CN104894237A - Integrated fluidic chip for identification of fruit fly type and use thereof - Google Patents

Integrated fluidic chip for identification of fruit fly type and use thereof Download PDF

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CN104894237A
CN104894237A CN201510219803.0A CN201510219803A CN104894237A CN 104894237 A CN104894237 A CN 104894237A CN 201510219803 A CN201510219803 A CN 201510219803A CN 104894237 A CN104894237 A CN 104894237A
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trypetid
seq
primer
probe
probe group
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CN104894237B (en
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李志红
姜帆
朱水芳
付伟
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Chinese Academy of Inspection and Quarantine CAIQ
China Agricultural University
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China Agricultural University
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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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    • C12Q2600/16—Primer sets for multiplex assays

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Abstract

The invention discloses an integrated fluidic chip for identification of a fruit fly type and a use thereof. The integrated microfluidic chip comprises specific primers for fruit fly type identification and a TaqMan-MGB probe. The invention provides a fruit fly type molecular identification technology utilizing the integrated fluidic chip. A test proves that the integrated microfluidic chip-based molecular identification technology realizes fast identification of a fruit fly type, has good specificity, high flux, a low cost and simple processes, realizes identification of a type of non-adult fruit fly or fruit fly adult residual body, provides a fast identification tool and technology for import and export plant quarantine and nationwide fruit fly epidemic monitoring, effectively prevents invasion and diffusion of dangerous fruit fly, protects agricultural production and ecological safety and promotes trade and economic development in China.

Description

A kind of integrated fluidic chip and application thereof identifying trypetid kind
Technical field
The invention belongs to biological technical field, be specifically related to a kind of integrated fluidic chip and the application thereof of identifying trypetid kind.
Background technology
Trypetid (Fruit Flies) belongs to Diptera (Diptera) Tephritidae (Tephritidae), and the whole world extensively distributes, and mostly occurs in subtropical and tropical zones.Tephritidae about has 500 genus 4500 kinds, and the trypetid with Important Economic meaning reaches more than 250 and plants, and some trypetid can cause crushing disaster to Production of fruit.The trypetid in the world with Economic Importance mainly contains 5 genus, is by Anastrepha (Anastrepha), Bactrocera (Bactrocera), little bar Anastrepha (Ceratitis), few hair on the neck Anastrepha (Dacus) and around Anastrepha (Rhagoletis) respectively.
In the inward Plant Quarantine harmful organism register that China is existing, quarantine fruit fly comprises by Anastrepha, Bactrocera, little bar Anastrepha, few hair on the neck trypetid (non-China seed), hammers 10 kinds such as abdomen trypetid, muskmelon fan trypetid and papaya long-tail trypetid into shape in fact around trypetid (non-China seed), Europe-Africa jujube fly, jujube fly, tangerine; National Agricultural quarantine fruit fly and forestry quarantine fruit fly respectively have a kind respectively, i.e. bactrocera tsuneonis and jujube fly; In the state key management Invasive Alien Species register that the Ministry of Agriculture issues, comprise 3 kinds of Fruit flies, be respectively citrus fruit fly, melon trypetid and jujube fly.
Trypetid due to intercept and capture mostly is non-adult form form being difficult to distinguish, and generally need raise as by precise Identification, greatly reducing the ageing of quarantine after adult through 1-2 week.Traditional trypetid qualification work mainly relies on morphological feature, but because trypetid kind is many, the morphological differences distinguished between different trypetid kind is often very trickle and complicated, some kinds also could need be distinguished by distribution and host's information, the classification position of some kinds is still comparatively chaotic, and trypetid newly belongs to the description of novel species also in continuous increase, particularly also there is dispute in the classification of some complex body kinds, and the existence of these problems is identified all to trypetid traditional form and brought great obstruction.Along with the development of international fruits and vegetables trade, the risk that Fruit fly imports China into significantly increases, and the demand of its quarantine identification particularly Rapid identification is more urgent.
Integrated fluidic (Integrated Fluidic Circuit, IFC) chip is that a kind of utilization has elastomeric polydimethyl siloxane material, multilayer Soft lithograph technology is adopted to design on chip and be processed into the micro-valve arrangement of highdensity Micropump, by controlling closedown and the unlatching of the micro-valve of Micropump, rapidly and accurately reaction solution is divided into some rising amount order reaction unit of independently receiving, realizes the PCR that the parallel flux carried out of multistep is high, volume is little and react platform.
Integrated fluid path technology, since appearance, has received the especially favor of scientific circles, and each major company in the whole world in this field has also released one after another commercially produced product.2006, Fluidigm company of the U.S. took the lead in releasing the BioMark based on IFC technology tMgene alaysis system, this system is the technology platform that integrated IFC technology, Real-Time round pcr and genetic analysis software combine, by BioMark tMreal-Time PCR system, IFC chip, IFC controller and data analysis software 4 part are formed.Wherein, 48.48IFC dynamic chip can carry out 2304 real-time fluorescence PCR reactions simultaneously.
At present, detect at medical diagnosis, gene type, unicellular genetic expression, detection GMOs, environmental microorganism with the multiple round pcr that IFC chip is reaction platform, be widely used in order-checking of future generation etc., but its application in insect Molecular Identification has no report.
Summary of the invention
An object of the present invention is to provide a kind of primer set for the identification of trypetid kind and probe.
Primer set for the identification of trypetid kind provided by the invention and probe are made up of 27 primed probe groups.
