is characterised in that its nucleotide sequence such as seq
Shown in id no.1 and seq id no.2；
Described quarantine fruit fly refer to by Anastrepha, Bactrocera, click Anastrepha, little bar Anastrepha, few hair on the neck Anastrepha and/or
Around Anastrepha；
Described quarantine fruit fly can be opened by described specific primer with other Tephritidaes and Diptera nearly edge species differentiation.
A kind of quarantine fruit fly multiple target rapid identification method is it is characterised in that comprise the steps:
(1) extract the genome dna of testing sample；
(2) with genome dna as template, carry out pcr using above-mentioned specific primer, obtain amplified production；
(3) detected through gel electrophoresis amplified production, if the specific band of 603bp, it is real that this sample belongs to quarantine
Fly；Conversely, then this sample is not belonging to quarantine fruit fly.
Preferably, the reaction system of described pcr is: 2 × taq pcr mastermix 12.5 μ l, dna template 1 μ l, 10 μ
M forward primer 0.5 μ l, 10 μm of reverse primers 0.5 μ l, ddh2o 10.5μl.
Preferably, the reaction condition of described pcr is: 95 DEG C of denaturations 3min；95 DEG C of degeneration 15s, 60 DEG C of annealing extend
1min, 35 circulations；60 DEG C of extension 1min.
A kind of quarantine fruit fly multiple target Rapid identification test kit is it is characterised in that include liquid or powder upper
State specific primer.
Preferably, described test kit also includes the required other conventional reagent of pcr reaction.
The present invention be meet China enter the territory plant quarantine harmful organism register in by trypetid superior classification rank unit in units of
Detection demand, retrieved the mitochondrial genome sequence of all dipteral insect species from genbank data base and carry out
Multiple Sequence Alignment, has screened the conserved positions of Important Economic Anastrepha (i.e. quarantine fruit fly).Nucleoside with conserved positions
Acid sequence is the multipair primer of stencil design and it is estimated, and finally screening obtains distinguishing quarantine fruit fly and other are real
Specific primer, its nucleotide sequence such as seq id no.1 and the seq id no.2 institute of the nearly edge species such as Nuscidae and Diptera
Show.
Described quarantine fruit fly refer to by Anastrepha, Bactrocera, click Anastrepha, little bar Anastrepha, few hair on the neck Anastrepha and/or
Around Anastrepha.
The quarantine fruit fly multiple target rapid identification method that the present invention provides, step is simple and easy to do, only need to extract and treat test sample
The genome dna of product；Carry out pcr using the specific primer that the present invention provides；Detected through gel electrophoresis pcr product, if occur
The specific band of 603bp, then this sample is quarantine fruit fly；Conversely, then this sample is not belonging to quarantine fruit fly.Methods described
It is capable of the sample of the non-adult form of quick detection or adult form, whole qualification process only needs a few hours, need not raise through 1-2 week and be into
Identify again after worm, improve the efficiency of quarantine, without labor intensive material resources, larva is raised, reduce and quarantine into
This；Can in multiple trypetid species, including by Anastrepha, Bactrocera, click Anastrepha, little bar Anastrepha, few hair on the neck Anastrepha and
Specific band is detected in Anastrepha, these trypetids are distinguished with non-quarantine fruit fly and other diptera species, and
Tool versatility and specificity；Can detectable concentration as little as 0.1ng/ul genome dna, sensitivity is high.
In methods described, optimal pcr reaction system and reaction condition are:
2 × taq pcr mastermix 12.5 μ l, dna template 1 μ l, 10 μm of forward primer 0.5 μ l, 10 μm of reverse primers
0.5 μ l, ddh2o 10.5μl.
95 DEG C of denaturations 3min；95 DEG C of degeneration 15s, 60 DEG C of annealing extend 1min, 35 circulations；60 DEG C of extension 1min.
To sum up, the present invention provides specific primer and quarantine fruit fly multiple target rapid identification method, with existing reality
Fly method for quick is compared, and has the advantage that
(1) easy quick: only need to extract sample dna, whether pcr, gel electrophoresiss can Rapid identification testing sample belong to
Quarantine fruit fly, is detected for after adult without cultivating larva again.
