CN102517388A - Kit for identifying fruit flies and special primers therefor - Google Patents
Kit for identifying fruit flies and special primers therefor Download PDFInfo
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Abstract
The invention discloses a kit for identifying fruit flies and special primers therefor. The primers provided in the invention comprise eleven primer pairs from a primer pair 1 to a primer pair 11. Experiments of the invention prove that a method in the invention has the following advantages: researches in the invention combine the monitoring case, port interception information, host information and the like, main economic flies in Chinese mainland and Taiwan area are contained, and the kind representativeness of the contained flies is strong, so the identification result accuracy and the technological practicality are ensured.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of test kit and primer special thereof of identifying trypetid.
Background technology
Anastrepha Diptera (Diptera) Tephritidae (Tephritidae), the whole world extensively distributes, and mostly occurs in the torrid zone and subtropical zone.Tephritidae has 500 to belong to 4500 kinds approximately, kind surplus the trypetid with important economic implications reaches 150, and some trypetid can be caused crushing disaster to fruit production.The trypetid that has Economic Importance in the world mainly contains 5 genus, is respectively to belong to (Bactrocera), little Anastrepha (Ceratitis), few hair on the neck Anastrepha (Dacus) and around Anastrepha (Rhagoletis) by Anastrepha (Anastrepha), fruit fly.Whole nation trypetid monitoring and surveying result shows that China mainland and the trypetid with important economic implications that the Taiwan took place are mainly fruit fly and belong to kind.
Acquisition of information and national trypetid monitoring situation according to the inward Plant Quarantine property harmful organism register of the new revision of China, national agricultural plants quarantine harmful organisms list, China port quarantine property trypetid; And combining expansion Taiwan fruit, vegetables and the fishery products access continent kind bulletin of State General Administration for Quality Supervision in issue in 2006, citrus fruit fly (B. (B.) dorsalis), capsicum trypetid (B. (B.) latifrons), piscidia trypetid (B. (B.) correcta), face band trypetid (B. (B.) cilifera), melon trypetid (B. (Z.) cucurbitae), South Asia fruit fly (B. (Z.) tau), tool bar trypetid (B. (Z.) scutellata), black line trypetid (B. (Z.) caudata), calabash melon trypetid (B. (Z.) nubila), bactrocera tsuneonis (B. (T.) tsuneoni) and the big trypetid of tangerine (B. (T.) minax) belong to kind for a main economic type fruit fly.
The kind of trypetid identifies that common morphological feature with adult is main foundation, requires sample to have the complete morphology structure, and needs the staff to have abundant kind evaluation experience.Simultaneously; Trypetid can carry out remote dispersal by man with host's fruit, packing material and transportation means etc. with ovum, larva, pupa, adult; In the quarantine of the fruits and vegetables that enter the territory, intercepting and capturing are often arranged, and be main, need it is raised and just can carry out kind to adult and identify with non-adult form samples such as ovum, larva, pupas.Therefore, traditional needs that can not satisfy port quarantine and domestic trypetid monitoring based on the trypetid kind authenticate technology of morphological feature.Development along with Protocols in Molecular Biology; Utilizing modern molecular biology technique to carry out the trypetid kind identifies; Do not receive the influence of trypetid sample etap and envrionment conditions; Sample and the incomplete adult sample of morphological structure to non-adult form all can obtain accurately from DNA, reliable authentication information.For the trypetid of non-adult form, need not it is raised to adult especially, just can realize the accurate evaluation of kind, can improve the travelling speed and quarantine quality of port quarantine greatly.
The birth of round pcr makes Protocols in Molecular Biology obtain unprecedented development; This technology has become the basis of Protocols in Molecular Biology; Be widely used in the middle of the research and clinical application of biological and medical science every field, also very extensive in Molecular Identification and the application in the phyletic evolution research of trypetid.The principle of round pcr is similar to DNA natural reproduction process in vivo, constitutes by sex change-annealing-three primitive reaction steps of extension, and be under the effect of Taq archaeal dna polymerase; With dNTP is raw material; Target sequence is a template, according to base complementrity pair principle and semiconservative replication principle, and synthetic new and a template DNA chain complementary chain; Every completion DNA chain that once circulates just increases one times; And can be used as next round-robin template, recirculation sex change-annealing-three processes of extension can obtain more dna double chain.
The special primer round pcr that species are identified is similar with the regular-PCR amplification; Just when carrying out design of primers according to target sequence; Need take into full account the specificity of primer; With the unknown template of the primer amplification of high specificity,, reach and differentiate the purpose that comes to target species and nontarget species through detecting having or not of target fragment.
Because biological gene group quantity is huge, is not easy to carry out comprehensive nucleic acid sequence analysis, need to carry out primary study to the individual characteristics gene fragment in the genome.Therefore, selecting suitable molecular marker gene fragment is the key point of Molecular Identification.A segment length is that the fragment of 648bp is used as one of gene the most commonly used in the zoological taxonomy in the COI gene, promptly in zoological taxonomy, is used as the DNA barcode of standard gene.
Summary of the invention
An object of the present invention is to provide a kind of primer that detects trypetid.