In above-mentioned primer set and probe,
Described primed probe group 1 forms for the primer 1-2 shown in primer 1-1, SEQ ID No.2 shown in SEQ ID No.1 and the probe 1 shown in SEQ ID No.3;
Described primed probe group 2 forms for the primer 2-2 shown in the primer 2-1 shown in SEQ ID No.4, SEQ ID No.5 and the probe 2 shown in SEQ ID No.6;
Described primed probe group 3 forms for the primer 3-2 shown in primer 3-1, SEQ ID No.8 shown in SEQ ID No.7 and the probe 3 shown in SEQ ID No.9;
Described primed probe group 4 forms for the primer 4-2 shown in primer 4-1, SEQ ID No.11 shown in SEQ ID No.10 and the probe 4 shown in SEQ ID No.12;
Described primed probe group 5 forms for the primer 5-2 shown in primer 5-1, SEQ ID No.14 shown in SEQ ID No.13 and the probe 5 shown in SEQ ID No.15;
Described primed probe group 6 forms for the primer 6-2 shown in primer 6-1, SEQ ID No.17 shown in SEQ ID No.16 and the probe 6 shown in SEQ ID No.18;
Described primed probe group 7 forms for the primer 7-2 shown in primer 7-1, SEQ ID No.20 shown in SEQ ID No.19 and the probe 7 shown in SEQ ID No.21;
Described primed probe group 8 forms for the primer 8-2 shown in primer 8-1, SEQ ID No.23 shown in SEQ ID No.22 and the probe 8 shown in SEQ ID No.24;
Described primed probe group 9 forms for the primer 9-2 shown in primer 9-1, SEQ ID No.26 shown in SEQ ID No.25 and the probe 9 shown in SEQ ID No.27;
Described primed probe group 10 forms for the primer 10-2 shown in primer 10-1, SEQ ID No.29 shown in SEQ ID No.28 and the probe 10 shown in SEQ ID No.30;
Described primed probe group 11 forms for the primer 11-2 shown in primer 11-1, SEQ ID No.32 shown in SEQ ID No.31 and the probe 11 shown in SEQ ID No.33;
Described primed probe group 12 forms for the primer 12-2 shown in primer 12-1, SEQ ID No.35 shown in SEQ ID No.34 and the probe 12 shown in SEQ ID No.36;
Described primed probe group 13 forms for the primer 13-2 shown in primer 13-1, SEQ ID No.38 shown in SEQ ID No.37 and the probe 13 shown in SEQ ID No.39;
Described primed probe group 14 forms for the primer 14-2 shown in primer 14-1, SEQ ID No.41 shown in SEQ ID No.40 and the probe 14 shown in SEQ ID No.42;
Described primed probe group 15 forms for the primer 15-2 shown in primer 15-1, SEQ ID No.44 shown in SEQ ID No.43 and the probe 15 shown in SEQ ID No.45;
Described primed probe group 16 forms for the primer 16-2 shown in primer 16-1, SEQ ID No.47 shown in SEQ ID No.46 and the probe 16 shown in SEQ ID No.48;
Described primed probe group 17 forms for the primer 17-2 shown in primer 17-1, SEQ ID No.50 shown in SEQ ID No.49 and the probe 17 shown in SEQ ID No.51;
Described primed probe group 18 forms for the primer 18-2 shown in primer 18-1, SEQ ID No.53 shown in SEQ ID No.52 and the probe 18 shown in SEQ ID No.54;
Described primed probe group 19 forms for the primer 19-2 shown in primer 19-1, SEQ ID No.56 shown in SEQ ID No.55 and the probe 19 shown in SEQ ID No.57;
Described primed probe group 20 forms for the primer 2 0-2 shown in primer 2 0-1, SEQ ID No.59 shown in SEQ ID No.58 and the probe 20 shown in SEQ ID No.60;
Described primed probe group 21 forms for the primer 2 1-2 shown in primer 2 1-1, SEQ ID No.62 shown in SEQ ID No.61 and the probe 21 shown in SEQ ID No.63;
Described primed probe group 22 forms for the primer 2 2-2 shown in primer 2 2-1, SEQ ID No.65 shown in SEQ ID No.64 and the probe 22 shown in SEQ ID No.66;
Described primed probe group 23 forms for the primer 2 3-2 shown in primer 2 3-1, SEQ ID No.68 shown in SEQ ID No.67 and the probe 23 shown in SEQ ID No.69;
Described primed probe group 24 forms for the primer 2 4-2 shown in primer 2 4-1, SEQ ID No.71 shown in SEQ ID No.70 and the probe 24 shown in SEQ ID No.72;
Described primed probe group 25 forms for the primer 2 5-2 shown in primer 2 5-1, SEQ ID No.74 shown in SEQ ID No.73 and the probe 25 shown in SEQ ID No.75;
Described primed probe group 26 forms for the primer 2 6-2 shown in primer 2 6-1, SEQ ID No.77 shown in SEQ ID No.76 and the probe 26 shown in SEQ ID No.78;
Described primed probe group 27 forms for the primer 2 7-2 shown in primer 2 7-1, SEQ ID No.80 shown in SEQ ID No.79 and the probe 27 shown in SEQ ID No.81.
In above-mentioned primer set and probe, the amount of substance of the primer in described primed probe group and probe is than being 9:20.
In above-mentioned primer set and probe, described trypetid kind is any one in following 27 kinds: Mexico is by trypetid, western India is by trypetid, Portugal fruit fly, Bactrocera correcta, Carambola fruit trypetid, citrus fruit fly, pepper fruit fly, rust haw trypetid, macadamia nut trypetid, short-tail fruit fly, breadfruit tree trypetid, Peach fruits fly, olea europaea fruit trypetid, the large trypetid of tangerine, bactrocera tsuneonis, calabash melon trypetid, two band fruit flies, melon trypetid, broadband fruit fly, selective propensity of Bactrocera tau, jujube fly, Mediterranean fruitfly, the little bar trypetid of mango, the little bar trypetid of Natta ear, the few hair on the neck trypetid of cucurbit, cherry around trypetid and apple around trypetid.
Another object of the present invention is to provide a kind of PCR reagent for the identification of trypetid kind.
PCR reagent for the identification of trypetid kind provided by the invention is by PCR reagent 1, PCR reagent 2, PCR reagent 3, PCR reagent 4, PCR reagent 5, PCR reagent 6, PCR reagent 7, PCR reagent 8, PCR reagent 9, PCR reagent 10, PCR reagent 11, PCR reagent 12, PCR reagent 13, PCR reagent 14, PCR reagent 15, PCR reagent 16, PCR reagent 17, PCR reagent 18, PCR reagent 19, PCR reagent 20, PCR reagent 21, PCR reagent 22, PCR reagent 23, PCR reagent 24, PCR reagent 25, PCR reagent 26 and PCR reagent 27 form,
Described PCR reagent 1 comprises the primed probe group 1 in above-mentioned primer set and probe;
Described PCR reagent 2 comprises the primed probe group 2 in above-mentioned primer set and probe;
Described PCR reagent 3 comprises the primed probe group 3 in above-mentioned primer set and probe;
Described PCR reagent 4 comprises the primed probe group 4 in above-mentioned primer set and probe;
Described PCR reagent 5 comprises the primed probe group 5 in above-mentioned primer set and probe;
Described PCR reagent 6 comprises the primed probe group 6 in above-mentioned primer set and probe;
Described PCR reagent 7 comprises the primed probe group 7 in above-mentioned primer set and probe;
Described PCR reagent 8 comprises the primed probe group 8 in above-mentioned primer set and probe;
Described PCR reagent 9 comprises the primed probe group 9 in above-mentioned primer set and probe;
Described PCR reagent 10 comprises the primed probe group 10 in above-mentioned primer set and probe;
Described PCR reagent 11 comprises the primed probe group 11 in above-mentioned primer set and probe;
Described PCR reagent 12 comprises the primed probe group 12 in above-mentioned primer set and probe;
Described PCR reagent 13 comprises the primed probe group 13 in above-mentioned primer set and probe;
Described PCR reagent 14 comprises the primed probe group 14 in above-mentioned primer set and probe;
Described PCR reagent 15 comprises the primed probe group 15 in above-mentioned primer set and probe;
Described PCR reagent 16 comprises the primed probe group 16 in above-mentioned primer set and probe;
Described PCR reagent 17 comprises the primed probe group 17 in above-mentioned primer set and probe;
Described PCR reagent 18 comprises the primed probe group 18 in above-mentioned primer set and probe;
Described PCR reagent 19 comprises the primed probe group 19 in above-mentioned primer set and probe;
Described PCR reagent 20 comprises the primed probe group 20 in above-mentioned primer set and probe;
Described PCR reagent 21 comprises the primed probe group 21 in above-mentioned primer set and probe;
Described PCR reagent 22 comprises the primed probe group 22 in above-mentioned primer set and probe;
Described PCR reagent 23 comprises the primed probe group 23 in above-mentioned primer set and probe;
Described PCR reagent 24 comprises the primed probe group 24 in above-mentioned primer set and probe;
Described PCR reagent 25 comprises the primed probe group 25 in above-mentioned primer set and probe;
Described PCR reagent 26 comprises the primed probe group 26 in above-mentioned primer set and probe;
Described PCR reagent 27 comprises the primed probe group 27 in above-mentioned primer set and probe.
