CN106367506B - A kind of quarantine fruit fly multiple target rapid identification method - Google Patents
A kind of quarantine fruit fly multiple target rapid identification method Download PDFInfo
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Abstract
The present invention relates to trypetid Quarantine Techniques, and in particular to a kind of quarantine fruit fly multiple target rapid identification method includes the following steps: the genomic DNA for extracting sample to be tested；Using genomic DNA as template, PCR is carried out using specific primer shown in SEQ ID NO.1 and SEQ ID NO.2；Detected through gel electrophoresis PCR product, if there is the specific band of 603bp, which belongs to quarantine fruit fly；Conversely, then the sample is not belonging to quarantine fruit fly.The quarantine fruit fly refers to by Anastrepha, Bactrocera, click Anastrepha, small Anastrepha, few hair on the neck Anastrepha and/or around Anastrepha；The specific primer can open quarantine fruit fly edge species differentiation close with other Tephritidaes and Diptera, versatile, be the incoming and diffusion of effective prevention and control quarantine fruit fly, and protection China's ecological safety provides strong technical support.
Description
Technical field
The present invention relates to trypetid Quarantine Techniques, and in particular to a kind of quarantine fruit fly multiple target rapid identification method.
Background technique
Trypetid Fruit Flies belongs to Diptera Diptera Tephritidae Tephritidae insect, and the whole world is widely distributed,
It mostly occurs in subtropical and tropical zones.The world is known, and there are about 500 4500 kinds of categories, wherein about 1500 kinds are related with various fruits,
Trypetid with economic significance is up to more than 250.Host range is wide for trypetid, with adult water fruits and vegetables fruit surface lay eggs shape
At hole, larva feeding inside fruit is caused harm, and fruit rot or shedding are caused, cause harm can cause up to 100% economy
Loss not only produces fruits and vegetables and constitutes serious harm, but also brings and seriously affect to foreign trade.
Mainly there are 5 categories in the whole world by the trypetid of quarantine concern, that is, presses Anastrepha Anastrepha, Bactrocera
Bactrocera, small Anastrepha Ceratitis, widow hair on the neck Anastrepha Dacus and around Anastrepha Rhagoletis.It is real in China
Fly invasion prevention and control are highly valued.In 2007 by the Ministry of Agriculture and State Administration for Quality Supervision and Inspection and Quarantine's joint publication
" People's Republic of China (PRC) enter the territory plant quarantine harmful organism register " in, quarantine fruit fly includes to press Anastrepha, fruit fly
Category, small Anastrepha, few hair on the neck trypetid (non-China seed), around 10 kinds such as trypetids (non-China seed)；2 months 2013, the Ministry of Agriculture
It has issued " state key manages alien species register (first) " (No. 1897 bulletin), has included 3 kinds of trypetid classes in the register
Pest, respectively citrus fruit fly B.dorsalis, melonfly B.cucurbitae and jujube fly Carpomya vesuviana.Together
When, the acquisition of information of China port last decade is shown, often has trypetid intercepting and capturing in passenger's belongings and cargo quarantine, and type is numerous
It is more.
The trypetid of intercept and capture is mostly non-adult form, could need to greatly be dropped by precise Identification after raising in 1-2 weeks is adult
The low timeliness of quarantine.With the rapid development of international trade, tourism and traffic, the risk of trypetid invasion increasingly increases
Add, easily outburst causes disaster and leads to huge economic loss after invasion, and whole world invasion has attracted great attention, quarantine identification
The demand of especially Rapid identification is more urgent.
Nearly ten years, different kinds of molecules biology techniques have been applied to the Rapid identification of trypetid, such as: DNA bar code technology
(DNA Barcoding), round pcr, restriction fragment length polymorphism technology (Restriction Fragment Length
Polymorphism, RFLP), random amplified polymorphic DNA technique (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 individual trypetid species
Method still fails to fully meet in the inward plant quarantine harmful organism register in China as unit of trypetid superior classification rank member
Detection demand.
It is desirable to the multiple target Rapid Identification system of Tephritidae Important Economic trypetid be established, to improve China
Border Quarantine Techniques are horizontal, more fully and effectively take precautions against the invasion of quarantine fruit fly, it is inhibited to colonize and spread sprawling, ensure
China's agricultural and forestry production and ecological safety.
Summary of the invention
The limitation of individual trypetids can only be detected in order to make up existing trypetid rapid identification method, the present invention provides one kind
Trypetid multiple target rapid identification method and its specific primer have broad applicability, quarantine means and measure have been reformed, to have
The incoming and diffusion for imitating prevention and control quarantine fruit fly protects China's agricultural production and ecology across external plant quarantine technical barrier
Safety provides strong technical support.
