CN110004244A

CN110004244A - Marker group, composition and the application of comprehensive screening transgene component

Info

Publication number: CN110004244A
Application number: CN201910204523.0A
Authority: CN
Inventors: 付伟; 王晨光; 朱鹏宇; 朱水芳
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2019-03-18
Filing date: 2019-03-18
Publication date: 2019-07-12

Abstract

This application discloses marker group, composition and the applications of a kind of comprehensive screening transgene component.The marker group includes: 7 kinds of screening elements: PCAMV35S, TCAMV35S, TNOS, PRbcS4, TPINII, TE9, PAT, further includes the strain specificity sequence of corn DAS40278, soybean DP305423 and soybean CV127.The composition is the reagent for detecting the marker group, preferably primer and probe shown in No.1-33 SEQ ID.The present invention can cover 66 kinds of commercialized transgenic strains on the market, have the advantages that reproducible, high-throughput, sensitive, accurately and quickly, have a good application prospect in terms of detection of GMOs.

Description

Marker group, composition and the application of comprehensive screening transgene component

Technical field

The present invention relates to field of biotechnology, the marker group of specifically a kind of comprehensive screening transgene component, combination Object and application.

Background technique

Genetically modified crops have been commercialized more than two decades, and cultivated area has had reached 1.851 hundred million hectares at 2016, are more than Genetically modified crops have been planted by 26 countries.With genetically modified crops large-scale plantation in the world and consumption, increasingly More countries and regions come into effect transgenic product mark management system.Add in view of genetically modified crops in plantation harvesting, production It will appear during work and transport consumption etc. and inevitably mix, have a countries and regions more than 60 to transgenic product mark at present Know and is provided with threshold value.Relevant departments, China, which are also being studied, to be introduced into Transgene-safty management system.Identification thresholds Using bringing more scientific and reasonable technical measures for Transgene-safty management, while also quantifying to the accurate of transgene component Propose urgent and specific demand.

Currently, transgene component quantitative detection generally uses real-time fluorescent PCR technology, it is by drawing standard substance concentration Curve calculates gm content by detection threshold value, but limitation is very big in practical applications, most importantly The preparation of standard curve is difficult to accomplish accurately normal concentration gradient first, secondly in operating procedure and drafting curve procedures It can be inevitably generated error, digital pcr can avoid this point well and can not be influenced by amplification efficiency, realize It is accurately quantitative.

Digital pcr (hereinafter referred to as dPCR) is a kind of PCR method that monomolecular amplification is realized based on Water-In-Oil structure, can be real Copy number now is detected independent of standard curve, calculates transgene component using reference gene and copy number of foreign gene ratio Content, to realize highly sensitive quantify.DPCR according to sample dispersing mode mainly have droplet type digital pcr (ddPCR) and Two kinds of chip type digital pcr (cdPCR).

Currently, detection of GMOs is certain genes for certain kinds mostly, there are no fully assess all turns The method of gene element or transgenic strain.

Summary of the invention

In view of the defects existing in the prior art, the purpose of the present invention is to provide a kind of marks of comprehensive screening transgene component Will object group, composition and application.The present invention can cover 66 kinds of commercialized transgenic strains on the market, have reproducible, high Flux, it is sensitive, accurately and quickly the advantages of, had a good application prospect in terms of detection of GMOs.

To achieve the above objectives, the technical solution adopted by the present invention is that:

Present invention firstly provides a marker groups, including following 7 kinds of screening elements: pea ribulose -1,5- diphosphonic acid carboxylic Change enzyme small subunit E9 gene terminator (TE9), rouge alkali synthetase gene terminator (TNOS), cauliflower mosaic virus promoter (PCAMV35S), green streptomyces chromogenes encoding phosphinothricin acetyl transferase gene (PAT), potato proteinase inhibitor II are whole Only sub (TPINII), arabidopsis RbcS4 gene promoter (PRbcS4) and cauliflower mosaic virus terminator (TCAMV35S)；

Optional, the marker group further includes strain specificity sequence, and/or the soybean of corn DAS40278 The strain specificity sequence of DP305423, and/or the strain specificity sequence of soybean CV127；

Standard items such as plasmid standard, stranded DNA molecule segment etc. can be made in the above marker group, for transgenosis at It is used when sorting is surveyed as referring to such as positive criteria product.

The present invention protects application of the marker group described above in (quantitative or qualitative) detection transgene component,

Preferably, from genetically modified plants and/or its processed goods, (processed goods can extract the transgene component To the DNA for not occurring to change)；

The genetically modified plants include but is not limited to following plant: corn and soybean, rape, potato, beet, clover, And/or rice；

It is furthermore preferred that the genetically modified plants include but is not limited to any one or a few following transgenic strain and its spread out Health product kind: corn MON810, corn NK603, corn MON89034, corn MON88017, corn MON87460, corn MON87427, modified corn MON 863, corn Bt11, corn 3272, corn 5307, corn MIR604, corn GA21, corn MIR162, corn Bt176, corn TC1507, corn 59122, corn 4114, corn T25, corn 98140, corn VCO- 01981-5, soybean GTS40-3-2, soybean MON89788, soybean MON87701, soybean MON87705, soybean MON87708, greatly Beans MON87769, soybean MON87751, soybean DP356043, soybean A5547-127, soybean A2704-12, soybean SYHT0H2, Soybean FG72, soybean DAS44406-6, soybean DAS68416-4, soybean DAS81419, modified rape GT 73, rape MON88302, oil Dish 73496, rape RF3, rape MS8, rape Topas-19/2, rape OXY-235, rape T45, rape MS1, rape RF1, oil Dish RF2, beet H7-1, clover J101, clover J163, clover KK179, oryza sativa l. LRICE62, rice 114-7-2, rice BT63, Rice Huahui No.1, rice KMD, rice KF6, rice KF8, rice T2A-1, rice T1c-19, potato AM04-1020, Potato EH92-527-1, potato PH05-026-0048, potato AV-43-6-G7；

Optional, the genetically modified plants are further selected from any one or a few following transgenic strain and its derived varieties: Corn DAS40278, soybean DP305423, soybean CV127.