The primer of detection provided by the invention trypetid, by following 11 kinds of primers to forming: primer to the 1-primer to 11;
Said primer is made up of primer 1 and primer 21; Said primer is made up of primer 3 and primer 42; Said primer is made up of primer 5 and primer 63; Said primer is made up of primer 7 and primer 84; Said primer is made up of primer 9 and primer 10 5; Said primer is made up of primer 11 and primer 12 6; Said primer is made up of primer 13 and primer 14 7; Said primer is made up of primer 15 and primer 16 8; Said primer is made up of primer 17 and primer 18 9; Said primer is made up of primer 19 and primer 20 10; Said primer is made up of primer 21 and primer 22 11;
The nucleotides sequence of said primer 1 is classified the sequence 1 in the sequence table as; The nucleotides sequence of said primer 2 is classified the sequence 2 in the sequence table as; The nucleotides sequence of said primer 3 is classified the sequence 3 in the sequence table as; The nucleotides sequence of said primer 4 is classified the sequence 4 in the sequence table as; The nucleotides sequence of said primer 5 is classified the sequence 5 in the sequence table as; The nucleotides sequence of said primer 6 is classified the sequence 6 in the sequence table as; The nucleotides sequence of said primer 7 is classified the sequence 7 in the sequence table as; The nucleotides sequence of said primer 8 is classified the sequence 8 in the sequence table as; The nucleotides sequence of said primer 9 is classified the sequence 9 in the sequence table as; The nucleotides sequence of said primer 10 is classified the sequence 10 in the sequence table as; The nucleotides sequence of said primer 11 is classified the sequence 11 in the sequence table as; The nucleotides sequence of said primer 12 is classified the sequence 12 in the sequence table as; The nucleotides sequence of said primer 13 is classified the sequence 13 in the sequence table as; The nucleotides sequence of said primer 14 is classified the sequence 14 in the sequence table as; The nucleotides sequence of said primer 15 is classified the sequence 15 in the sequence table as; The nucleotides sequence of said primer 16 is classified the sequence 16 in the sequence table as; The nucleotides sequence of said primer 17 is classified the sequence 17 in the sequence table as; The nucleotides sequence of said primer 18 is classified the sequence 18 in the sequence table as; The nucleotides sequence of said primer 19 is classified the sequence 19 in the sequence table as; The nucleotides sequence of said primer 20 is classified the sequence 20 in the sequence table as; The nucleotides sequence of said primer 21 is classified the sequence 21 in the sequence table as; The nucleotides sequence of said primer 22 is classified the sequence 22 in the sequence table as.
In the above-mentioned primer, said trypetid is at least a in citrus fruit fly, capsicum trypetid, piscidia trypetid, face band trypetid, melon trypetid, South Asia fruit fly, tool bar trypetid, black line trypetid, calabash melon trypetid, bactrocera tsuneonis and the big trypetid of tangerine.
Another object of the present invention provides a kind of PCR reagent that detects trypetid.
PCR reagent provided by the invention is made up of PCR reagent 1-PCR reagent 11;
Said PCR reagent 1 by above-mentioned primer to 1, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 2 by above-mentioned primer to 2, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 3 by above-mentioned primer to 3, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 4 by above-mentioned primer to 4, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 5 by above-mentioned primer to 5, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 6 by above-mentioned primer to 6, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 7 by above-mentioned primer to 7, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 8 by above-mentioned primer to 8, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 9 by above-mentioned primer to 9, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 10 by above-mentioned primer to 10, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 11 by above-mentioned primer to 11, dNTP, archaeal dna polymerase and PCR damping fluid form;
The concentration of each primer of above-mentioned primer centering in the above-mentioned PCR reagent of correspondence is 0.6 μ M.
In the above-mentioned PCR reagent, said trypetid is at least a in citrus fruit fly, capsicum trypetid, piscidia trypetid, face band trypetid, melon trypetid, South Asia fruit fly, tool bar trypetid, black line trypetid, calabash melon trypetid, bactrocera tsuneonis and the big trypetid of tangerine.
The 3rd purpose of the present invention provides a kind of test kit that detects trypetid.
Test kit provided by the invention comprises above-mentioned PCR reagent;
Above-mentioned trypetid is specially at least a in citrus fruit fly, capsicum trypetid, piscidia trypetid, face band trypetid, melon trypetid, South Asia fruit fly, tool bar trypetid, black line trypetid, calabash melon trypetid, bactrocera tsuneonis and the big trypetid of tangerine.
The application in evaluation or assistant identification trypetid of above-mentioned primer or above-mentioned PCR reagent or mentioned reagent box also is the scope that the present invention protects; Said trypetid is specially at least a in citrus fruit fly, capsicum trypetid, piscidia trypetid, face band trypetid, melon trypetid, South Asia fruit fly, tool bar trypetid, black line trypetid, calabash melon trypetid, bactrocera tsuneonis and the big trypetid of tangerine.
The 4th purpose of the present invention provides the method for a kind of evaluation or assistant identification trypetid to be measured.
Method provided by the invention comprises the steps:
1) with the said 11 kinds of primers in above-mentioned primer or above-mentioned PCR reagent or the mentioned reagent box to respectively trypetid to be measured being carried out pcr amplification, obtain amplified production, detect amplified production,
If said primer is 216bp to the size of 1 amplified production, trypetid then to be measured is or the candidate is a citrus fruit fly;
If said primer is 211bp to the size of 2 amplified productions, trypetid then to be measured is or the candidate is the capsicum trypetid;
If said primer is 275bp to the size of 3 amplified productions, trypetid then to be measured is or the candidate is the piscidia trypetid;
If said primer is 159bp to the size of 4 amplified productions, trypetid then to be measured is or the candidate is a face band trypetid;
If said primer is 416bp to the size of 5 amplified productions, trypetid then to be measured is or the candidate is the melon trypetid;
If said primer is 284bp to the size of 6 amplified productions, trypetid then to be measured is or the candidate is the South Asia fruit fly;
If said primer is 317bp to the size of 7 amplified productions, trypetid then to be measured is or the candidate is a tool bar trypetid;
If said primer is 181bp to the size of 8 amplified productions, trypetid then to be measured is or the candidate is black line trypetid;
If said primer is 362bp to the size of 9 amplified productions, trypetid then to be measured is or the candidate is a calabash melon trypetid;
If said primer is 337bp to the size of 10 amplified productions, trypetid then to be measured is or the candidate is a bactrocera tsuneonis;
If said primer is 499bp to the size of 11 amplified productions, trypetid then to be measured is or the candidate is the big trypetid of tangerine.