The claimed technical solution of the present invention is as follows:
Specific primer for quarantine fruit fly multiple target Rapid identification, which is characterized in that its nucleotide sequence such as SEQ
Shown in ID NO.1 and SEQ ID NO.2；
The quarantine fruit fly refer to by Anastrepha, Bactrocera, click Anastrepha, small Anastrepha, few hair on the neck Anastrepha and/or
Around Anastrepha；
The specific primer can open quarantine fruit fly edge species differentiation close with other Tephritidaes and Diptera.
A kind of quarantine fruit fly multiple target rapid identification method, which comprises the steps of:
(1) genomic DNA of sample to be tested is extracted；
(2) using genomic DNA as template, PCR is carried out using above-mentioned specific primer, obtains amplified production；
(3) detected through gel electrophoresis amplified production, if there is the specific band of 603bp, which belongs to quarantine reality
Fly；Conversely, then the sample is not belonging to quarantine fruit fly.
Preferably, the reaction system of the PCR are as follows: 2 × Taq PCR MasterMix, 12.5 μ l, DNA profiling 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 the PCR are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 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 kit, which is characterized in that including liquid or powdered upper
State specific primer.
Preferably, the kit further includes other conventional reagents needed for PCR reaction.
The present invention is to meet in the inward plant quarantine harmful organism register in China as unit of trypetid superior classification rank member
Detection demand, retrieved the mitochondrial genomes sequence of all dipteral insect species from GenBank database and carry out
Multiple Sequence Alignment, has screened the conserved positions of Important Economic Anastrepha (i.e. quarantine fruit fly).With the nucleosides of conserved positions
Acid sequence is the multipair primer of stencil design and assesses it that finally screening obtains that quarantine fruit fly and other realities can be distinguished
The specific primer of the nearly edge species such as Nuscidae and Diptera, nucleotide sequence such as SEQ ID NO.1 and SEQ ID NO.2 institute
Show.
The quarantine fruit fly refer to by Anastrepha, Bactrocera, click Anastrepha, small Anastrepha, few hair on the neck Anastrepha and/or
Around Anastrepha.
Quarantine fruit fly multiple target rapid identification method provided by the invention, step is simple and easy to do, need to only extract to test sample
The genomic DNA of product；PCR is carried out using specific primer provided by the invention；Detected through gel electrophoresis PCR product, if occurring
The specific band of 603bp, then the sample is quarantine fruit fly；Conversely, then the sample is not belonging to quarantine fruit fly.The method
The sample of non-adult form or adult form can quickly be detected, entire qualification process only needs a few hours, without through 1-2 weeks raising at
Identified again after worm, improve the efficiency of quarantine, larva is raised without spending human and material resources, reduce quarantine at
This；Can in multiple trypetid species, including by Anastrepha, Bactrocera, click Anastrepha, small Anastrepha, few hair on the neck Anastrepha and
Specific band is detected in Anastrepha, these trypetids and non-quarantine fruit fly and other diptera species are distinguished, it is simultaneous
Have versatility and specificity；Detectable genomic DNA of the concentration down to 0.1ng/ul, high sensitivity.
In the method, optimal PCR reaction system and reaction condition are as follows:
2 × Taq PCR MasterMix, 12.5 μ l, DNA profiling 1 μ l, 10 μM of 0.5 μ l of forward primer, 10 μM of reverse primers
0.5 μ l, ddH2O 10.5μl。
95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 60 DEG C of annealing extend 1min, 35 circulations；60 DEG C of extension 1min.
To sum up, specific primer and quarantine fruit fly multiple target rapid identification method provided by the invention, with existing reality
Fly rapid detection method is compared, and is had the advantage that
(1) it is easy quickly: need to only extract sample DNA, PCR, gel electrophoresis can Rapid identification sample to be tested whether belong to
Quarantine fruit fly detects after adult again without cultivating larva.
(2) high specificity: the primer can distinguish quarantine fruit fly and the nearly edge species such as other Tephritidaes and Diptera,
Qualification result accuracy is high.
(3) high sensitivity: the method for the present invention is able to detect that concentration down to the genomic DNA of 0.1ng/ul.
(4) versatile: the method can detect multiple trypetid species, overcome existing method just for individual trypetid objects
The limitation of kind, not needing Multiple experiments can identify whether sample to be tested belongs to quarantine fruit fly.