(2) high specificity: described primer can distinguish quarantine fruit fly and the nearly edge species such as other Tephritidaes and Diptera,
Qualification result accuracy is high.
(3) sensitivity is high: the inventive method is able to detect that the genome dna of concentration as little as 0.1ng/ul.
(4) highly versatile: methods described can detect multiple trypetid species, overcomes existing method just for indivedual trypetid things
The limitation planted is it is not necessary to Multiple experiments can identify whether testing sample belongs to quarantine fruit fly.
Brief description
Fig. 1. the specificity verification result figure of Important Economic trypetid universal primer,
Wherein, dna marker is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom successively；
Swimming lane 1～44 represents Important Economic trypetid species, non-economy trypetid species, other diptera species and feminine gender respectively
The amplified band of control sample, be followed successively by South America by trypetid a.fraterculus, melon by trypetid a.grandis, Mexico by reality
Trypetid a.obliqua, Jambul fruit fly b.albistrigata, pitch black face fruit fly are pressed by fly a.ludens, western India
B.atrifacies, calabash courd trypetid b.bezziana, Carambola fruit trypetid b.carambolae, common fruit fly b.caudata, two
Band fruit fly b.cilifera, Bactrocera correcta b.correcta, melonfly b.cucurbitae, different face fruit fly
B.diversa, citrus fruit fly b.dorsalis, He Shi China trypetid b.hochii, Sri Lanka fruit fly b.kandiensis, peppery
Green pepper fruit fly b.latifrons, Fructus Citri tangerinae big trypetid b.minax, olea europaea fruit trypetid b.oleae, rust Fructus Pyracanthae trypetid b.rubigina,
India fruit fly b.scutellaris, broadband fruit fly b.scutellata, in reach fruit fly b.synnephes, South Asia fruit
Fly b.tau, Thailand fruit fly b.thailandica, macadamia nut trypetid b.tryoni, bactrocera tsuneonis b.tsuneonis, face
Bag fruit fly b.umbrosa, Wuzhi Mountain fruit fly b.wuzhishana, Yao Shi fruit fly b.yoshimotoi, peach fruit trypetid
B.zonata, jujube fly c.vesuviana, Mediterranean fruitfly c.capitata, Fructus Mangifera Indicae little bar trypetid c.cosyra, Baunatal ear are little
Bar trypetid c.rosa, calabash widow hair on the neck trypetid d.bivittatus, Ethiopia widow hair on the neck trypetid d.ciliatus, angle Fructus Luffae rod abdomen
Trypetid d.longicornis, Fructus Pruni pseudocerasi are around trypetid r.cerasi, Herba Marsileae Quadrifoliae around trypetid r.pomonella, Herba Lycopi trypetid
Procecidochares utilis, Drosophila melanogaster drosophila melanogaster, striped fit Qiang fly adapsilia
Striatis and ddh2o.
Fig. 2. the sensitivity technique result figure of Important Economic trypetid Rapid identification system,
Wherein, Fig. 2 a is by Anastrepha sensitivity technique result, and Fig. 2 b is Bactrocera sensitivity technique result, and Fig. 2 c is
Click Anastrepha sensitivity technique result, Fig. 2 d is little bar Anastrepha sensitivity technique result, and Fig. 2 e is few hair on the neck Anastrepha sensitivity
Testing result, Fig. 2 f is around Anastrepha sensitivity technique result；
Dna marker is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom successively；
Swimming lane 1～7 represent respectively sample concentration be respectively 100ng/ul, 10ng/ul, 1ng/ul, 0.1ng/ul,
Genome dna and ddh of 0.01ng/ul, 0.001ng/ul2o.
Specific embodiment
Below by specific embodiment, the present invention is described in more detail, it is to be understood that, following embodiments are only
As explaining and illustrating, limit the scope of the present invention never in any form.
Biomaterial
Important Economic Anastrepha listed by table 1 (presses Anastrepha, Bactrocera, click Anastrepha, little bar Anastrepha, few hair on the neck
Anastrepha and around Anastrepha) species and other Tephritidaes and Diptera nearly edge species are test sample, are maintained in China's inspection
Quarantine research institute.
Each sample is respectively placed in dehydrated alcohol, -20 DEG C save backup.