Above-mentioned each PCR reagent is made up of Assays system and Sample system; Assays system described in every 5ul is by forward primer (100 μMs) 0.45 μ l, reverse primer (100 μMs) 0.45 μ l, probe (10 μMs) 1 μ l, 2 × Assay Loading Reagent2.5 μ l, 50 × ROX Reference Dye II 0.25 μ l and ddH 2o 0.35 μ l forms; Sample system described in every 5ul is made up of 2 × Premix Ex Taq 2.5 μ l and 20 × GE Sample Loading Reagent 0.25 μ l.
A further object of the invention is to provide a kind of integrated fluidic chip for the identification of trypetid kind.
Integrated fluidic chip for the identification of trypetid kind provided by the invention comprises above-mentioned primer set and probe or above-mentioned PCR reagent.
In above-mentioned integrated fluidic chip, described integrated fluidic chip is each PCR reagent above-mentioned be divided in each micro-reaction chamber of integrated fluidic chip, obtains the integrated fluidic chip containing above-mentioned primer set and probe or above-mentioned PCR reagent.
A further object of the invention is to provide a kind of test kit for the identification of trypetid kind.
Test kit for the identification of trypetid kind provided by the invention comprises above-mentioned primer set and probe or above-mentioned PCR reagent or above-mentioned integrated fluidic chip.
Above-mentioned primer set and probe or above-mentioned PCR reagent or above-mentioned integrated fluidic chip or mentioned reagent box are being identified or whether assistant identification trypetid to be measured is that application in 27 kinds of trypetids in any one also belongs to protection scope of the present invention.
Above-mentioned primer set and probe or above-mentioned PCR reagent or above-mentioned integrated fluidic chip or mentioned reagent box identify or assistant identification trypetid to be measured be any in 27 kinds of trypetids in application also belong to protection scope of the present invention.
In above-mentioned application, described 27 kinds of trypetids are that Mexico is by trypetid, western India is by trypetid, Portugal fruit fly, Bactrocera correcta, Carambola fruit trypetid, citrus fruit fly, pepper fruit fly, rust haw trypetid, macadamia nut trypetid, short-tail fruit fly, breadfruit tree trypetid, Peach fruits fly, olea europaea fruit trypetid, the large trypetid of tangerine, bactrocera tsuneonis, calabash melon trypetid, two band fruit flies, melon trypetid, broadband fruit fly, selective propensity of Bactrocera tau, jujube fly, Mediterranean fruitfly, the little bar trypetid of mango, the little bar trypetid of Natta ear, the few hair on the neck trypetid of cucurbit, cherry around trypetid and apple around trypetid.
Last object of the present invention is to provide a kind of method of qualification or assistant identification trypetid kind to be measured.
The method of qualification provided by the invention or assistant identification trypetid to be measured kind comprises the steps: the genomic dna of trypetid to be measured to add in each micro-reaction chamber of above-mentioned integrated fluidic chip, carries out real-time fluorescence PCR, detects each micro-reaction chamber PCR primer;
If on described integrated fluidic chip certain primed probe group place micro-reaction chamber in carry out positive amplification, then trypetid to be measured is or candidate is target trypetid corresponding to this primed probe group;
If on described integrated fluidic chip certain primed probe group place micro-reaction chamber in be non-positive amplification, then trypetid to be measured is not or candidate is not target trypetid corresponding to this primed probe group.
In aforesaid method, described positive amplification is that micro-reaction chamber produces fluorescence and CT value≤20.0; Described non-positive amplification refers to that micro-reaction chamber does not produce fluorescence or produces fluorescence and CT value > 20.0.
In aforesaid method,
The trypetid of described primed probe group 1 correspondence is that Mexico is by trypetid;
The trypetid of described primed probe group 2 correspondence is that western India is by trypetid;
The trypetid of described primed probe group 3 correspondence is Portugal fruit fly;
The trypetid of described primed probe group 4 correspondence is Bactrocera correcta;
The trypetid of described primed probe group 5 correspondence is Carambola fruit trypetid;
The trypetid of described primed probe group 6 correspondence is citrus fruit fly;
The trypetid of described primed probe group 7 correspondence is pepper fruit fly;
The trypetid of described primed probe group 8 correspondence is rust haw trypetid;
The trypetid of described primed probe group 9 correspondence is macadamia nut trypetid;
The trypetid of described primed probe group 10 correspondence is short-tail fruit fly;
The trypetid of described primed probe group 11 correspondence is breadfruit tree trypetid;
The trypetid of described primed probe group 12 correspondence is Peach fruits fly;
The trypetid of described primed probe group 13 correspondence is olea europaea fruit trypetid;
The trypetid of described primed probe group 14 correspondence is the large trypetid of tangerine;
The trypetid of described primed probe group 15 correspondence is bactrocera tsuneonis;
The trypetid of described primed probe group 16 correspondence is calabash melon trypetid;
The trypetid of described primed probe group 17 correspondence is two band fruit flies;
The trypetid of described primed probe group 18 correspondence is melon trypetid;
The trypetid of described primed probe group 19 correspondence is broadband fruit fly;
The trypetid of described primed probe group 20 correspondence is selective propensity of Bactrocera tau;
The trypetid of described primed probe group 21 correspondence is jujube fly;
The trypetid of described primed probe group 22 correspondence is Mediterranean fruitfly;
The trypetid of described primed probe group 23 correspondence is the little bar trypetid of mango;
The trypetid of described primed probe group 24 correspondence is the little bar trypetid of Natta ear;
The trypetid of described primed probe group 25 correspondence is the few hair on the neck trypetid of cucurbit;
The trypetid of described primed probe group 26 correspondence is that cherry is around trypetid;
The trypetid of described primed probe group 27 correspondence is that apple is around trypetid;
Described trypetid kind is any one in following 27 kinds: Mexico is by trypetid, western India is by trypetid, Portugal fruit fly, Bactrocera correcta, Carambola fruit trypetid, citrus fruit fly, pepper fruit fly, rust haw trypetid, macadamia nut trypetid, short-tail fruit fly, breadfruit tree trypetid, Peach fruits fly, olea europaea fruit trypetid, the large trypetid of tangerine, bactrocera tsuneonis, calabash melon trypetid, two band fruit flies, melon trypetid, broadband fruit fly, selective propensity of Bactrocera tau, jujube fly, Mediterranean fruitfly, the little bar trypetid of mango, the little bar trypetid of Natta ear, the few hair on the neck trypetid of cucurbit, cherry around trypetid and apple around trypetid.