Detailed description of the invention
The specificity verification result figure of Fig. 1 Important Economic trypetid universal primer,
Wherein, DNA Marker is successively 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom；
Swimming lane 1~44 respectively indicates Important Economic trypetid species, non-economy trypetid species, other diptera species and feminine gender
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, Portugal fruit fly B.albistrigata, pitch black face fruit fly press in fly A.ludens, western India
B.atrifacies, calabash melonfly 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
It is B.diversa, citrus fruit fly B.dorsalis, He Shi China trypetid B.hochii, Sri Lanka fruit fly B.kandiensis, peppery
The big trypetid B.minax of green pepper fruit fly B.latifrons, tangerine, olea europaea fruit trypetid B.oleae, rust red fruit 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
Packet fruit fly B.umbrosa, Wuzhi Mountain fruit fly B.wuzhishana, Yao Shi fruit fly B.yoshimotoi, Peach fruits fly
B.zonata, jujube fly C.vesuviana, Mediterranean fruitfly C.capitata, small trypetid C.cosyra of mango, Natta ear are small
Trypetid C.rosa, cucurbit widow hair on the neck trypetid D.bivittatus, Ethiopia widow hair on the neck trypetid D.ciliatus, angle sponge gourd rod abdomen
Trypetid D.longicornis, cherry are around trypetid R.cerasi, apple around trypetid R.pomonella, Herba Lycopi trypetid
Procecidochares utilis, Drosophila melanogaster Drosophila melanogaster, striped fit Qiang fly Adapsilia
Striatis and ddH2O。
The sensitivity technique result figure of Fig. 2 Important Economic trypetid Rapid identification system,
Wherein, Fig. 2A is by Anastrepha sensitivity technique as a result, Fig. 2 B is Bactrocera sensitivity technique as a result, Fig. 2 C is
Click Anastrepha sensitivity technique is as a result, Fig. 2 D is small Anastrepha sensitivity technique as a result, 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 successively 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom；
Swimming lane 1~7 respectively indicate sample concentration be respectively 100ng/ul, 10ng/ul, 1ng/ul, 0.1ng/ul,
The genomic 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 explanation and illustration, do not limit the scope of the invention in any way.
Biomaterial
Important Economic Anastrepha listed by table 1 is (i.e. by Anastrepha, Bactrocera, click Anastrepha, small Anastrepha, few hair on the neck
Anastrepha with around Anastrepha) species edge species close with other Tephritidaes and Diptera be test sample, be maintained in Chinese inspection
Quarantine research institute.
Each sample is respectively placed in dehydrated alcohol, -20 DEG C save backup.
Table 1 is for trying trypetid sample type
Main agents
2 × Taq PCR MasterMix is purchased from TIANGEN Biotech (Beijing) Co., Ltd.；
Blood/cell/tissue genome DNA extracting reagent kit is purchased from TIANGEN Biotech (Beijing) Co., Ltd..
In following embodiment of the present invention, not specified biological chemical reagent is this field conventional reagent, can be with
It is obtained according to conventional method in that art preparation or commercially available, specification is the pure grade in laboratory.
The design of 1. Tephritidae Important Economic trypetid universal primer of embodiment
The mitochondrial genomes sequence of all dipteral insect species is retrieved in GenBank database, amounts to 146 kinds.
More sequences are carried out with DNAMAN software to the mitochondrial genomes sequence of all 146 kinds of dipteral insect species of acquisition
Column compare.
The Important Economic trypetid known to 18 kinds of mitochondrial genomes sequences (is adhered to separately by Anastrepha, Bactrocera, click trypetid
Category, small Anastrepha, few hair on the neck Anastrepha and around Anastrepha) trypetid species in screen and can specifically distinguish Important Economic
The conserved positions of trypetid and the nearly edge species such as other Tephritidaes and Diptera.
Screening is obtained conserved positions (such as citrus fruit fly B.dorsalis, mitochondrial genomes sequence
GenBank number is DQ845759, and the position of conserved positions is 3142bp-3744bp) engineer's primer is soft using Oligo
Part assesses primer, and the specific primer for finally screening acquisition is as shown in table 2.
The universal primer of 2 specificity identification Tephritidae Important Economic trypetid of table
2. Important Economic trypetid multiple target rapid identification method of embodiment
1, DNA is extracted:
Biological sample:
40 Important Economic trypetid species in table 1, including trypetid species known to 15 kinds of mitochondrial genomes sequences and
The unpub trypetid species of 25 kinds of mitochondrial genomes sequences, for verifying the versatility of specific primer of the present invention；
Herba Lycopi trypetid Procecidochares utilis, Drosophila melanogaster Drosophila in table 1
It is other Tephritidaes and the nearly edge species of Diptera that melanogaster, striped, which fit Qiang fly Adapsilia striatis, for testing
Demonstrate,prove the specificity of specific primer of the present invention.