Table 1 is for examination trypetid sample type
Main agents
2 × taq pcr mastermix, purchased from TIANGEN Biotech (Beijing) Co., Ltd.；
Blood/cell/tissue genome dna extracts kit, purchased from TIANGEN Biotech (Beijing) Co., Ltd..
In following examples of the present invention, not specified biological chemical reagent is this area conventional reagent, permissible
Prepare according to this area conventional method and obtain or commercially available, specification is the pure level of laboratory.
The design of embodiment 1. Tephritidae Important Economic trypetid universal primer
The mitochondrial genome sequence of all dipteral insect species is retrieved in genbank data base, 146 kinds altogether.
With dnaman software, many sequences are carried out to the mitochondrial genome sequence of all 146 kinds of dipteral insect species obtaining
Row compare.
(adhere to separately by Anastrepha, Bactrocera, click trypetid from Important Economic trypetid known to 18 kinds of mitochondrial genome sequences
Genus, little bar Anastrepha, few hair on the neck Anastrepha and around Anastrepha) trypetid species in screen and can specifically distinguish Important Economic
Trypetid and the conserved positions of the nearly edge species such as other Tephritidaes and Diptera.
To screening obtain conserved positions (such as citrus fruit fly b.dorsalis, its mitochondrial genome sequence
It is dq845759 that genbank numbers, and the position of conserved positions is 3142bp-3744bp) engineer's primer is soft using oligo
Part is estimated to primer, and the specific primer finally screening acquisition is as shown in table 2.
The universal primer of table 2 specificity identification Tephritidae Important Economic trypetid
Embodiment 2. Important Economic trypetid multiple target rapid identification method
1st, dna extracts:
Biological sample:
40 Important Economic trypetid species in table 1, including trypetid species known to 15 kinds of mitochondrial genome sequences and
The unpub trypetid species of 25 kinds of mitochondrial genome sequences, for verifying the versatility of specific primer of the present invention；
Herba Lycopi trypetid procecidochares utilis in table 1, Drosophila melanogaster drosophila
It is other Tephritidaes and Diptera nearly edge species that melanogaster, striped fit Qiang fly adapsilia striatis, is used for testing
Demonstrate,prove the specificity of specific primer of the present invention.
Using " blood/cell/tissue genome dna extracts kit " respectively to single head sample (non-adult form or one-tenth
Worm state) carry out genome dna extraction, concrete operations flow process is with reference to kit specification.
2nd, primer versatility and specificity verification:
As template, the primer using design in embodiment 1 carries out pcr amplification to the genome dna being extracted with step 1, leads to
Cross and continue to optimize pcr reaction reagent consumption and the parameter such as annealing temperature, time, the optimal reaction system of acquisition and reaction condition
As follows.
After amplified reaction terminates, take 5 μ l pcr amplified productions that electrophoresis detection is carried out on 1.5% agarose gel, eb contaminates
Color 15 minutes, observed result under uviol lamp.
Result is as shown in figure 1, all amplify specific band, Er Feijing from the sample of all Important Economic trypetid species
In Ji trypetid and other diptera species, amplified band does not occur.
Respectively specific band is carried out cutting glue reclaim, send company of Sangon Biotech (Shanghai) Co., Ltd. to survey
Sequence, result shows, the size expanding the specific band obtaining from 40 Important Economic trypetid species samples is 603bp,
Sequence alignment result shows, the nucleotide of the amplified band of Important Economic trypetid species of mitochondrial genome sequence known to 15 kinds
The nucleotide sequence of the mitochondrial genome conserved positions that sequence is obtained with embodiment 1 screening is completely the same.
Therefore, the tfdeg/tr primer pair of the present invention is under above-mentioned pcr reaction system and reaction condition, to and only to all
Important Economic trypetid species occur positive reaction, produce 603bp specificity purpose band, to remaining non-economy trypetid and other
Diptera species are negative reaction, and no specific band produces.
3rd, sensitivity technique:
From each Important Economic Anastrepha, in the genome dna extracting in step 1, select the base of trypetid species
Because organizing dna, it is serially diluted with te buffer respectively, is obtained the diluent of 6 Concentraton gradient, wherein genome dna's is dense
Degree is respectively as follows: 100ng/ μ l, 10ng/ μ l, 1ng/ μ l, 0.1ng/ μ l, 0.01ng/ μ l, 0.001ng/ μ l.Respectively with each concentration
The genome dna diluent of gradient is template (distilled water be negative control), with designing in embodiment 1 and that screening obtains is special
Property primer carries out pcr amplification respectively to template dna of variable concentrations, and pcr reaction system and reaction condition are with step 2.