In aforesaid method, the reaction conditions of described real-time fluorescence PCR is 95 DEG C of denaturation 30s; 95 DEG C of sex change 5s of 40 circulations, 60 DEG C of annealing extend 34s; 60 DEG C extend 1min.
Advantage of the present invention:
(1) the quarantine fruit fly category that covers of the present invention is wide, can meet the actual demand of China's Plant Quarantine work completely.
(2) utilize custom-designed stream and valve, by computer control, chip completes the separatory of biological sample and reaction reagent automatically, greatly simplify pipetting, can antipollutionly occur again.
(3) react flux high, and its reaction system receiving rising amount level is high-throughout detection analysis has saved great amount of cost.
(4) DNA molecular of single copy can be detected, fundamentally improve sensitivity and the accuracy of detection.
(5) the Identification of Species problem of non-adult form trypetid and the residual body of trypetid adult is solved.
(6) high specificity, efficiency are high; 1/48 is reduced to than the operating process of traditional real-time fluorescence PCR; The reagent that the reaction system receiving upgrading is reacted than normal PCR and real-time fluorescence PCR expends minimizing 100 times.
The present invention is belonging on the basis of 179 kinds 2252 trypetid mtDNA COI bar code sequences comparison to 6, for 27 kinds of quarantine fruit flies, namely Mexico is by trypetid, western India is by trypetid, Portugal fruit fly, Bactrocera correcta, Carambola fruit trypetid, citrus fruit fly, pepper fruit fly, rust haw trypetid, macadamia nut trypetid, short-tail fruit fly, breadfruit tree trypetid, Peach fruits fly, olea europaea fruit trypetid, the large trypetid of tangerine, bactrocera tsuneonis, calabash melon trypetid, two band fruit flies, melon trypetid, broadband fruit fly, selective propensity of Bactrocera tau, jujube fly, Mediterranean fruitfly, the little bar trypetid of mango, the little bar trypetid of Natta ear, the few hair on the neck trypetid of cucurbit, cherry around trypetid and apple around trypetid, design and screen species-specific primer and TaqMan-MGB probe, develop the Molecular Identification technology based on integrated fluidic chip.Proved by test: the Molecular Identification technology based on integrated fluidic chip of the present invention can realize the qualification to trypetid kind fast; and specificity is good, flux is high, cost is low, simple to operate; solve the Identification of Species problem of non-adult form trypetid and the residual body of trypetid adult; the tools and techniques of Rapid identification can be provided for pass in and out Plant Quarantine and national trypetid epidemic monitoring, effectively can prevent invasion and the diffusion of dangerous trypetid, protecting agriculture produces and ecological safety, the economic trade development of promotion China.
Accompanying drawing explanation
Fig. 1 is 4848 dynamic chip.
Fig. 2 is 27 kinds of trypetid primers and probe combinations integrated fluidic chip specific detection result.
Fig. 3 is that integrated fluidic chip identifies non-adult form trypetid result figure.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The name of the 48.48IFC dynamic chip in following embodiment is called Dynamic Array IFCs; Model is 48.48; Producer is Fluidigm (U.S.).
Control Line Fluid is the product of Fluidigm (U.S.) company;
2 × Premix Ex Taq and 50 × ROX Reference Dye II is the product of TaKaRa, catalog number: RR390A;
2 × Assay Loading Reagent (PN 85000736) and 20 × GE Sample Loading Reagent (PN 85000735) is the product of Fluidigm (U.S.) company.
The Auele Specific Primer of embodiment 1, qualification 27 kinds of trypetids is to the acquisition with probe
One, the acquisition of trypetid mtDNA COI gene order
1, as shown in table 1 for examination trypetid sample, sample is all placed in dehydrated alcohol, and-20 DEG C save backup.
Table 1, for examination trypetid sample type and locality information
2, (most of sample is got monopodia and is extracted to adopt " blood/cell/tissue genome DNA extracting reagent kit " (TIANGEN Biotech (Beijing) Co., Ltd.) to extract the genomic dna of each trypetid sample in table 1 respectively, a few sample is that single head extracts), concrete operations flow process, with reference to specification sheets, obtains the genomic dna of each trypetid sample in table 1 respectively.
Respectively with the genomic dna of each trypetid sample in table 1 for template, use universal primer LCO1490/HCO2198 to carry out pcr amplification, obtain the pcr amplification product of each trypetid sample in table 1 respectively.
The total system of pcr amplification (25 μ l): 2 × Taq PCR MasterMix (TIANGEN Biotech (Beijing) Co., Ltd., catalog number (Cat.No.): KT201) 12.5 μ l, upstream and downstream primer (10 μMs, AudioCodes prosperous bio tech ltd in Beijing synthesizes) each 1 μ l, ddH 2o 9.5 μ l, DNA profiling 1 μ l;
Pcr amplification condition: 94 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 50 DEG C of annealing 1min, 72 DEG C extend 1min 35 circulation; 72 DEG C extend 10min.
3, get 5 μ l pcr amplification products and carry out electrophoresis detection respectively, directly transfer to AudioCodes prosperous bio tech ltd in Beijing to carry out purifying and two-way order-checking the qualified pcr amplification product of detection.Utilize Chromas software to check order-checking peak figure, with DNAMAN software, sequencing result is spliced.
Download published economic Anastrepha all trypetid kinds sequence (by May 17th, 2013) in BOLD, and filter out the mtDNA COI bar code sequence that length is not shorter than 600bp.Download the trypetid mtDNA COI bar code sequence obtaining 6 genus 179 kinds 2252 altogether by experiment with in BOLD.
Two, 27 species-specific primers are to the design with TaqMan-MGB probe
Belong to trypetid mtDNA COI bar code sequence design primer and the probe of 179 kinds 2252 according to 6 of above-mentioned acquisition, finally obtain 27 pairs of primers and probe, 27 pairs of primers and 27 corresponding TaqMan-MGB probe (5 '-FAM; 3 '-MGB) as shown in table 2, by the English Weihe River, prompt base (Shanghai) trade Co., Ltd synthesizes.
Table 2,27 kinds of quarantine fruit fly special primers and probe
The preparation of the integrated fluidic chip of embodiment 2, qualification 27 kinds of trypetids and specific detection thereof
One, the preparation of integrated fluidic chip
1, the initialize of 48.48 dynamic chip
(1) open packing chip, and use in 24h;
(2) Control Line Fluid is injected respectively the collector at 48.48 dynamic chip two ends, note Control Line Fluid not being instilled Assay Inlets or Sample Inlets;
(3) blue protection film of IFC chip bottom is removed;
(4) chip is proceeded to IFC Controller, run Primer (113x) program and carry out initialize to chip, initialization time is 10min; After initialize completes, in 60min, complete loading operation.