The present invention also provides a kind of for detecting the composition of transgene component, and the composition includes for more than detection The PCR primer combination of any marker group.

In a preferred embodiment, the PCR primer combination includes following primer pair:

For the primer pair 1 of specific detection screening element TE9, the upstream primer of the primer pair 1 such as SEQ ID No.1 institute Show, downstream primer is as shown in SEQ ID No.2；

For the primer pair 2 of specific detection screening element TNOS, the upstream primer of the primer pair 2 such as SEQ ID No.4 Shown, downstream primer is as shown in SEQ ID No.5；

For the primer pair 3 of specific detection screening element PCAMV35S, the upstream primer of the primer pair 3 such as SEQ ID Shown in No.7, downstream primer is as shown in SEQ ID No.8；

For the primer pair 4 of specific detection screening element PAT, the upstream primer of the primer pair 4 such as SEQ ID No.10 Shown, downstream primer is as shown in SEQ ID No.11；

For the primer pair 5 of specific detection screening element TPINII, the upstream primer of the primer pair 5 such as SEQ ID Shown in No.13, downstream primer is as shown in SEQ ID No.14；

For the primer pair 6 of specific detection screening element PRbcS4, the upstream primer of the primer pair 6 such as SEQ ID Shown in No.16, downstream primer is as shown in SEQ ID No.17；Or the upstream primer of the primer pair 6 such as SEQ ID No.31 institute Show, downstream primer is as shown in SEQ ID No.32；

For the primer pair 7 of specific detection screening element TCAMV35S, the upstream primer of the primer pair 7 such as SEQ ID Shown in No.19, downstream primer is as shown in SEQ ID No.20；

Draw the upstream of the primer pair 8 of strain specificity sequence for specific detection corn DAS40278, the primer pair 8 Object is as shown in SEQ ID No.22, and downstream primer is as shown in SEQ ID No.23；

The primer pair 9 of strain specificity sequence for specific detection soybean CV127, the upstream primer of the primer pair 9 As shown in SEQ ID No.25, downstream primer is as shown in SEQ ID No.26；

The primer pair 10 of strain specificity sequence for specific detection soybean DP305423, the upstream of the primer pair 10 Primer is as shown in SEQ ID No.28, and downstream primer is as shown in SEQ ID No.29；

Preferably, the PCR primer combination further includes detecting the probe groups of the amplified production of the primer pair；

It is furthermore preferred that the probe groups include:

For detection primer to the probe 1 of 1 amplified production, the sequence of the probe 1 is SEQ ID No.3 or its complementary sequence Column；

For detection primer to the probe 2 of 2 amplified productions, the sequence of the probe 2 is SEQ ID No.6 or its complementary sequence Column；

For detection primer to the probe 3 of 3 amplified productions, the sequence of the probe 3 is SEQ ID No.9 or its complementary sequence Column；

For detection primer to the probe 4 of 4 amplified productions, the sequence of the probe 4 is SEQ ID No.12 or it is complementary Sequence；

For detection primer to the probe 5 of 5 amplified productions, the sequence of the probe 5 is SEQ ID No.15 or it is complementary Sequence；

For detection primer to the probe 6 of 6 amplified productions, the sequence of the probe 6 is SEQ ID No.18 or it is complementary Sequence；Or the sequence of the probe 6 is SEQ ID No.33 or its complementary series；

For detection primer to the probe 7 of 7 amplified productions, the sequence of the probe 7 is SEQ ID No.21 or it is complementary Sequence；

For detection primer to the probe 8 of 8 amplified productions, the sequence of the probe 8 is SEQ ID No.24 or it is complementary Sequence；

For detection primer to the probe 9 of 9 amplified productions, the sequence of the probe 9 is SEQ ID No.27 or it is complementary Sequence；

For detection primer to the probe 10 of 10 amplified productions, the sequence of the probe 10 is SEQ ID No.30 or it is mutual Complementary series.

In one embodiment, the probe is Taqman probe；Preferably, 5 ' ends of the probe are glimmering using reporting Light group FAM, VIC, HEX, Texas Red or CY5 are modified, the probe 3 ' end using quenching group TAMRA, BHQ1, BHQ2 or BHQ3 are modified.

The present invention protects application of any description above composition in qualitatively or quantitatively detection transgene component；

Preferably, the transgene component derives from genetically modified plants and/or its processed goods；

Preferably, the genetically modified plants include but is not limited to following plant: corn and soybean, rape, potato, beet, Clover, and/or rice；

The present invention also provides a kind of PCR kit of (qualitative or quantitative) detection transgene component, the kit includes Any description above composition,

Optional, the kit further includes the marker group of any description above；

Preferably, the kit further includes regular-PCR, real-time fluorescence PCR or digital pcr amplifing reagent (including buffering Liquid, relevant ions, dNTP, archaeal dna polymerase)；

It is furthermore preferred that the digital pcr amplifing reagent includes American AB I Life Technology company The chip of 2 × MasterMix of reagent and Fluidigm company, the U.S. in Universal Master Mix II kit AssayLoading Reagent and 20 × GE Sample Loading Reagent.

The present invention also provides a kind of PCR methods of (qualitative or quantitative) detection transgene component, and the method includes as follows Step:

S1, the genomic DNA for acquiring sample to be tested；

S2, using the DNA of step S1 as template, use any description above composition or the kit to carry out PCR amplification；

S3, according to step S2's as a result, judging in sample to be tested whether containing foreign gene or to determine institute in sample to be tested State the copy number of foreign gene.