In the aforesaid method, said primer is classified the sequence 23 or 24 in the sequence table as to the nucleotides sequence of 1 amplified production;
Said primer is classified the sequence 25 or 26 in the sequence table as to the nucleotides sequence of 2 amplified productions;
Said primer is classified the sequence 27 or 28 in the sequence table as to the nucleotides sequence of 3 amplified productions;
Said primer is classified the sequence 29 or 30 in the sequence table as to the nucleotides sequence of 4 amplified productions;
Said primer is classified the sequence 31 or 32 in the sequence table as to the nucleotides sequence of 5 amplified productions;
Said primer is classified the sequence 33 or 34 in the sequence table as to the nucleotides sequence of 6 amplified productions;
Said primer is classified the sequence 35 or 36 in the sequence table as to the nucleotides sequence of 7 amplified productions;
Said primer is classified the sequence 37 in the sequence table as to the nucleotides sequence of 8 amplified productions;
Said primer is classified the sequence 38 in the sequence table as to the nucleotides sequence of 9 amplified productions;
Said primer is classified the sequence 39 or 40 in the sequence table as to the nucleotides sequence of 10 amplified productions;
Said primer is classified the sequence 41 or 42 in the sequence table as to the nucleotides sequence of 11 amplified productions.
In the aforesaid method, in pcr amplification, be template with the genomic dna of trypetid to be measured;
The annealing temperature of above-mentioned pcr amplification is 55 ℃.
In the aforesaid method, the method for said detection pcr amplification product is an agarose gel electrophoresis.
The method of above-mentioned detection pcr amplification product also can be order-checking.
Of the present inventionly experiment showed, that method of the present invention has following advantage:
(1) this research combines monitoring situation, port acquisition of information, host's information etc., has related to and has comprised the main economic trypetid kind in China's Mainland and Taiwan, and the trypetid kind that is comprised is representative strong, has guaranteed the accuracy of qualification result and the practicality of technology; The present invention has realized the kind Rapid identification to 11 kinds of main economic type trypetids of China mainland and Taiwan; The kind that has solved non-adult form trypetid sample is identified problem; Rapid identification instrument and the technology of the main economic type trypetid of China mainland and Taiwan can be provided for pass in and out Plant Quarantine and national trypetid epidemic monitoring, can effectively prevent the invasion of dangerous trypetid and diffusion, protection agriculture prodn and ecological security, promotion both sides of the Straits economic trade to develop;
(2) with DNA barcode COI gene as target gene sequences, make this Rapid identification technology have more stdn;
(3) technological highly versatile is less demanding to plant and instrument, in real work, applies easily;
(4) qualification process is easy, quick, economical, only needs about 7 hours from sample preparation to the whole qualification process that goes out the result.
Description of drawings
Fig. 1 is citrus fruit fly primer (primer is to 1) PCR specific detection gel electrophoresis result
Fig. 2 is capsicum trypetid primer (primer is to 2) PCR specific detection gel electrophoresis result
Fig. 3 is piscidia trypetid primer (primer is to 3) PCR specific detection gel electrophoresis result
Fig. 4 is face band trypetid primer (primer is to 4) PCR specific detection gel electrophoresis result
Fig. 5 is melon trypetid primer (primer is to 5) PCR specific detection gel electrophoresis result
Fig. 6 is South Asia fruit fly primer (primer is to 6) PCR specific detection gel electrophoresis result
Fig. 7 is tool bar trypetid primer (primer is to 7) PCR specific detection gel electrophoresis result
Fig. 8 is black line trypetid primer (primer is to 8) PCR specific detection gel electrophoresis result
Fig. 9 is calabash melon trypetid primer (primer is to 9) PCR specific detection gel electrophoresis result
Figure 10 is bactrocera tsuneonis primer (primer is to 10) PCR specific detection gel electrophoresis result
Figure 11 is the big trypetid primer of tangerine (primer is to 11) PCR specific detection gel electrophoresis result
Figure 12 is a citrus fruit fly primer sensitivity detected result
Figure 13 is a capsicum trypetid primer sensitivity detected result
Figure 14 is a piscidia trypetid primer sensitivity detected result
Figure 15 is a face band trypetid primer sensitivity detected result
Figure 16 is a melon trypetid primer sensitivity detected result
Figure 17 is a South Asia fruit fly primer sensitivity detected result
Figure 18 is a tool bar trypetid primer sensitivity detected result
Figure 19 is black line trypetid primer sensitivity detected result
Figure 20 is a calabash melon trypetid primer sensitivity detected result
Figure 21 is a bactrocera tsuneonis primer sensitivity detected result
Figure 22 is the big trypetid primer of a tangerine sensitivity detected result
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
1, the acquisition of trypetid DNA barcode
Can obtain the open sequence of Barcode of citrus fruit fly, capsicum trypetid, piscidia trypetid, melon trypetid and South Asia fruit fly through inquiry in BOLD and GenBank system.The open sequence number of template sequence is respectively citrus fruit fly (DQ116269), capsicum trypetid (DQ116296), piscidia trypetid (DQ116264), melon trypetid (DQ116242), South Asia fruit fly (GU323773.1).