2, the preparation of standard reaction system and sample mix liquid
(1) prepare the 10 × Assays of 27 kinds of trypetids according to the test system shown in table 3 respectively, comprise upstream and downstream primer, probe, 2 × Assay Loading Reagent, 50 × ROX II Reference Dye II and ddH 2o, mixing is also centrifugal.
Table 3, test system
10×Assays
(2) prepare often kind of trypetid sample mix liquid respectively according to the sample system shown in table 4, comprise the DNA (table 5) of 2 × Premix Ex Taq, 20 × GE Sample Loading Reagent and often kind of trypetid sample, mixing is also centrifugal.
Table 4, sample system
The trypetid species that table 5, Assay Inlets and Sample Inlets are corresponding
3, chip loading
(1) after chip initiation, it is taken out from IFC Controller, in the corresponding pipeline of Assay Inlets and Sample Inlets, inject the 5 μ l 10 × Assays reagent mixed and trypetid sample mix liquid respectively; No Assay Inlets, adds 2.5 μ l 2 × Assay Loading Reagent, 0.25 μ l 50 × ROX II and 2.25 μ l ddH 2o; No Sample Inlets, adds 2.5 μ l 2 × Premix Ex Taq, 0.25 μ l 20 × GE Sample Loading Reagent and 2.25 μ l ddH 2o.In loading process, the generation of bubble should be avoided, use during pipettor and slowly beat liquid to the first grade.
(2) put back to by chip in IFC Controller, run Load Mix (113x) program, assays and samples is automatically imported in IFC chip by this program carries out separatory, and working time is 1h;
(3) after Load Mix EP (end of program), chip is taken out from IFC Controller, with impurity such as the dusts of the slight sticky removing chip surface of adhesive tape, and in 4h, start the real-time fluorescence PCR of chip.
4, real-time fluorescence PCR
(1) chip is put into BioMark real-time fluorescence PCR system, select Gene Expression type used, ROX reference fluorescent, FAM-MGB probe type, under auto exposure mode, run real-time fluorescence PCR reaction according to the reaction conditions set up.Reaction conditions is as shown in table 5.
Table 5, reaction conditions
(2), after real-time fluorescence PCR reaction terminates, chip is taken out from BioMark real-time fluorescence PCR system, with Fluidigm Real-Time pcr analysis software, experimental data is analyzed.
By checking the Heat Map of reaction result, 27 kinds of quarantine fruit fly species-specific primers designed by judgement to the applicability of probe under the reaction platform of integrated fluidic chip and specificity.In 2304 micro-reaction chambers on chip, have micro-reaction chamber of amplified reaction then can produce fluorescence, micro-reaction chamber corresponding in Heat Map can present with colour side's point form, and the fluorescence color that the reaction of different CT value sends is different.
Two, the specific detection result of integrated fluidic chip
The principle of specificity verification is that the primer of often kind of trypetid and probe combinations only produce Positive fluorescence signal in micro-reaction chamber of the target trypetid corresponding with it, and other micro-reaction chambers equal unstressed configuration signal produces or is non-positive amplification result, namely this Auele Specific Primer and probe are under set up quarantine fruit fly integrated fluidic chip authenticate technology system, can identify target trypetid species specifically, setting CT≤20.0 are positive amplification.
If certain Auele Specific Primer and micro-reaction chamber corresponding to probe produce fluorescence and CT value≤20.0, then trypetid to be measured is or candidate is the target trypetid that this Auele Specific Primer and probe are corresponding;
If certain Auele Specific Primer and micro-reaction chamber unstressed configuration signal corresponding to probe or produce fluorescence and CT value > 20.0, then trypetid to be measured is not or candidate is not the target trypetid that this Auele Specific Primer and probe are corresponding.
The target trypetid of primer 1-1, primer 1-2 and probe 1 correspondence is that Mexico is by trypetid;
Primer 2-1, primer 2-2 and probe 2 the target trypetid of correspondence be that western India is by trypetid;
The target trypetid of primer 3-1, primer 3-2 and probe 3 correspondence is Portugal fruit fly;
The target trypetid of primer 4-1, primer 4-2 and probe 4 correspondence is Bactrocera correcta;
The target trypetid of primer 5-1, primer 5-2 and probe 5 correspondence is trypetid is Carambola fruit trypetid;
The target trypetid of primer 6-1, primer 6-2 and probe 6 correspondence is citrus fruit fly;
The target trypetid of primer 7-1, primer 7-2 and probe 7 correspondence is pepper fruit fly;
The target trypetid of primer 8-1, primer 8-2 and probe 8 correspondence is rust haw trypetid;
The target trypetid of primer 9-1, primer 9-2 and probe 9 correspondence is macadamia nut trypetid;
The target trypetid of primer 10-1, primer 10-2 and probe 10 correspondence is short-tail fruit fly;
The target trypetid of primer 11-1, primer 11-2 and probe 11 correspondence is breadfruit tree trypetid;
The target trypetid of primer 12-1, primer 12-2 and probe 12 correspondence is Peach fruits fly;
The target trypetid of primer 13-1, primer 13-2 and probe 13 correspondence is olea europaea fruit trypetid;
The target trypetid of primer 14-1, primer 14-2 and probe 14 correspondence is the large trypetid of tangerine;
The target trypetid of primer 15-1, primer 15-2 and probe 15 correspondence is bactrocera tsuneonis;
The target trypetid of primer 16-1, primer 16-2 and probe 16 correspondence is calabash melon trypetid;
The target trypetid of primer 17-1, primer 17-2 and probe 17 correspondence is two band fruit flies;
The target trypetid of primer 18-1, primer 18-2 and probe 18 correspondence is melon trypetid;
The target trypetid of primer 19-1, primer 19-2 and probe 19 correspondence is broadband fruit fly;
The target trypetid of primer 2 0-1, primer 2 0-2 and probe 20 correspondence is selective propensity of Bactrocera tau;
The target trypetid of primer 2 1-1, primer 2 1-2 and probe 21 correspondence is jujube fly;
The target trypetid of primer 2 2-1, primer 2 2-2 and probe 22 correspondence is Mediterranean fruitfly;
The target trypetid of primer 2 3-1, primer 2 3-2 and probe 23 correspondence is the little bar trypetid of mango;
The target trypetid of primer 2 4-1, primer 2 4-2 and probe 24 correspondence is the little bar trypetid of Natta ear;
The target trypetid of primer 2 5-1, primer 2 5-2 and probe 25 correspondence is the few hair on the neck trypetid of cucurbit;
The target trypetid of primer 2 6-1, primer 2 6-2 and probe 26 correspondence is that cherry is around trypetid;
The target trypetid of primer 2 7-1, primer 2 7-2 and probe 27 correspondence is that apple is around trypetid.