In a preferred embodiment, the PCR amplification is digital pcr amplification,

Preferably, the concentration of template DNA is 2-3ng/ μ L in the digital pcr amplified reaction, it is furthermore preferred that 2.5ng/ μ L；

Preferably, expand or detect in the digital pcr amplified reaction upstream primer of same target gene, downstream primer and The molar ratio of probe is (5-1): (5-1): 1, it is furthermore preferred that 4.5:4.5:1；

It is furthermore preferred that expanding or detecting upstream primer, the downstream primer of same target gene in the digital pcr amplified reaction Concentration with probe is respectively 2.25 μM, 2.25 μM and 0.5 μM.

In a preferred embodiment, the program of the digital pcr amplification are as follows: 50 DEG C of hot activation 300s；95 DEG C pre- It is denaturalized 120s；50 circulations: 95 DEG C of denaturation 15s, 60 DEG C of annealing and extension 60s；In 60 DEG C of acquisition fluorescence signals.

Beneficial effects of the present invention are as follows:

(1) present invention as 7 screening element PCAMV35S, TCAMV35S of marker group, TNOS, PRbcS4, TPINII, TE9 and PAT are covered on the market except comprising corn DAS40278 strain, soybean DP305423 strain and soybean CV127 All commercialized transgenic strains (63 kinds) except three strains of strain, as long as tentatively judging that unknown sample is more than removing Strain except three strains, 7 screening elements by detection as marker group can be in accurate judgement unknown sample It is no to contain transgene component.The present invention as 7 screening element PCAMV35S, TCAMV35S of marker group, TNOS, The strain specificity sequence of PRbcS4, TPINII, TE9, PAT and corn DAS40278, soybean DP305423 and soybean CV127 Column cover all commercialized transgenic strains (66 kinds, as shown in table 2) on the market, make just without the strain to unknown sample Step judgement, directly by detection as 7 screening elements and/or corn DAS40278 of marker group and/or soybean The strain specificity sequence of DP305423 and/or soybean CV127 can in accurate judgement unknown sample whether containing transgenosis at Point.

(2) present invention is for as 7 screening elements and/or corn DAS40278 of marker group and/or soybean The a plurality of primer and probe of the strain specificity sequence design of DP305423 and/or soybean CV127, and a large amount of screenings have been carried out, Its comprehensive stability, specificity, sensitivity, pairing composite amplification influence each other and different primers probe combinations and number The suitability of PCR amplification kit, finishing screen select specificity it is good, high sensitivity, stability is good and can detect 7 screenings simultaneously The primer of the strain specificity sequence of element and/or corn DAS40278 and/or soybean DP305423 and/or soybean CV127 and Probe combinations.

(3) present invention establishes the digital pcr inspection of detection transgene component on the basis of the combination of aforementioned primer and probe Survey method, can be simultaneously to 7 screening elements and/or corn DAS40278 and/or soybean DP305423 and/or soybean CV127 Strain specificity sequence carry out qualitative and quantitative detection, and it is reproducible, high-throughput, sensitive, accurately and quickly the advantages of, turn Gene element context of detection has a good application prospect.

Detailed description of the invention

Fig. 1-17 is respectively that different primers and probe amplification contain the same sample group of corresponding target genes (screening element) not With the result of sample.Wherein, abscissa is recurring number, and ordinate is Δ R_n。

Figure 18 is primed probe group-specific amplification schematic diagram.Horizontally-arranged 1-33 respectively represents the screening element of amplification successively Are as follows: PCAMV35S, TCAMV35S, TNOS, PAT, TPINII, TE9, PRbcS4, DAS40278, DP305423, CV127, each Screening element amplification sets 3 in parallel；Horizontally-arranged 34-48 respectively represents the reference gene of amplification successively are as follows: corn Adh1 (alcohol Dehydrogenase alcohol dehydrogenase), soybean lectin (lectin genes), rice sps (sucrose phosphate Synthase sucrose synthase), rape PEP (phosphoenolpyruvate carboxylase gene, phosphoenolpyruvate third Keto acid carboxylase gene), potato UPGase (UDP-glucose pyrophosphorylase (cytoplasm marker) UDPglucose pyrophosphorylase), (Alfalfa Acetyl-CoA carboxylase clover acetyl is auxiliary by clover ACC Enzyme a carboxylase), beet GS (glutamine synthetase glutamine synthelase), each reference gene amplification sets 2 and puts down Row；Vertical setting of types 1-48 represents the sample of amplification successively are as follows: corn MON810, corn NK603, corn MON89034, corn MON88017, corn MON87460, corn MON87427, modified corn MON 863, corn Bt11, corn 3272, corn 5307, corn MIR604, corn GA21, corn MIR162, corn Bt176, corn TC1507, corn 59122, corn 98140, corn T25, Corn DAS40278, soybean GTS40-3-2, soybean MON89788, soybean MON87701, soybean MON87705, soybean MON87708, soybean MON87769, soybean DP305423, soybean DP356043, soybean A5547-127, soybean A2704-12, greatly Beans SYHT0H2, soybean FG72, soybean DAS44406-6, soybean DAS68416-4, soybean DAS81419, soybean CV127, rape GT73, rape MON88302, rape 73496, rape RF3, rape MS8, rape Topas-19/2, rape OXY-235, rape T45, rape RF2, potato PH05-026-0048, beet H7-1, clover KK179, soybean MON87751；Right side is different in figure The numerical value that gray scale represents is CT value.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Used reaction reagent CTAB, Tris alkali, NaCl, Na in following embodiments₂EDTA, ethyl alcohol, chloroform and isopropyl Alcohol, is purchased from Sigma company, and purity is that analysis is pure.

Used water, is the level-one water for meeting GB/T 6682 in following embodiments.