To the target gene sequences that in DB, does not retrieve, use universal primer LCO-1490 (5 '-GGTCAACAAATCATAAAGATATTG-3 ') and HCO-2198 (5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 ') the trypetid genomic dna that has extracted is carried out the purpose fragment amplification.
2, primer design
Compare with the DNA barcode of DNAMAN software to the target species trypetid; According to analytical results; To the artificial design of target gene sequences special primer; And with Oligo software primer is assessed, inspection primer Tm, GC%, mispairing, dimer and hairpin structure etc., and with the Blast programmed inspection homologous sequence that provides among the GenBank.
11 kinds of primers of design are to as shown in table 1 below:
Table 1 is the special primer table look-up
The evaluation of embodiment 2, trypetid
Gather citrus fruit fly, capsicum trypetid, piscidia trypetid, face band trypetid, melon trypetid, South Asia fruit fly, tool bar trypetid, black line trypetid, calabash melon trypetid, bactrocera tsuneonis and the big trypetid of tangerine from different places, wherein,
Citrus fruit fly Bactrocera (Bactrocera) dorsalis (Hendel) is documented in Zhang Bin, Liu Yinghong, Zhao Lanlan, all rising suns. the citrus fruit fly progress, and Chinese agronomy circular, 2008,24 (11): among the 391-397., the public can obtain from China Agricultural University.
Capsicum trypetid Bactrocera (Bactrocera) latifrons (Hendel) is documented in Huang and shakes; Huang Kehui. form, harm and the Quarantine Countermeasures of quarantine harmful organisms---capsicum trypetid, Wuyi science, 2009; 25:21-23. the public can obtain from China Agricultural University.
Piscidia trypetid Bactrocera (Bactrocera) correcta (Bezzi) is documented in Liu Jiaqi, Deng Yuliang, Li Zhihong; Bai Yonghua, Huang Ligong. Fructus psidii guajavae immaturus trypetid Progress on Molecular Biology, plant protection; 2009,35 (2): 11-14., the public can obtain from China Agricultural University.
Face band trypetid Bactrocera (Bactrocera) cilifera (Hendel) is documented in woods Mingguang City, Yang Zujiang, Wang Xingjian; Li Jiyong; Li Weidong. and Hainan few hair on the neck trypetid subfamily sort research (Diptera: Tephritidae), insect journal, 2006; 49 (2): 310-314., the public can obtain from China Agricultural University.
Melon trypetid Bactrocera (Zeugodacus) cucurbitae (Coquillett) is documented in Kong Lingbin, Lin Wei, Li Zhihong; Incomparably great, Wang Zhiling, yellow hat worn by a Taoist priest wins. based on the potential geographical forecast of distribution of the melon trypetid of CLIMEX and DIVA-GIS; The plant protection journal; 2008,35 (2): 148-154., the public can obtain from China Agricultural University.
South Asia fruit fly Bactrocera (Zeugodacus) tau (Walker) is documented in Shi Wei. and South Asia fruit fly molecular biology research overview, 2010,24 (6): 40-41., the public can obtain from China Agricultural University.
Tool bar trypetid Bactrocera (Zeugodacus) scutellata (Hendel) is documented in Wu Shaoying, Xie Guiying, Han Shiping; Mao Hongyan, Zhao Zhongyi, former state brightness. the monitoring of Henan Province's tool bar trypetid population dynamics; Agricultural University Of He'nan's journal; 2009,43 (6): 642-651., the public can obtain from China Agricultural University.
Black line trypetid Bactrocera (Zeugodacus) caudata (Fabricius) is documented in Wu Jiajiao, Liang Fan, Liang Guangqin. and trypetid class important pests is identified atlas [M], Guangdong science and technology press, 2009:66., the public can obtain from China Agricultural University.
Calabash melon trypetid Bactrocera (Zeugodacus) nubila (Hendel) is documented in Liang Guangqin, Yang Guohai, Liang Fan, Si Tubaolu, Liang Xiaodan. Asian-Pacific area bactrocera oligochaeta [M], and Guangdong science and technology press, 1996:236., the public can obtain from China Agricultural University.
Bactrocera tsuneonis Bactrocera (Tetradacus) tsuneoni (Miyake) is documented in Wang Junwei, Li Zhihong, and Chen Hongjun, Geng builds, and Wang Zhiling is incomparably great. and bactrocera tsuneonis is in the right natural disposition research of China, Plant Quarantine, 2009,23 (1): 1-4.
The big trypetid Bactrocera of tangerine (Tetradacus) minax (Enderlein) is documented in Yin Sufen, Zhang Zengchuan, Zhang Xianping, Deng Gensheng; Wang Xiaoe, Liang Qiuxia, Han Ding. the domestic research of citrus fruit fly, Shaanxi agricultural sciences; 2011, (5): 265-267., the public can obtain from China Agricultural University.
Mediterranean fruitfly Ceratitis (Ceratitis) capitata (Wiedemann) is documented in Lin Lili, Ceng Ling, Liang Guangwen; Wu Jiajiao, Gu Yujuan, bang army; Hu Xuenan. the research of Mediterranean fruitfly geographical population genetic variation and genetic differentiation, environmental entomology newspaper, 2010; 32 (4): 469-475., the public can obtain from China Agricultural University.