After amplified reaction terminates, take 5ul pcr amplified production that electrophoresis detection is carried out on 1.5% agarose gel, eb contaminates
Color 15 minutes, observed result under uviol lamp.Result as shown in Fig. 2 the detectable template concentrations of the primer of the present invention all up to
To 0.1ng/ul.
Claims (6)
1. it is used for the specific primer of quarantine fruit fly multiple target Rapid identification it is characterised in that its nucleotide sequence such as seq
Shown in id no.1 and seq id no.2；
Described quarantine fruit fly refers to by Anastrepha, Bactrocera, click Anastrepha, little bar Anastrepha, few hair on the neck Anastrepha and/or around reality
Fly belongs to；
Described quarantine fruit fly can be opened by described specific primer with other Tephritidaes and Diptera nearly edge species differentiation.
2. a kind of quarantine fruit fly multiple target rapid identification method is it is characterised in that comprise the steps:
(1) extract the genome dna of testing sample；
(2) with genome dna as template, carry out pcr using the specific primer described in claim 1, obtain amplified production；
(3) detected through gel electrophoresis amplified production, if the specific band of 603bp, this sample belongs to quarantine fruit fly；Instead
It, then this sample is not belonging to quarantine fruit fly.
3. method according to claim 2 is it is characterised in that the reaction system of described pcr is: 2 × taq pcr
Mastermix 12.5 μ l, dna template 1 μ l, 10 μm of forward primer 0.5 μ l, 10 μm of reverse primers 0.5 μ l, ddh2o 10.5μl.
4. according to the method in claim 2 or 3 it is characterised in that the reaction condition of described pcr is: 95 DEG C of denaturations
3min；95 DEG C of degeneration 15s, 60 DEG C of annealing extend 1min, 35 circulations；60 DEG C of extension 1min.
5. a kind of quarantine fruit fly multiple target Rapid identification test kit is it is characterised in that include liquid or powder right
Require the specific primer described in 1.
6. test kit according to claim 5 is it is characterised in that also include the required other conventional reagent of pcr reaction.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055572A (en) * | 2018-09-07 | 2018-12-21 | 中国检验检疫科学研究院 | Primer and method for Bactrocera multiple target Rapid identification |
| CN112322745A (en) * | 2020-08-28 | 2021-02-05 | 上海海关动植物与食品检验检疫技术中心 | Specific primer pair, kit and method for rapidly identifying Bactrocera cucurbitae |
| CN114740134A (en) * | 2022-02-25 | 2022-07-12 | 中国检验检疫科学研究院 | Effective heat treatment method for identifying quarantine fruit flies and application thereof |
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| CN101845495A (en) * | 2010-04-07 | 2010-09-29 | 中华人民共和国四川出入境检验检疫局 | Gene chip of inspection and quarantine fruit fly |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055572A (en) * | 2018-09-07 | 2018-12-21 | 中国检验检疫科学研究院 | Primer and method for Bactrocera multiple target Rapid identification |
| CN112322745A (en) * | 2020-08-28 | 2021-02-05 | 上海海关动植物与食品检验检疫技术中心 | Specific primer pair, kit and method for rapidly identifying Bactrocera cucurbitae |
| CN112322745B (en) * | 2020-08-28 | 2024-04-12 | 上海海关动植物与食品检验检疫技术中心 | Specific primer pair, kit and method for rapidly identifying pumpkin fruit fly |
| CN114740134A (en) * | 2022-02-25 | 2022-07-12 | 中国检验检疫科学研究院 | Effective heat treatment method for identifying quarantine fruit flies and application thereof |
| CN114740134B (en) * | 2022-02-25 | 2024-05-14 | 中国检验检疫科学研究院 | Method for identifying quarantine fruit fly for effective heat treatment and application thereof |
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| Publication number | Publication date |
|---|---|
| CN106367506B (en) | 2019-10-25 |
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