Solution in following embodiments is formulated as follows:

CTAB extracting solution: by CTAB, NaCl, Tris alkali, Na₂EDTA and water, which mix, is adjusted to 8.0 for pH with hydrochloric acid later, respectively Component final concentration is respectively as follows: CTAB 20g/L, NaCl 1.4mol/L, Tris alkali 0.1mol/L, Na₂EDTA 0.02mol/L。

CTAB precipitated liquid: CTAB, NaCl and water are mixed, and each component final concentration is respectively as follows: CTAB 5g/L, NaCl 40mmol/L。

Proteinase K Solution (20mg/mL): take 20mg Proteinase K be dissolved in 1mL Tris-HCl containing 50mmol/L (pH8.0), 1.5mmol/L calcium acetate (CaCl₂) and the solution by sterilizing of 50% (volume ratio) glycerol in, slight mix makes Proteinase K It is completely dissolved, it is stand-by to be placed in -20 DEG C of storages after packing.

NaCl solution: concentration is the aqueous solution containing NaCl of 1.2mol/L.

The template preparation method that PCR amplification uses in following embodiments is as follows:

1, sample treatment

(1) it will be dried in the hot wind of sample to be tested (such as crop seeds) merging lower temperature, reduce moisture in seed Content and the degradation for avoiding genome；

(2) sample after drying is put into ball milling instrument and is crushed, crushing program is to crush 20s 500rpm/s, is placed in room The lower 1min of temperature, makes the sample crushed be down to room temperature to reduce degradation of the genome in crushing process；

(3) the crushing program in step (2) repeats 10 circulations, obtains the powder of sample to be tested.

2, extracting genome DNA

CTAB method is taken to extract genome, the specific steps are as follows:

(1) it takes the powder of 50mg sample to be tested to be put into 2mL centrifuge tube, and the CTAB extraction that 1mL is preheated to 65 DEG C is added Liquid and 4 μ L Proteinase K Solutions (20mg/mL) are placed in water-bath 1h in 65 DEG C of water-baths, and midway, which is taken out, to be mixed for several times；

(2) system after water-bath is placed in a centrifuge, 13000g be centrifuged 15min after, draw supernatant be transferred to it is new from In heart pipe；

(3) supernatant is mixed with 500 μ L chloroforms, is placed in vortex 30s on vortex oscillation instrument and mixes well, is placed in centrifuge Middle 13000g is centrifuged 15min；

(4) supernatant is transferred in new 1.5mL centrifuge tube, the CTAB precipitated liquid of 2 times of volumes, room temperature preservation is added 1h；

(5) previous step centrifuge tube is placed in a centrifuge 13000g centrifugation 5min, abandons supernatant；

(6) 350 μ L NaCl solutions and 350 μ L chloroforms are added in centrifuge tube one step up, are placed in whirlpool concussion instrument After vortex 30s is mixed well, 13000g is centrifuged 10min；

(7) supernatant is transferred in new 1.5mL centrifuge tube, the isopropanol of 0.8 times of volume is added, jog mixes, room temperature Lower placement 20min；

(8) previous step system is placed in a centrifuge 13000g centrifugation 10min, abandons supernatant, and 500 μ L 75% are added Ethyl alcohol, vortex 30s sufficiently wash the precipitating in pipe, and 13000g is centrifuged 10min；

(9) supernatant is abandoned, and precipitating is dried, 50 μ L ultrapure waters are added and sufficiently dissolve, gene is measured by NanoDrop Group concentration and the identification for carrying out genomic DNA purity；

(10) genome 50ng/ μ L is diluted to be placed at -20 DEG C for use.Experiment is 20ng/ μ L with genome concentration.

Following digital pcrs use Fluidigm digital pcr platform, amplifing reagent source explanation: 2 × MasterMix of reagent in Universal Master Mix II kit is purchased from American AB I Life Technology Company, chip Assay Loading Reagent and 20 × GE Sample Loading Reagent related with digital pcr It is purchased from Fluidigm company, the U.S..

Embodiment 1, the design of primer and probe and screening

According to a large amount of market survey data, the transgenic strain of real commercial growths all on the market is first determined, then Determine screening of the following 7 kinds of screening elements as covering in addition to corn DAS40278, soybean DP305423 and soybean CV127 strain Element: PCAMV35S, TCAMV35S, TNOS, PRbcS4, TPINII, TE9 and PAT；Finally inquires again and determine three kinds of transgenosis Strain: the strain specificity sequence of corn DAS40278, soybean DP305423 and soybean CV127.

With the specific sequence (totally 10 kinds of sequences) of above-mentioned 7 kinds of screening element sequences and above-mentioned three kinds of transgenic strains for base Plinth collects the insetion sequence information of commercialization transgenic strain, and to every group, (each screening element or strain are special based on this Anisotropic sequence is classified as one group, totally 10 groups) sequence separately designs multipair primer and its probe, and primer and its probe are steady between considering 10 groups Qualitative, specific, sensitivity, pairing composite amplification influence each other and different primers and its probe combinations and digital pcr expansion Increase the suitability of kit, first the preferable two pairs of primers of expanding effect and its probe is filtered out from every group, using containing primer The genomic DNA of same group of sample of corresponding target genes is template, carries out real-time fluorescence PCR, selects the amplification capabilities such as specificity Best primer and its probe.Each group primer and its detecting probe information and corresponding amplification are as shown in table 1.

7 kinds of screening elements are respectively as follows:

PCAMV35S: cauliflower mosaic virus promoter；

TCAMV35S: cauliflower mosaic virus terminator；

TNOS: rouge alkali synthetase gene terminator；

PRbcS4: arabidopsis RbcS4 gene promoter；

TPINII: potato proteinase inhibitor II terminator；

TE9: pea ribulose -1,5- diphosphonic acid carboxylase small subunit TE9 gene terminator；

PAT: green streptomyces chromogenes encoding phosphinothricin acetyl transferase gene；

The screening element contained in all commercialized transgenic strains is as shown in table 2.