Collection is numbered the trypetid of 1-22, and concrete collection ground and acquisition time are as shown in table 2:
The employed trypetid sample of this research of table 2 (all having identified its kind)
1, the extraction of trypetid DNA
" blood/cell/tissue genome DNA extracting reagent kit " of employing TIANGEN Biotech (Beijing) Co., Ltd. extracts the genomic dna of the single head trypetid sample that is numbered 1-22 in the above-mentioned table 2; According to the test kit service manual; Slightly adjust, concrete process for extracting is following:
Above-mentioned sample is cleaned 5 times with distilled water, and thieving paper blots and is placed in the buffer pipe that liquid nitrogen is housed, and uses stirring rod that polypide is smashed rapidly and is cell suspension, adds 200 μ l damping fluid GA, and concussion is to thoroughly suspending; Add 30 μ l Proteinase K solution mixings, 56 ℃ of water-bath 3h per hour shake 3 times, and are briefly centrifugal; Add 200 μ l damping fluid GB, fully put upside down mixing, place 10min for 70 ℃, briefly centrifugal; Add 200 μ l absolute ethyl alcohols, fully shake mixing 15s, briefly centrifugal; To go up step gained solution and flocks and all add among the adsorption column CB3, the centrifugal 30s of 12000rpm outwells waste liquid, and adsorption column CB3 is put back in the collection tube; In adsorption column CB3, add 500 μ l damping fluid GD, the centrifugal 30s of 12000rpm outwells waste liquid, and CB3 puts into collection tube with adsorption column; In adsorption column CB3, add 700 μ l rinsing liquid PW, the centrifugal 30s of 12000rpm outwells waste liquid, and CB3 puts into collection tube with adsorption column; In adsorption column CB3, add 500 μ l rinsing liquid PW, the centrifugal 30s of 12000rpm outwells waste liquid; Adsorption column CB3 is put back in the collection tube, and the centrifugal 2min of 12000rpm outwells waste liquid.Adsorption column CB3 uncapped place room temperature to place 5min; Adsorption column CB3 is changed in the clean centrifuge tube, and to the unsettled Dropwise 50 μ l elution buffer TE in the middle part of adsorption film, room temperature is placed 2-5min, the centrifugal 2min of 12000rpm; The centrifugal solution that obtains is added among the adsorption column CB3 again, and room temperature is placed 2min, and the centrifugal 2min of 12000rpm collects solution in the centrifuge tube; 4 ℃ of to be measured or preservations under-20 ℃; Obtain being numbered the genomic dna of the trypetid of 1-22 respectively.
2, PCR specific amplification
Be template with the 1 trypetid genomic dna that is numbered 1-22 that extracts respectively, with the primer in the table 1 to carrying out pcr amplification respectively.
The PCR reaction system is all following:
The pcr amplification system is 52 μ l, wherein comprises ddH
2O 35.6 μ l, 10xBuffer (contain Mg
2+) each 3.0 μ l of 6.0 μ l, 2.5mM dNTPs (the final concentration 0.1mM of dNTPs in reaction system) 2.0 μ l, 0.01mM primer (final concentration of upstream and downstream primer is 0.0006mM), 2.5U/ μ l Taq enzyme 0.4 μ l (the final concentration 0.02U/ μ l of Taq enzyme in reaction system), template DNA 2.0 μ l.
The pcr amplification condition is: 94 ℃ of heating 3 minutes, carry out 30 PCR circulation then: and 94 ℃ of sex change 1 minute, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, and last 72 ℃ were extended 5 minutes.
Amplified reaction is got 22 kind of 5 special amplified production of μ l PCR respectively and on 1.5% sepharose, is carried out electrophoresis detection after finishing, EB dyeing 15 minutes, observations under uv lamp.
The result shown in Fig. 1-11, wherein, M:DNA relative molecular weight standard; 1: the citrus fruit fly that picks up from Hainan; 2: the citrus fruit fly that picks up from Fujian; 3: the capsicum trypetid of picking up from Thailand; 4: pick up from Malay capsicum trypetid; 5: pick up from the brave awake face band trypetid in Yunnan; 6: the face band trypetid of picking up from the Jinghong, Yunnan; 7: the melon trypetid of picking up from Fujian; 8: the melon trypetid of picking up from Yunnan; 9: the South Asia fruit fly that picks up from Zhejiang; 10: the South Asia fruit fly that picks up from Guizhou; 11: the tool bar trypetid of picking up from Yunnan; 12: the tool bar trypetid of picking up from Guizhou; 13: the black line trypetid of picking up from Thailand; 14: pick up from the bactrocera tsuneonis that Guizhou is all spared; 15: the bactrocera tsuneonis that picks up from Yibin, Sichuan; 16: pick up from the big trypetid of the tangerine of all sparing in Guizhou; 17: the big trypetid of tangerine of picking up from Chengdu, Sichuan; 18: the piscidia trypetid of picking up from the Jinghong, Yunnan; 19: the piscidia trypetid of picking up from Yunnan Ban Na; 20: from the Mediterranean fruitfly of Chile; 21: from the Mediterranean fruitfly of Egypt; 22: the calabash melon trypetid of picking up from Guizhou;
Fig. 1 is citrus fruit fly primer (primer is to 1) PCR specific detection gel electrophoresis result, and the result can find out, is numbered the PCR product that 1 and 2 citrus fruit fly obtains 216bp; Through order-checking, the nucleotides sequence that is numbered 1 PCR product is classified the sequence 23 in the sequence table as; The nucleotides sequence that is numbered 2 PCR product is classified the sequence 24 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for special evaluation citrus fruit fly to 1.