Specific detection result is as shown in Figure 2: in 35 kinds of trypetid samples, (Mexico is by trypetid with 27 kinds of trypetids, western India is by trypetid, Portugal fruit fly, Bactrocera correcta, Carambola fruit trypetid, citrus fruit fly, pepper fruit fly, rust haw trypetid, macadamia nut trypetid, short-tail fruit fly, breadfruit tree trypetid, Peach fruits fly, olea europaea fruit trypetid, the large trypetid of tangerine, bactrocera tsuneonis, calabash melon trypetid, two band fruit flies, melon trypetid, broadband fruit fly, selective propensity of Bactrocera tau, jujube fly, Mediterranean fruitfly, the little bar trypetid of mango, the little bar trypetid of Natta ear, the few hair on the neck trypetid of cucurbit, cherry around trypetid with apple around trypetid) micro-reaction chamber of the Auele Specific Primer trypetid sample corresponding with probe species all have issued fluorescent signal and CT value≤20.0, unstressed configuration signal or produce fluorescence and CT value > 20.0 in micro-reaction chamber of not corresponding with the Auele Specific Primer of 27 kinds of trypetids and probe species trypetid sample.The Rapid identification of 27 kinds of quarantine fruit flies disposablely can be realized by trypetid integrated fluidic chip of the present invention.
The application in qualification trypetid of embodiment 3, integrated fluidic chip
One, trypetid sample to be measured
For following non-adult form trypetid sample, the integrated fluidic chip described in Application Example 2 identifies its kind.
The trypetid larva 3 that in May, 2012, Beijing Administration for Entry-Exit Inspection and Quarantine intercepted and captured from the wax-apple of Thailand's import;
The trypetid larva 2 that in May, 2014, Beijing Administration for Entry-Exit Inspection and Quarantine intercepted and captured from the mango of Tanzania's import;
Pick up from the trypetid larva 2 in the balsam pear decayed fruit of In Nanning Area, China Guangxi Zhuang Autonomous Region in July, 2009;
The host of Sichuan Province of China's in October, 2011 Chengdu plant test station for plant protection censorship is the trypetid pupa 2 of oranges and tangerines;
The pepper fruit fly blow 3 pieces that in August, 2011 country of Inspection and Quarantine Technic Center, Guangdong Entry-Exit Inspection and Qu trypetid quarantine key lab raises.
Two, authentication method
Adopt RNA isolation kit to extract the genomic dna of the trypetid sample to be measured in above-mentioned steps one, leaching process carries out with reference to specification sheets.
The integrated fluidic chip described in embodiment 2 and the genomic dna of authentication method to above-mentioned trypetid sample to be measured thereof is utilized to detect.
10 × Assays of each species-specific primer and probe combinations is totally 5 μ l, comprising each 0.45 μ l of upstream and downstream primer (100 μMs), probe (10 μMs) 1 μ l, 2 × Assay Loading Reagent 2.5 μ l, 50 × ROX II 0.25 μ l, ddH 2o 0.35 μ l.
Each sample be totally 5 μ l, comprising 2 × Premix Ex Taq 2.5 μ l, 20 × GE Sample Loading Reagent 0.25 μ l, DNA 2.25 μ l.
Reaction conditions: 95 DEG C of denaturation 30s; 95 DEG C of sex change 5s of 40 circulations, 60 DEG C of annealing extend 34s; 60 DEG C extend 1min.
After reaction terminates, by directly observing Heat Map (CT≤20.0 are positive amplification) judged result.
Three, qualification result
Result is as shown in Figure 3: 3 the trypetid larvas intercepted and captured from Thailand's wax-apple all produce Positive fluorescence signal in micro-reaction chamber of the Bactrocera correcta primer corresponding with it and probe combinations, CT value is respectively 17.29,19.77 and 14.40, and other micro-reaction chambers equal unstressed configuration signal produces or is the non-positive amplification of CT > 20.0, therefore identify that its kind is Bactrocera correcta; 2 the trypetid larvas intercepted and captured from Tanzania's mango all produce Positive fluorescence signal in micro-reaction chamber of the mango corresponding with it little bar trypetid primer and probe combinations, CT value is respectively 10.69 and 12.98, and other micro-reaction chambers equal unstressed configuration signal produces or is the non-positive amplification of CT > 20.0, therefore identify that its kind is the little bar trypetid of mango; All in micro-reaction chamber of the selective propensity of Bactrocera tau primer corresponding with it and probe combinations, Positive fluorescence signal is produced from 2 trypetid larvas China Nanning Region Guangxi balsam pear decayed fruit, CT value is respectively 11.17 and 13.33, and other micro-reaction chambers equal unstressed configuration signal produces or is the non-positive amplification of CT > 20.0, therefore identify that its kind is selective propensity of Bactrocera tau; The trypetid pupa being oranges and tangerines from 2 hosts of Chengdu, Sichuan plant test station for plant protection censorship all produces Positive fluorescence signal in micro-reaction chamber of the tangerine corresponding with it large trypetid primer and probe combinations, CT value is respectively 10.98 and 11.66, and other micro-reaction chambers equal unstressed configuration signal produces or is the non-positive amplification of CT > 20.0, therefore identify that its kind is the large trypetid of tangerine; 3 pieces of pepper fruit fly blows of raising from trypetid quarantine key lab of inspection and quarantine technique center, Guangdong country all produce Positive fluorescence signal in micro-reaction chamber of the pepper fruit fly primer corresponding with it and probe combinations, CT value is respectively 13.03,15.41 and 17.08, and other micro-reaction chambers equal unstressed configuration signal produces or is the non-positive amplification of CT > 20.0, therefore qualification result is all pepper fruit fly, illustrates that integrated fluidic chip technology can be successfully applied to the Molecular Identification of trypetid ovum.
Above test-results describes the validity of the real-time fluorescence PCR technical evaluation non-adult form trypetid sample type based on integrated fluidic chip.