Table 1, primer and probe sequence information and amplification

Note: primer or probe title consist of three parts: target gene or transgenic strain symbol-upstream or downstream primer or Probe symbol and group #, wherein F represents upstream primer；R represents downstream primer；P represents probe；As the sequence of probe, 5 ' End label reporter fluorescence group FAM, 3 ' end label quenching group TAMRA.

Table 2, all commercialized transgenic strain theories contain screening element/strain specificity sequence situation and summarize

Note: the "+" that blank in table 2 represents in this transgenic strain without corresponding screening element, in table, which represents, to exist There is corresponding screening element in this strain.

The method of above-mentioned real-time fluorescence PCR is as follows:

Reaction system: template is 2-5ng/ μ L；12.5 μ L TaqMan gene expression master mix, 0.4 μ M upstream primer；0.4 μM of downstream primer；0.2 μM of probe；Water is mended to 25 μ L.

Response procedures: 50 DEG C/2min；95℃/10min；95 DEG C/15s, 60 DEG C/60s, it is greater than or equal to 40 circulations.

Real-time fluorescence PCR result: as shown in Fig. 1-17；Each pair of primed probe group is tested with positive template respectively, often Group setting two is parallel.It can be seen that, amplification efficiency is different in the two pairs of primers and its probe of every group of design, together from amplification figure When positive amplification number it is also different, choosing in the primer and probe of every group of design has obvious amplification curve and whole samples have obviously A pair of amplification curve is as preferred primer and its probe, if two pairs of primers and its probe have obvious amplification curve, and CT Value is approximate, then any to choose a pair.Finally pick out totally 10 primer pairs and probe combinations, be respectively as follows: TE9-F1/R1/P1, TNOS-F1/R1/P1、PCAMV35S-F1/R1/P1、PAT-F1/R1/P1、TPINII-F/R/P、PRbcS4-F1/R1/P1、 TCAMV35S-F1/R1/P1,DAS40278-F1/R1/P1,CV127-F/R/P,DP305423-F/R/P.In addition, PRbcS4- F1/R1/P1 (Figure 10 and Figure 11) identical as the amplification of PRbcS4-F2/R2/P2, it is also possible to PRbcS4-F2/R2/P2 replacement PRbcS4-F1/R1/P1。

Embodiment 2 uses the method for digital pcr detection transgene component

The present embodiment judges whether biological sample contains transgene component according to the result that digital pcr expands, and this method makes Obtained 10 primer and probes combination is screened with embodiment 1 to carry out.

Using the genome of sample to be tested as template, each sample to be tested is arranged three in parallel, is obtained using the screening of embodiment 1 The combination (SEQ ID No.1-30) of primer and probe carry out digital pcr amplification, while negative control and blank control are set, Wherein negative control: with negative genes group (i.e. the genomic DNA of non-transgenic sample) for template, blank control: using water as mould Plate.

Specific step is as follows for digital pcr amplification:

(1) Prime step: blue protection film is removed from digital pcr chip, and Control Line Fluid is injected It in the hole of the upper and lower two sides of chip, is placed in IFC Controller MX, executes Prime (167 ×) operation.

(2) the genome system and primed probe system difference of digital pcr reaction are as shown in Table 3 and Table 4:

The genome system of table 3, digital pcr amplified reaction

Component	Volume/μ L	Final concentration/additive amount
			2×MasterMix	2	1×
20×GE Sample Loading Reagent	0.2	1×
			Genomic DNA	1	20ng
Ultrapure water	0.8
			Total volume	4

The primed probe system of table 4, digital pcr amplified reaction

Up/down trip primer and probe in table 4 is respectively selected from any one group in following 10 groups:

TE9-F1/R1/P1、TNOS-F1/R1/P1、PCAMV35S-F1/R1/P1、PAT-F1/R1/P1、TPINII-F/R/ P、PRbcS4-F1/R1/P1、TCAMV35S-F1/R1/P1、DAS40278-F1/R1/P1、CV127-F/R/P、DP305423-F/ R/P。

(3) after the completion of digital pcr reaction system is prepared, genome system is transferred to the template of Fluidigm digit chip In well, primed probe system is transferred in the assay well of Fluidigm digit chip, to be kept away during sample-adding Exempt to generate bubble in the sky.

(4) after the completion of being loaded, Fluidigm digit chip is put back in IFC Controller MX and executes Load (167 ×) operate, after Load process, chip is taken out, removes the dust of chip surface, and chip is put into BioMark HD In System.

(5) digital pcr amplification program are as follows: 50 DEG C of hot activation 300s；95 DEG C of initial denaturation 120s；50 circulations: 95 DEG C of denaturation 15s, 60 DEG C of annealing and extension 60s；In 60 DEG C of acquisition fluorescence signals.

(6) fluorescence signal acquisition process is FAM single channel fluorescent collecting, collects fluorescence signal automatically by instrument and generates The amplification curve in each hole.

(7) qualitative analysis: analyzing the amplification curve in each hole, and the threshold cycle of the amplification curve in each hole is arranged.When Amplification curve regular shape, and when CT value is less than or equal to 40, then be judged as positive, this represent in sample to be tested containing with primer and The corresponding screening element of probe combinations or strain specificity sequence, namely contain transgene component；If Ct value is greater than 40, then judge For feminine gender, this, which represents to be free of in sample to be tested, combines corresponding screening element or strain specificity sequence with primer and probe.