Fig. 2 is capsicum trypetid primer (primer is to 2) PCR specific detection gel electrophoresis result, and the result can find out, is numbered the PCR product that 3 and 4 capsicum trypetid obtains 211bp; Through order-checking, the nucleotides sequence that is numbered 3 PCR product is classified the sequence 25 in the sequence table as; The nucleotides sequence that is numbered 4 PCR product is classified the sequence 26 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for special evaluation capsicum trypetid to 2.
Fig. 3 is piscidia trypetid primer (primer is to 3) PCR specific detection gel electrophoresis result, and the result can find out, is numbered the PCR product that 18 and 19 piscidia trypetid obtains 275bp; Through order-checking, the nucleotides sequence that is numbered 18 PCR product is classified the sequence 27 in the sequence table as; The nucleotides sequence that is numbered 19 PCR product is classified the sequence 28 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for special evaluation piscidia trypetid to 3.
Fig. 4 is face band trypetid primer (primer is to 4) PCR specific detection gel electrophoresis result, and the result can find out, is numbered the PCR product that 5 and 6 face band trypetid obtains 159bp; Through order-checking, the nucleotides sequence that is numbered 5 PCR product is classified the sequence 29 in the sequence table as; The nucleotides sequence that is numbered 6 PCR product is classified the sequence 30 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for special evaluation face band trypetid to 4.
Fig. 5 is melon trypetid primer (primer is to 5) PCR specific detection gel electrophoresis result, and the result can find out, is numbered the PCR product that 7 and 8 melon trypetid obtains 416bp; Through order-checking, the nucleotides sequence that is numbered 7 PCR product is classified the sequence 31 in the sequence table as; The nucleotides sequence that is numbered 8 PCR product is classified the sequence 32 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for special evaluation melon trypetid to 5.
Fig. 6 is South Asia fruit fly primer (primer is to 6) PCR specific detection gel electrophoresis result, and the result can find out, is numbered the PCR product that 9 and 10 South Asia fruit fly obtains 284bp; Through order-checking, the nucleotides sequence that is numbered 9 PCR product is classified the sequence 33 in the sequence table as; The nucleotides sequence that is numbered 10 PCR product is classified the sequence 34 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for special evaluation South Asia fruit fly to 6.
Fig. 7 is tool bar trypetid primer (primer is to 7) PCR specific detection gel electrophoresis result, and the result can find out, is numbered the PCR product that 11 and 12 tool bar trypetid obtains 317bp; Through order-checking, the nucleotides sequence that is numbered 11 PCR product is classified the sequence 35 in the sequence table as; The nucleotides sequence that is numbered 12 PCR product is classified the sequence 36 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for special evaluation tool bar trypetid to 7.
Fig. 8 be for black line trypetid primer (primer is to 8) PCR specific detection gel electrophoresis result, result can find out, is numbered the PCR product that 13 black line trypetid obtains 181bp; Through order-checking, the nucleotides sequence that is numbered 13 PCR product is classified the sequence 37 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for the black line trypetid of special evaluation to 8.
Fig. 9 is calabash melon trypetid primer (primer is to 9) PCR specific detection gel electrophoresis result, and the result can find out, is numbered the PCR product that 22 calabash melon trypetid obtains 362bp; Through order-checking, the nucleotides sequence that is numbered 22 PCR product is classified the sequence 38 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for special evaluation calabash melon trypetid to 9.
Figure 10 is bactrocera tsuneonis primer (primer is to 10) PCR specific detection gel electrophoresis result, and the result can find out, is numbered the PCR product that 14 and 15 bactrocera tsuneonis obtains 337bp; The nucleotides sequence that is numbered 14 PCR product is classified the sequence 39 in the sequence table as; The nucleotides sequence that is numbered 15 PCR product is classified the sequence 40 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for special evaluation bactrocera tsuneonis to 10.
Figure 11 is the big trypetid primer of tangerine (primer is to 11) PCR specific detection gel electrophoresis result, and the result can find out, is numbered 16 and 17 the big trypetid of the tangerine PCR product to 499bp; Through order-checking, the nucleotides sequence that is numbered 16 PCR product is classified the sequence 41 in the sequence table as; The nucleotides sequence that is numbered 17 PCR product is classified the sequence 42 in the sequence table as; Other trypetids do not have the PCR product, explain that primer can be used for the big trypetid of special evaluation tangerine to 11.
Above-mentioned experimental result shows that the primer of table 1 can be realized the Rapid identification of the main economic type trypetid of 11 kinds of China to can be used as the combination primer sets.
3, the right sensitivity of primer detects
With above-mentioned 1 obtain being numbered 1 citrus fruit fly, be numbered 3 capsicum trypetid, be numbered 18 piscidia trypetid, be numbered 5 face band trypetid, be numbered 7 melon trypetid, be numbered 9 South Asia fruit fly, be numbered 11 tool bar trypetid, be numbered 13 black line trypetid, be numbered 22 calabash melon trypetid, be numbered 14 bactrocera tsuneonis, the genomic dna that is numbered 16 the big trypetid of tangerine all carries out serial dilution with the TE damping fluid, obtains 8 kinds of diluents; Genomic dna concentration in 8 kinds of diluents is not for being: 100ng/ μ l, 50ng/ μ l, 25ng/ μ l, 10ng/ μ l, 1ng/ μ l, 0.1ng/ μ l, 0.01ng/ μ l, 0.001ng/ μ l.Be template (contrast of water belongs with yin property) with every kind of diluent respectively, with above-mentioned 11 pairs of special primers to respectively its special kind being carried out pcr amplification.