Claims (10)

1., for the identification of primer set and the probe of trypetid kind, be made up of 27 primed probe groups;
Described primed probe group 1 forms for the primer 1-2 shown in primer 1-1, SEQ ID No.2 shown in SEQ ID No.1 and the probe 1 shown in SEQ ID No.3;
Described primed probe group 2 forms for the primer 2-2 shown in the primer 2-1 shown in SEQ ID No.4, SEQ ID No.5 and the probe 2 shown in SEQ ID No.6;
Described primed probe group 3 forms for the primer 3-2 shown in primer 3-1, SEQ ID No.8 shown in SEQ ID No.7 and the probe 3 shown in SEQ ID No.9;
Described primed probe group 4 forms for the primer 4-2 shown in primer 4-1, SEQ ID No.11 shown in SEQ ID No.10 and the probe 4 shown in SEQ ID No.12;
Described primed probe group 5 forms for the primer 5-2 shown in primer 5-1, SEQ ID No.14 shown in SEQ ID No.13 and the probe 5 shown in SEQ ID No.15;
Described primed probe group 6 forms for the primer 6-2 shown in primer 6-1, SEQ ID No.17 shown in SEQ ID No.16 and the probe 6 shown in SEQ ID No.18;
Described primed probe group 7 forms for the primer 7-2 shown in primer 7-1, SEQ ID No.20 shown in SEQ ID No.19 and the probe 21 shown in SEQ ID No.21;
Described primed probe group 8 forms for the primer 8-2 shown in primer 8-1, SEQ ID No.23 shown in SEQ ID No.22 and the probe 8 shown in SEQ ID No.24;
Described primed probe group 9 forms for the primer 9-2 shown in primer 9-1, SEQ ID No.26 shown in SEQ ID No.25 and the probe 9 shown in SEQ ID No.27;
Described primed probe group 10 forms for the primer 10-2 shown in primer 10-1, SEQ ID No.29 shown in SEQ ID No.28 and the probe 10 shown in SEQ ID No.30;
Described primed probe group 11 forms for the primer 11-2 shown in primer 11-1, SEQ ID No.32 shown in SEQ ID No.31 and the probe 11 shown in SEQ ID No.33;
Described primed probe group 12 forms for the primer 12-2 shown in primer 12-1, SEQ ID No.35 shown in SEQ ID No.34 and the probe 12 shown in SEQ ID No.36;
Described primed probe group 13 forms for the primer 13-2 shown in primer 13-1, SEQ ID No.38 shown in SEQ ID No.37 and the probe 13 shown in SEQ ID No.39;
Described primed probe group 14 forms for the primer 14-2 shown in primer 14-1, SEQ ID No.41 shown in SEQ ID No.40 and the probe 14 shown in SEQ ID No.42;
Described primed probe group 15 forms for the primer 15-2 shown in primer 15-1, SEQ ID No.44 shown in SEQ ID No.43 and the probe 15 shown in SEQ ID No.45;
Described primed probe group 16 forms for the primer 16-2 shown in primer 16-1, SEQ ID No.47 shown in SEQ ID No.46 and the probe 16 shown in SEQ ID No.48;
Described primed probe group 17 forms for the primer 17-2 shown in primer 17-1, SEQ ID No.50 shown in SEQ ID No.49 and the probe 17 shown in SEQ ID No.51;
Described primed probe group 18 forms for the primer 18-2 shown in primer 18-1, SEQ ID No.53 shown in SEQ ID No.52 and the probe 18 shown in SEQ ID No.54;
Described primed probe group 19 forms for the primer 19-2 shown in primer 19-1, SEQ ID No.56 shown in SEQ ID No.55 and the probe 19 shown in SEQ ID No.57;
Described primed probe group 20 forms for the primer 2 0-2 shown in primer 2 0-1, SEQ ID No.59 shown in SEQ ID No.58 and the probe 20 shown in SEQ ID No.60;
Described primed probe group 21 forms for the primer 2 1-2 shown in primer 2 1-1, SEQ ID No.62 shown in SEQ ID No.61 and the probe 21 shown in SEQ ID No.63;
Described primed probe group 22 forms for the primer 2 2-2 shown in primer 2 2-1, SEQ ID No.65 shown in SEQ ID No.64 and the probe 22 shown in SEQ ID No.66;
Described primed probe group 23 forms for the primer 2 3-2 shown in primer 2 3-1, SEQ ID No.68 shown in SEQ ID No.67 and the probe 23 shown in SEQ ID No.69;
Described primed probe group 24 forms for the primer 2 4-2 shown in primer 2 4-1, SEQ ID No.71 shown in SEQ ID No.70 and the probe 24 shown in SEQ ID No.72;
Described primed probe group 25 forms for the primer 2 5-2 shown in primer 2 5-1, SEQ ID No.74 shown in SEQ ID No.73 and the probe 25 shown in SEQ ID No.75;
Described primed probe group 26 forms for the primer 2 6-2 shown in primer 2 6-1, SEQ ID No.77 shown in SEQ ID No.76 and the probe 26 shown in SEQ ID No.78;
Described primed probe group 27 forms for the primer 2 7-2 shown in primer 2 7-1, SEQ ID No.80 shown in SEQ ID No.79 and the probe 27 shown in SEQ ID No.81.
2. primer set according to claim 1 and probe, is characterized in that: the primer in described primed probe group is 9:20 with the amount of substance ratio of probe.
3. primer set according to claim 1 and 2 and probe, is characterized in that:
Described trypetid kind is any one in following 27 kinds: Mexico is by trypetid, western India is by trypetid, Portugal fruit fly, Bactrocera correcta, Carambola fruit trypetid, citrus fruit fly, pepper fruit fly, rust haw trypetid, macadamia nut trypetid, short-tail fruit fly, breadfruit tree trypetid, Peach fruits fly, olea europaea fruit trypetid, the large trypetid of tangerine, bactrocera tsuneonis, calabash melon trypetid, two band fruit flies, melon trypetid, broadband fruit fly, selective propensity of Bactrocera tau, jujube fly, Mediterranean fruitfly, the little bar trypetid of mango, the little bar trypetid of Natta ear, the few hair on the neck trypetid of cucurbit, cherry around trypetid and apple around trypetid.
4., for the identification of a PCR reagent for trypetid kind, be made up of PCR reagent 1, PCR reagent 2, PCR reagent 3, PCR reagent 4, PCR reagent 5, PCR reagent 6, PCR reagent 7, PCR reagent 8, PCR reagent 9, PCR reagent 10, PCR reagent 11, PCR reagent 12, PCR reagent 13, PCR reagent 14, PCR reagent 15, PCR reagent 16, PCR reagent 17, PCR reagent 18, PCR reagent 19, PCR reagent 20, PCR reagent 21, PCR reagent 22, PCR reagent 23, PCR reagent 24, PCR reagent 25, PCR reagent 26 and PCR reagent 27;
Described PCR reagent 1 comprises the primed probe group 1 in primer set according to claim 1 and probe;
Described PCR reagent 2 comprises the primed probe group 2 in primer set according to claim 1 and probe;
Described PCR reagent 3 comprises the primed probe group 3 in primer set according to claim 1 and probe;
Described PCR reagent 4 comprises the primed probe group 4 in primer set according to claim 1 and probe;
Described PCR reagent 5 comprises the primed probe group 5 in primer set according to claim 1 and probe;
Described PCR reagent 6 comprises the primed probe group 6 in primer set according to claim 1 and probe;
Described PCR reagent 7 comprises the primed probe group 7 in primer set according to claim 1 and probe;
Described PCR reagent 8 comprises the primed probe group 8 in primer set according to claim 1 and probe;
Described PCR reagent 9 comprises the primed probe group 9 in primer set according to claim 1 and probe;
Described PCR reagent 10 comprises the primed probe group 10 in primer set according to claim 1 and probe;
Described PCR reagent 11 comprises the primed probe group 11 in primer set according to claim 1 and probe;
Described PCR reagent 12 comprises the primed probe group 12 in primer set according to claim 1 and probe;
Described PCR reagent 13 comprises the primed probe group 13 in primer set according to claim 1 and probe;
Described PCR reagent 14 comprises the primed probe group 14 in primer set according to claim 1 and probe;
Described PCR reagent 15 comprises the primed probe group 15 in primer set according to claim 1 and probe;
Described PCR reagent 16 comprises the primed probe group 16 in primer set according to claim 1 and probe;
Described PCR reagent 17 comprises the primed probe group 17 in primer set according to claim 1 and probe;
Described PCR reagent 18 comprises the primed probe group 18 in primer set according to claim 1 and probe;
Described PCR reagent 19 comprises the primed probe group 19 in primer set according to claim 1 and probe;
Described PCR reagent 20 comprises the primed probe group 20 in primer set according to claim 1 and probe;
Described PCR reagent 21 comprises the primed probe group 21 in primer set according to claim 1 and probe;
Described PCR reagent 22 comprises the primed probe group 22 in primer set according to claim 1 and probe;
Described PCR reagent 23 comprises the primed probe group 23 in primer set according to claim 1 and probe;
Described PCR reagent 24 comprises the primed probe group 24 in primer set according to claim 1 and probe;
Described PCR reagent 25 comprises the primed probe group 25 in primer set according to claim 1 and probe;
Described PCR reagent 26 comprises the primed probe group 26 in primer set according to claim 1 and probe;
Described PCR reagent 27 comprises the primed probe group 27 in primer set according to claim 1 and probe.