Embodiment 3, sensitivity technique

Sensitivity verifying has been carried out according to the method using digital pcr detection transgene component that embodiment 2 is established.Detection The sensitivity of system, which is defined as the detection architecture, can carry out the minimum copy number of stable detection, and the present embodiment, which uses, contains 7 A screening element PCAMV35S, TCAMV35S, TNOS, PRbcS4, TPINII, TE9, PAT and corn DAS40278, soybean The transgenic sample of the strain specificity sequence of DP305423, soybean CV127 contains different quality by artificial dilution preparation Then the homogeneous sample of percentage transgene component carries out digital pcr amplification according to the method for embodiment 2, the specific steps are as follows:

(1) test sample: select transgenic sample: corn MON87427 is (as detection screening element " PCAMV35S " Sample), corn 59122 (as detection screening element " TPINII " sample), corn MON89034 (as detection screening element The sample of " TNOS "), soybean MON87705 (sample as detection screening element " TE9 "), soybean DAS44406-6 is (as inspection Survey screening element " PAT " sample), corn 59122 (as detection screening element " TCAMV35S " sample), soybean MON87701 (sample as detection screening element " PRbcS4 "), corn DAS40278 are (as detection screening element " corn The sample of the strain specificity sequence of DAS40278 "), (as detection screening element, " strain of soybean CV127 is special by soybean CV127 The sample of anisotropic sequence ") and soybean DP305423 (as detection screening element " the strain specificity sequence of soybean DP305423 " Sample), be maintained in China Inst. of Quarantine Inspection Sciences.Screening element to be measured in the test sample is single copy, Be conducive to copy number calculating in this way.

Non-transgenic sample includes: corn non-transgenic sample and soybean non-transgenic sample, is maintained in Chinese inspection Quarantine research institute.

(2) genomic DNA of sample is extracted respectively.

(3) respectively using the absolute copy number of genomic DNA of the method detection each sample of embodiment 2.

(4) each sample is diluted to the transgenic sample of copy number 50,20,10,5,4,3,2,1.

(5) each sample in step (4) is placed on blending instrument at a slow speed, mixing overnight is to guarantee to configure the uniform of sample Property.

(6) it records and detects most using the genomic DNA of the method detection each sample various concentration of embodiment 2 respectively The stability of low copy number.

As a result: non-transgenic sample is without amplification, and the results are shown in Table 5 for the sensitivity technique of transgenic sample.

Table 5, the sensitivity technique result containing different screening elements

Element in 5 gauge outfit of table represents screening element or strain specificity sequence, and Detected cp., which is represented, to be detected The minimum copy number arrived, digital 1-8 are eight parallel reactions of minimum copy number, and the data below 1-8 represent CT value, Average represents 8 parallel reaction mean CT-numbers, and STDEV represents standard deviation, and RSD (%) represents relative standard deviation.

Table 5 the result shows that, the sensitivity that the method for the embodiment of the present application 2 detects each screening element is respectively as follows: detection The sensitivity of TNOS is 4 copies/detection architecture；The sensitivity for detecting PCaMV35S is 5 copies/detection architecture；Detection The sensitivity of TCAMV35S is 5 copies/detection architecture；The sensitivity for detecting PAT is 4 copies/detection architecture；Detect the spirit of TE9 Sensitivity is 2 copies/detection architecture；The sensitivity for detecting TPINII is 10 copies/detection architecture；Detection PRbcS4 sensitivity be 5 copies/detection architecture；The sensitivity for detecting corn DAS40278 strain specificity sequence is 5 copies/detection architecture；Detection is big The sensitivity of beans CV127 strain specificity sequence is 4 copies/detection architecture；Detect soybean DP305423 strain specificity sequence Sensitivity be 5 copies/detection architecture.

Embodiment 4, specific detection

Validity, specificity and stability are to judge the whether feasible major criterion of a screening detection architecture.This implementation Example carries out the intersection amplification of the test sample of different lines to foreign gene as described in Example 2 to verify its validity, special Property and stability.

48 test samples: corn MON810, corn NK603, corn MON89034, corn MON88017, corn MON87460, corn MON87427, modified corn MON 863, corn Bt11, corn 3272, corn 5307, corn MIR604, corn GA21, corn MIR162, corn Bt176, corn TC1507, corn 59122, corn 98140, corn T25, corn DAS40278, soybean GTS40-3-2, soybean MON89788, soybean MON87701, soybean MON87705, soybean MON87708, greatly Beans MON87769, soybean DP305423, soybean DP356043, soybean A5547-127, soybean A2704-12, soybean SYHT0H2, Soybean FG72, soybean DAS44406-6, soybean DAS68416-4, soybean DAS81419, soybean CV127, modified rape GT 73, rape MON88302, rape 73496, rape RF3, rape MS8, rape Topas-19/2, rape OXY-235, rape T45, rape RF2, Potato PH05-026-0048, beet H7-1, clover KK179, soybean MON87751；Above-mentioned test sample is maintained in Chinese inspection Test quarantine research institute.

Experimental procedure is carried out according to embodiment 2, and every group of up/down trip primer and probe has corresponding small reaction system, often A small reaction system does three parallel, each test sample corresponding digital pcr amplification such as Figure 18 institutes for each test sample Show.

By the CT value and the positive and yin of the positive in 2 and negative judgment method to each test sample in conjunction with the embodiments of Figure 18 Property is summarized, and the testing result of table 6 is obtained.

Each screening element/strain specificity sequence testing result is directed in table 6, each test sample

Note: the blank representative of table 6 can not amplify corresponding screening element in this test sample, that is, be negative；In table "+" representative have corresponding screening element in this test sample, that is, be positive.

Through deck watch 6 and table 2 it is found that screening element/strain specificity the sequence for each test sample that the present embodiment is tested The amplification (table 6) of column and screening element/strain specificity sequence covering result (table of theoretic each transgenic strain 2) completely the same, this illustrates the number for the detection transgene component that the primer and probe combination of embodiment 1 and embodiment 2 are established The validity and specificity of PCR method are high, can accurately identify the transgene component in different lines；It is every simultaneously Three parallel results of a screening element are able to maintain consistency, this illustrates the primer and probe combination and reality of embodiment 1 The stability for applying the digital pcr method of the detection transgene component of the foundation of example 2 is very good.