Reaction system, condition are with 2.
Amplified reaction is got the special amplified production of 5 μ l PCR and on 1.5% sepharose, is carried out electrophoresis detection after finishing, EB dyeing 15 minutes, observations under uv lamp.
Each diluent pcr amplification product electrophoresis result is seen Figure 12-22, among the figure, and M:DNA relative molecular weight standard; Swimming lane 1-8 is followed successively by diluent 1 to diluent 8, and corresponding DNA concentration is respectively 100ng/ μ l, 50ng/ μ l, 25ng/ μ l, 10ng/ μ l, 1ng/ μ l, 0.1ng/ μ l, 0.01ng/ μ l, 0.001ng/ μ l; Swimming lane 9 negative contrasts.
Figure 12 is a citrus fruit fly primer sensitivity detected result, can find out, sensitivity is 1ng/ μ l; Figure 13 is a capsicum trypetid primer sensitivity detected result, can find out, sensitivity is 1ng/ μ l; Figure 14 is a piscidia trypetid primer sensitivity detected result, can find out, sensitivity is 1ng/ μ l; Figure 15 is a face band trypetid primer sensitivity detected result, can find out that sensitivity is 1ng/ μ l; Figure 16 is a melon trypetid primer sensitivity detected result, can find out, sensitivity is 0.1ng/ μ l; Figure 17 is a South Asia fruit fly primer sensitivity detected result, can find out, sensitivity is 1ng/ μ l; Figure 18 is a tool bar trypetid primer sensitivity detected result, can find out that sensitivity is 1ng/ μ l; Figure 19 can find out that for black line trypetid primer sensitivity detected result sensitivity is 0.1ng/ μ l; Figure 20 is a calabash melon trypetid primer sensitivity detected result, can find out that sensitivity is 1ng/ μ l; Figure 21 is a bactrocera tsuneonis primer sensitivity detected result, can find out, sensitivity is 0.1ng/ μ l; Figure 22 is the big trypetid primer of a tangerine sensitivity detected result, can find out, sensitivity is 1ng/ μ l;
The result can find out that detectable template concentrations all can reach 1ng/ μ l, have in addition can reach 0.1ng/ μ l, and after template DNA concentration arrived certain altitude, the brightness of purpose band did not increase with the rising of its concentration.
Claims (10)
1. primer that detects trypetid, by following 11 kinds of primers to forming: primer to the 1-primer to 11;
Said primer is made up of primer 1 and primer 21;
Said primer is made up of primer 3 and primer 42;
Said primer is made up of primer 5 and primer 63;
Said primer is made up of primer 7 and primer 84;
Said primer is made up of primer 9 and primer 10 5;
Said primer is made up of primer 11 and primer 12 6;
Said primer is made up of primer 13 and primer 14 7;
Said primer is made up of primer 15 and primer 16 8;
Said primer is made up of primer 17 and primer 18 9;
Said primer is made up of primer 19 and primer 20 10;
Said primer is made up of primer 21 and primer 22 11;
The nucleotides sequence of said primer 1 is classified the sequence 1 in the sequence table as;
The nucleotides sequence of said primer 2 is classified the sequence 2 in the sequence table as;
The nucleotides sequence of said primer 3 is classified the sequence 3 in the sequence table as;
The nucleotides sequence of said primer 4 is classified the sequence 4 in the sequence table as;
The nucleotides sequence of said primer 5 is classified the sequence 5 in the sequence table as;
The nucleotides sequence of said primer 6 is classified the sequence 6 in the sequence table as;
The nucleotides sequence of said primer 7 is classified the sequence 7 in the sequence table as;
The nucleotides sequence of said primer 8 is classified the sequence 8 in the sequence table as;
The nucleotides sequence of said primer 9 is classified the sequence 9 in the sequence table as;
The nucleotides sequence of said primer 10 is classified the sequence 10 in the sequence table as;
The nucleotides sequence of said primer 11 is classified the sequence 11 in the sequence table as;
The nucleotides sequence of said primer 12 is classified the sequence 12 in the sequence table as;
The nucleotides sequence of said primer 13 is classified the sequence 13 in the sequence table as;
The nucleotides sequence of said primer 14 is classified the sequence 14 in the sequence table as;
The nucleotides sequence of said primer 15 is classified the sequence 15 in the sequence table as;
The nucleotides sequence of said primer 16 is classified the sequence 16 in the sequence table as;
The nucleotides sequence of said primer 17 is classified the sequence 17 in the sequence table as;
The nucleotides sequence of said primer 18 is classified the sequence 18 in the sequence table as;
The nucleotides sequence of said primer 19 is classified the sequence 19 in the sequence table as;
The nucleotides sequence of said primer 20 is classified the sequence 20 in the sequence table as;
The nucleotides sequence of said primer 21 is classified the sequence 21 in the sequence table as;
The nucleotides sequence of said primer 22 is classified the sequence 22 in the sequence table as.
2. primer according to claim 1 is characterized in that:
Said trypetid is at least a in citrus fruit fly, capsicum trypetid, piscidia trypetid, face band trypetid, melon trypetid, South Asia fruit fly, tool bar trypetid, black line trypetid, calabash melon trypetid, bactrocera tsuneonis and the big trypetid of tangerine.