5., for the identification of an integrated fluidic chip for trypetid kind, comprise arbitrary described primer set and probe or PCR reagent according to claim 4 in claim 1-3.
6. integrated fluidic chip according to claim 5, it is characterized in that: described integrated fluidic chip is each PCR reagent in claim 4 be divided in each micro-reaction chamber of integrated fluidic chip, obtain the integrated fluidic chip containing described primer set arbitrary in claim 1-3 and probe or PCR reagent according to claim 4.
7., for the identification of a test kit for trypetid kind, to comprise in claim 1-3 arbitrary described primer set and probe or PCR reagent according to claim 4 or the integrated fluidic chip described in claim 5 or 6.
8. in claim 1-3, arbitrary described primer set and probe or PCR reagent according to claim 4 or the integrated fluidic chip described in claim 5 or 6 or test kit according to claim 7 are being identified or whether assistant identification trypetid to be measured is application in 27 kinds of trypetids in any one;
In claim 1-3 arbitrary described primer set and probe or PCR reagent according to claim 4 or the integrated fluidic chip described in claim 5 or 6 or test kit according to claim 7 identify or assistant identification trypetid to be measured be any in 27 kinds of trypetids in application;
Described 27 kinds of trypetids are that Mexico is by trypetid, western India is by trypetid, Portugal fruit fly, Bactrocera correcta, Carambola fruit trypetid, citrus fruit fly, pepper fruit fly, rust haw trypetid, macadamia nut trypetid, short-tail fruit fly, breadfruit tree trypetid, Peach fruits fly, olea europaea fruit trypetid, the large trypetid of tangerine, bactrocera tsuneonis, calabash melon trypetid, two band fruit flies, melon trypetid, broadband fruit fly, selective propensity of Bactrocera tau, jujube fly, Mediterranean fruitfly, the little bar trypetid of mango, the little bar trypetid of Natta ear, the few hair on the neck trypetid of cucurbit, cherry around trypetid and apple around trypetid.
9. the method for a qualification or assistant identification trypetid kind to be measured, comprise the steps: the genomic dna of trypetid to be measured to add in each micro-reaction chamber of the integrated fluidic chip described in claim 5 or 6, carry out real-time fluorescence PCR, detect each micro-reaction chamber PCR primer;
If on described integrated fluidic chip certain primed probe group place micro-reaction chamber in carry out positive amplification, then trypetid to be measured is or candidate is target trypetid corresponding to this primed probe group;
If on described integrated fluidic chip certain primed probe group place micro-reaction chamber in be non-positive amplification, then trypetid to be measured is not or candidate is not target trypetid corresponding to this primed probe group.
10. method according to claim 9, is characterized in that:
Described positive amplification is that micro-reaction chamber produces fluorescence and CT value≤20.0; Described non-positive amplification refers to that micro-reaction chamber does not produce fluorescence or produces fluorescence and CT value > 20.0;
The trypetid of described primed probe group 1 correspondence is that Mexico is by trypetid;
The trypetid of described primed probe group 2 correspondence is that western India is by trypetid;
The trypetid of described primed probe group 3 correspondence is Portugal fruit fly;
The trypetid of described primed probe group 4 correspondence is Bactrocera correcta;
The trypetid of described primed probe group 5 correspondence is Carambola fruit trypetid;
The trypetid of described primed probe group 6 correspondence is citrus fruit fly;
The trypetid of described primed probe group 7 correspondence is pepper fruit fly;
The trypetid of described primed probe group 8 correspondence is rust haw trypetid;
The trypetid of described primed probe group 9 correspondence is macadamia nut trypetid;
The trypetid of described primed probe group 10 correspondence is short-tail fruit fly;
The trypetid of described primed probe group 11 correspondence is breadfruit tree trypetid;
The trypetid of described primed probe group 12 correspondence is Peach fruits fly;
The trypetid of described primed probe group 13 correspondence is olea europaea fruit trypetid;
The trypetid of described primed probe group 14 correspondence is the large trypetid of tangerine;
The trypetid of described primed probe group 15 correspondence is bactrocera tsuneonis;
The trypetid of described primed probe group 16 correspondence is calabash melon trypetid;
The trypetid of described primed probe group 17 correspondence is two band fruit flies;
The trypetid of described primed probe group 18 correspondence is melon trypetid;
The trypetid of described primed probe group 19 correspondence is broadband fruit fly;
The trypetid of described primed probe group 20 correspondence is selective propensity of Bactrocera tau;
The trypetid of described primed probe group 21 correspondence is jujube fly;
The trypetid of described primed probe group 22 correspondence is Mediterranean fruitfly;
The trypetid of described primed probe group 23 correspondence is the little bar trypetid of mango;
The trypetid of described primed probe group 24 correspondence is the little bar trypetid of Natta ear;
The trypetid of described primed probe group 25 correspondence is the few hair on the neck trypetid of cucurbit;
The trypetid of described primed probe group 26 correspondence is that cherry is around trypetid;
The trypetid of described primed probe group 27 correspondence is that apple is around trypetid;
Described trypetid kind is any one in following 27 kinds: Mexico is by trypetid, western India is by trypetid, Portugal fruit fly, Bactrocera correcta, Carambola fruit trypetid, citrus fruit fly, pepper fruit fly, rust haw trypetid, macadamia nut trypetid, short-tail fruit fly, breadfruit tree trypetid, Peach fruits fly, olea europaea fruit trypetid, the large trypetid of tangerine, bactrocera tsuneonis, calabash melon trypetid, two band fruit flies, melon trypetid, broadband fruit fly, selective propensity of Bactrocera tau, jujube fly, Mediterranean fruitfly, the little bar trypetid of mango, the little bar trypetid of Natta ear, the few hair on the neck trypetid of cucurbit, cherry around trypetid and apple around trypetid.
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CN114058707A (en) * 2020-07-29 2022-02-18 中国农业大学 LAMP kit for identifying Bactrocera mediterranei and application thereof
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