Embodiment 5, coverage verifying

The present embodiment is to 7 screening elements and corn DAS40278, soybean DP305423 as marker group and greatly The strain specificity sequence of beans CV127 has carried out coverage verifying, and combines (SEQ ID to the primer and probe of embodiment 1 No.1-30), the method for the detection transgene component of embodiment 2 has carried out validity and specificity verification.

The test sample (transgenic sample) of use shares 66 kinds, covers all commercialized transgenic strains on the market, Sample is respectively 21 kinds of corn shown in table 2 (Maize), 17 kinds of soybean (Soybean), 11 kinds of rape (Canola), beet (Sugarbeet) a kind, 3 kinds of clover (Alfalfa), 9 kinds of rice (Rice) and 4 kinds of potato (Potato), all samples are protected There are China Inst. of Quarantine Inspection Sciences.

Experimental procedure is carried out according to embodiment 2, and every group of up/down trip primer and probe has corresponding small reaction system, often A small reaction system does three in parallel for each test sample, and summarizes to the positive and feminine gender of each test sample, obtains Testing result is consistent completely with the theoretical case in table 2.

Result of this example indicate that the present invention as marker group 7 screening element PCAMV35S, TCAMV35S, The strain of TNOS, PRbcS4, TPINII, TE9, PAT and corn DAS40278, soybean DP305423 and soybean CV127 are special Property sequential covering all commercialized transgenic strains (66 kinds) on the market, can directly utilize the primer and probe of embodiment 2 Combination and method detection are as 7 screening elements and corn DAS40278 of marker group, soybean DP305423 and soybean Whether the strain specificity sequence of CV127 can contain transgene component in accurate judgement unknown sample, this further illustrates The primer and probe combination of embodiment 2 and the validity and specificity of digital PCR method are high, and simultaneously for transgenosis at The detection divided has the characteristics that comprehensive.

In conclusion for the present invention, the length and type for the exogenous gene sequence that different transgenic strains turn are not Together, even if what is be transferred to is same screening element, can also exist in different transgenic strains and be shown because of base mutation Each species diversity, therefore primer and probe of the invention combination needs to consider in design: (1) whether the target area expanded is institute Whether the position in the region of the foreign gene turned, (2) design is in the conserved region for the foreign gene that these turn, and (3) avoid drawing The complex effect of object and primer, primer and probe, probe and probe；Actually not only to consider above-mentioned triple factors, but also Validity, specificity, stability and the susceptibility of verifying primer and probe combination are removed with practice, while meeting these limitation items Part is very difficult, and the present invention overcomes numerous difficulties, design primer and probe combination and set up embodiment 2 Detection transgene component digital pcr method, not only detection comprehensively, also have high validity, specificity, stability and Susceptibility has a good application prospect.

The content being not described in detail in this specification belongs to the prior art well known to professional and technical personnel in the field.More than Described is only embodiments herein, is not intended to limit this application.To those skilled in the art, the application can To there is various modifications and variations.All any modification, equivalent replacement, improvement and so within the spirit and principles of the present application, It should be included within the scope of the claims of this application.

SEQUENCE LISTING

<110>China Inst. of Quarantine Inspection Sciences

<120>marker group, composition and the application of screening transgene component comprehensively

<130> JH-CNP190093

<160> 33

<170> PatentIn version 3.5

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tcttgtacca tttgttgtgc ttgt 24

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ggaccatatc attcattaac tcttctcc 28

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cggttttcgc tatcgaactg tgaaatggaa atgg 34

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gcatgacgtt atttatgaga tggg 24

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tcctagtttg cgcgctatat tt 22

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agagtcccgc aattatacat ttaatacgcg 30

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ttccaaccac gtcttcaaag c 21

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ggaagggtct tgcgaaggat a 21

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ccactgacgt aagggatgac gcacaatcc 29

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ggagaggaga ccagttgaga ttag 24

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gtgtttgtgg ctctgtccta aag 23

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atcacaaacc gcggccatat cagctgc 27

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gacttgtcca tcttctggat tgg 23

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agtgattagc atgtcactat gtgtgcatcc 30

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ccactccacc atcacacaat ttc 23

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ggagaggtgt tgagaccctt atc 23

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acgtggcatt attccagcgg ttcaagcc 28

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tcaccagtct ctctctacaa atctatc 27

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caacacatga gcgaaaccct ataa 24

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tgtgtgagta gttcccagat aagggaatta gggt 34

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gtccagcacg aaccattgag 20

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tcattgtatt ctggctttgc tgta 24

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acaatcaaca gcaccgtacc ttgaagcgga 30

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cgttgagctt taagacgttt gg 22

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gtggcccttc tcgccttat 19

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agctgtccca tgcccatcaa agaagacagt 30

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agcttggtac taacatacga aatca 25

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aagcttatat atgccttccg ctaag 25

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acgagaaaga accaatttct ttgcattcgg c 31

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caagccgata agggtctcaa c 21

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atcggagggt ctaggataca tga 23

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Claims

1. a marker group, which is characterized in that the marker group includes following 7 kinds of screening elements: pea ribulose -1,5- bis- Phosphoric acid carboxylase small subunit E9 gene terminator (TE9), rouge alkali synthetase gene terminator (TNOS), cauliflower mosaic virus Promoter (PCAMV35S), green streptomyces chromogenes encoding phosphinothricin acetyl transferase gene (PAT), potato proteinase inhibit Agent II terminator (TPINII), arabidopsis RbcS4 gene promoter (PRbcS4) and cauliflower mosaic virus terminator (TCAMV35S)；

Optional, the marker group further includes the strain specificity sequence, and/or soybean DP305423 of corn DAS40278 The strain specificity sequence of strain specificity sequence, and/or soybean CV127.