3. a PCR reagent that detects trypetid is made up of PCR reagent 1-PCR reagent 11;
Said PCR reagent 1 by the primer in claim 1 or the 2 described primers to 1, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 2 by the primer in claim 1 or the 2 described primers to 2, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 3 by the primer in claim 1 or the 2 described primers to 3, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 4 by the primer in claim 1 or the 2 described primers to 4, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 5 by the primer in claim 1 or the 2 described primers to 5, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 6 by the primer in claim 1 or the 2 described primers to 6, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 7 by the primer in claim 1 or the 2 described primers to 7, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 8 by the primer in claim 1 or the 2 described primers to 8, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 9 by the primer in claim 1 or the 2 described primers to 9, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 10 by the primer in claim 1 or the 2 described primers to 10, dNTP, archaeal dna polymerase and PCR damping fluid form;
Said PCR reagent 11 by the primer in claim 1 or the 2 described primers to 11, dNTP, archaeal dna polymerase and PCR damping fluid form;
The concentration of each primer of said primer centering in the said PCR reagent of correspondence is 0.6 μ M.
4. reagent according to claim 3 is characterized in that: said trypetid is at least a in citrus fruit fly, capsicum trypetid, piscidia trypetid, face band trypetid, melon trypetid, South Asia fruit fly, tool bar trypetid, black line trypetid, calabash melon trypetid, bactrocera tsuneonis and the big trypetid of tangerine.
5. a test kit that detects trypetid comprises claim 3 or 4 described PCR reagent; Said trypetid is specially at least a in citrus fruit fly, capsicum trypetid, piscidia trypetid, face band trypetid, melon trypetid, South Asia fruit fly, tool bar trypetid, black line trypetid, calabash melon trypetid, bactrocera tsuneonis and the big trypetid of tangerine.
6. claim 1 or 2 said primers or claim 3 or 4 said PCR reagent or the said test kit of claim 5 application in evaluation or assistant identification trypetid; Said trypetid is specially at least a in citrus fruit fly, capsicum trypetid, piscidia trypetid, face band trypetid, melon trypetid, South Asia fruit fly, tool bar trypetid, black line trypetid, calabash melon trypetid, bactrocera tsuneonis and the big trypetid of tangerine.
7. identify or the method for assistant identification trypetid for one kind, comprise the steps:
1) with the said 11 kinds of primers in the primer in claim 1 or 2 said primers or claim 3 or 4 said PCR reagent or the said test kit of claim 5 to respectively trypetid to be measured being carried out pcr amplification, obtain amplified production,
Detect amplified production,
If said primer is 216bp to the size of 1 amplified production, trypetid then to be measured is or the candidate is a citrus fruit fly;
If said primer is 211bp to the size of 2 amplified productions, trypetid then to be measured is or the candidate is the capsicum trypetid;
If said primer is 275bp to the size of 3 amplified productions, trypetid then to be measured is or the candidate is the piscidia trypetid;
If said primer is 159bp to the size of 4 amplified productions, trypetid then to be measured is or the candidate is a face band trypetid;
If said primer is 416bp to the size of 5 amplified productions, trypetid then to be measured is or the candidate is the melon trypetid;
If said primer is 284bp to the size of 6 amplified productions, trypetid then to be measured is or the candidate is the South Asia fruit fly;
If said primer is 317bp to the size of 7 amplified productions, trypetid then to be measured is or the candidate is a tool bar trypetid;
If said primer is 181bp to the size of 8 amplified productions, trypetid then to be measured is or the candidate is black line trypetid;
If said primer is 362bp to the size of 9 amplified productions, trypetid then to be measured is or the candidate is a calabash melon trypetid;
If said primer is 337bp to the size of 10 amplified productions, trypetid then to be measured is or the candidate is a bactrocera tsuneonis;
If said primer is 499bp to the size of 11 amplified productions, trypetid then to be measured is or the candidate is the big trypetid of tangerine.
8. according to the method for claim 7, it is characterized in that:
Said primer is classified the sequence 23 or 24 in the sequence table as to the nucleotides sequence of 1 amplified production;
Said primer is classified the sequence 25 or 26 in the sequence table as to the nucleotides sequence of 2 amplified productions;
Said primer is classified the sequence 27 or 28 in the sequence table as to the nucleotides sequence of 3 amplified productions;
Said primer is classified the sequence 29 or 30 in the sequence table as to the nucleotides sequence of 4 amplified productions;
Said primer is classified the sequence 31 or 32 in the sequence table as to the nucleotides sequence of 5 amplified productions;
Said primer is classified the sequence 33 or 34 in the sequence table as to the nucleotides sequence of 6 amplified productions;
Said primer is classified the sequence 35 or 36 in the sequence table as to the nucleotides sequence of 7 amplified productions;
Said primer is classified the sequence 37 in the sequence table as to the nucleotides sequence of 8 amplified productions;
Said primer is classified the sequence 38 in the sequence table as to the nucleotides sequence of 9 amplified productions;
Said primer is classified the sequence 39 or 40 in the sequence table as to the nucleotides sequence of 10 amplified productions;
Said primer is classified the sequence 41 or 42 in the sequence table as to the nucleotides sequence of 11 amplified productions.
9. according to the method for claim 7 or 8, it is characterized in that:
In the said pcr amplification, be template with the genomic dna of trypetid to be measured;
The annealing temperature of said pcr amplification is 55 ℃.
10. according to arbitrary described method among the claim 7-9, it is characterized in that:
The method of said detection pcr amplification product is an agarose gel electrophoresis.
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-
2011
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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 |
| CN101805792A (en) * | 2010-04-08 | 2010-08-18 | 中华人民共和国上海出入境检验检疫局 | Real-time fluorescence PCR (Polymerase Chain Reaction) detecting method of Bactrocera philippinensis as well as primer and probe for detection |
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