Utilize " blood/cell/tissue genome DNA extracting reagent kit " respectively to single head sample (non-adult form or at
Worm state) extracting genome DNA is carried out, concrete operations process is referring to kit specification.
2, primer versatility and specificity verification:
Using the genomic DNA extracted in step 1 as template, PCR amplification is carried out using the primer designed in embodiment 1, is led to
It crosses and continues to optimize the parameters such as PCR reaction reagent dosage and annealing temperature, time, the optimal reaction system and reaction condition of acquisition
It is as follows.
After amplified reaction, 5 μ l pcr amplification products is taken to carry out electrophoresis detection, EB dye on 1.5% Ago-Gel
Color 15 minutes, result was observed in the UV lamp.
As a result as shown in Figure 1, amplifying specific band, Er Feijing from the sample of all Important Economic trypetid species
There is not amplified band in Ji trypetid and other diptera species.
Gel extraction is carried out to specific band respectively, company, Sangon Biotech (Shanghai) Co., Ltd. is sent to survey
Sequence, the results showed that, the size of the specific band expanded from 40 Important Economic trypetid species samples is 603bp,
Sequence alignment result shows the nucleotide of the amplified band of the Important Economic trypetid species of mitochondrial genomes sequence known to 15 kinds
Sequence and the nucleotide sequence that embodiment 1 screens obtained mitochondrial genomes conserved positions are completely the same.
Therefore, TFdeg/TR primer pair of the invention is under above-mentioned PCR reaction system and reaction condition, pair and only to all
Positive reaction occurs for Important Economic trypetid species, the specific purpose band of 603bp is generated, to remaining non-economy trypetid and other
Diptera species are negative reaction, and no specific band generates.
3, sensitivity technique:
In the genomic DNA extracted in step 1, the base of a trypetid species is selected from each Important Economic Anastrepha
It because of a group DNA, is serially diluted respectively with TE buffer, obtains the dilution of 6 concentration gradients, wherein genomic DNA 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 genomic DNA dilution of gradient is template (distilled water is negative control), is obtained with design in embodiment 1 and screening special
Property primer PCR amplification is carried out to the template DNA of various concentration respectively, PCR reaction system and reaction condition is the same as step 2.
After amplified reaction, 5ul pcr amplification product is taken to carry out electrophoresis detection, EB dye on 1.5% Ago-Gel
Color 15 minutes, result was observed in the UV lamp.As a result as shown in Fig. 2, the detectable template concentrations of primer of the invention are reachable
To 0.1ng/ul.
Claims (5)
1. being used for the specific primer of quarantine fruit fly multiple target Rapid identification, which is characterized in that its nucleotide sequence such as SEQ
Shown in ID NO.1 and SEQ ID NO.2；
The quarantine fruit fly refers to by Anastrepha, Bactrocera, click Anastrepha, small Anastrepha, few hair on the neck Anastrepha and/or around reality
Fly category；
The specific primer can open quarantine fruit fly edge species differentiation close with other Tephritidaes and Diptera.
2. a kind of quarantine fruit fly multiple target rapid identification method, which comprises the steps of:
(1) genomic DNA of sample to be tested is extracted；
(2) using genomic DNA as template, PCR is carried out using specific primer described in claim 1, obtains amplified production；
(3) detected through gel electrophoresis amplified production, if there is the specific band of 603bp, which belongs to quarantine fruit fly；Instead
It, then the sample is not belonging to quarantine fruit fly.
3. according to the method described in claim 2, it is characterized in that, the reaction system of the PCR are as follows: 2 × Taq PCR
12.5 μ l of MasterMix, DNA profiling 1 μ l, 10 μM of forward primers 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, which is characterized in that the reaction condition of the PCR are as follows: 95 DEG C of initial denaturations
3min；95 DEG C of denaturation 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 kit, which is characterized in that including liquid or powdered right
It is required that specific primer described in 1.
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| 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 |
| 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 |
| 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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| 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 |
| CN104232756A (en) * | 2014-02-14 | 2014-12-24 | 青岛农业大学 | Dangerous tephritidae pest gene detection chip and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104232756A (en) * | 2014-02-14 | 2014-12-24 | 青岛农业大学 | Dangerous tephritidae pest gene detection chip and preparation method thereof |
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| Title |
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| A high-throughput detection method for invasive fruit fly(Diptera:Tephritidae) species based on microfluidic dynamic array;Fan Jiang et al.;《Mol Ecol Resour》;20160705;第16卷(第6期);1378-1388 * |
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