2. application of the marker group described in claim 1 in detection transgene component；

It is furthermore preferred that the genetically modified plants include but is not limited to any one or a few following transgenic strain and its spin-off Kind: corn MON810, corn NK603, corn MON89034, corn MON88017, corn MON87460, corn MON87427, Modified corn MON 863, corn Bt11, corn 3272, corn 5307, corn MIR604, corn GA21, corn MIR162, corn Bt176, corn TC1507, corn 59122, corn 4114, corn T25, corn 98140, corn VCO-01981-5, soybean GTS40-3-2, soybean MON89788, soybean MON87701, soybean MON87705, soybean MON87708, soybean MON87769, greatly Beans MON87751, soybean DP356043, soybean A5547-127, soybean A2704-12, soybean SYHT0H2, soybean FG72, soybean DAS44406-6, soybean DAS68416-4, soybean DAS81419, modified rape GT 73, rape MON88302, rape 73496, rape RF3, rape MS8, rape Topas-19/2, rape OXY-235, rape T45, rape MS1, rape RF1, rape RF2, beet H7- 1, clover J101, clover J163, clover KK179, oryza sativa l. LRICE62, rice 114-7-2, rice BT63, rice Huahui No.1, rice KMD, rice KF6, rice KF8, rice T2A-1, rice T1c-19, potato AM04-1020, potato EH92- 527-1, potato PH05-026-0048, potato AV-43-6-G7；

3. a kind of for detecting the composition of transgene component, which is characterized in that the composition includes wanting for detecting right Seek the PCR primer combination of the 1 or 2 marker groups.

4. composition as claimed in claim 3, which is characterized in that the PCR primer combination includes following primer pair:

For the primer pair 1 of specific detection screening element TE9, the upstream primer of the primer pair 1 as shown in SEQ ID No.1, Downstream primer is as shown in SEQ ID No.2；

For the primer pair 2 of specific detection screening element TNOS, the upstream primer of the primer pair 2 as shown in SEQ ID No.4, Downstream primer is as shown in SEQ ID No.5；

For the primer pair 3 of specific detection screening element PCAMV35S, the upstream primer of the primer pair 3 such as SEQ ID No.7 Shown, downstream primer is as shown in SEQ ID No.8；

For the primer pair 4 of specific detection screening element PAT, the upstream primer of the primer pair 4 as shown in SEQ ID No.10, Downstream primer is as shown in SEQ ID No.11；

For the primer pair 5 of specific detection screening element TPINII, the upstream primer of the primer pair 5 such as SEQ ID No.13 institute Show, downstream primer is as shown in SEQ ID No.14；

For the primer pair 6 of specific detection screening element PRbcS4, the upstream primer of the primer pair 6 such as SEQ ID No.16 institute Show, downstream primer is as shown in SEQ ID No.17；Or the upstream primer of the primer pair 6 is as shown in SEQ ID No.31, downstream Primer is as shown in SEQ ID No.32；

For the primer pair 7 of specific detection screening element TCAMV35S, the upstream primer of the primer pair 7 such as SEQ ID No.19 Shown, downstream primer is as shown in SEQ ID No.20；

The primer pair 8 of strain specificity sequence for specific detection corn DAS40278, the upstream primer of the primer pair 8 is such as Shown in SEQ ID No.22, downstream primer is as shown in SEQ ID No.23；

The primer pair 9 of strain specificity sequence for specific detection soybean CV127, the upstream primer such as SEQ of the primer pair 9 Shown in ID No.25, downstream primer is as shown in SEQ ID No.26；

The primer pair 10 of strain specificity sequence for specific detection soybean DP305423, the upstream primer of the primer pair 10 As shown in SEQ ID No.28, downstream primer is as shown in SEQ ID No.29；

It is furthermore preferred that the probe groups include:

For detection primer to the probe 1 of 1 amplified production, the sequence of the probe 1 is SEQ ID No.3 or its complementary series；

For detection primer to the probe 2 of 2 amplified productions, the sequence of the probe 2 is SEQ ID No.6 or its complementary series；

For detection primer to the probe 3 of 3 amplified productions, the sequence of the probe 3 is SEQ ID No.9 or its complementary series；

For detection primer to the probe 4 of 4 amplified productions, the sequence of the probe 4 is SEQ ID No.12 or its complementary series；

For detection primer to the probe 5 of 5 amplified productions, the sequence of the probe 5 is SEQ ID No.15 or its complementary series；

For detection primer to the probe 6 of 6 amplified productions, the sequence of the probe 6 is SEQ ID No.18 or its complementary series； Or the sequence of the probe 6 is SEQ ID No.33 or its complementary series；

For detection primer to the probe 7 of 7 amplified productions, the sequence of the probe 7 is SEQ ID No.21 or its complementary series；

For detection primer to the probe 8 of 8 amplified productions, the sequence of the probe 8 is SEQ ID No.24 or its complementary series；

For detection primer to the probe 9 of 9 amplified productions, the sequence of the probe 9 is SEQ ID No.27 or its complementary series；

For detection primer to the probe 10 of 10 amplified productions, the sequence of the probe 10 is SEQ ID No.30 or its complementary sequence Column.

5. composition as described in claim 3 or 4, it is characterised in that: the probe is Taqman probe；Preferably, described 5 ' ends of probe are modified using reporter fluorescence group FAM, VIC, HEX, Texas Red or CY5, and 3 ' ends of the probe make It is modified with quenching group TAMRA, BHQ1, BHQ2 or BHQ3.

6. application of any composition in detection transgene component in claim 3-5,

Preferably, the genetically modified plants include but is not limited to following plant: corn and soybean, rape, potato, beet, lucerne Mu, and/or rice；

7. a kind of PCR kit for detecting transgene component, which is characterized in that the kit includes appointing in claim 3-5 One composition,

Optional, the kit further includes marker group of any of claims 1 or 2；

Preferably, the kit further includes regular-PCR, real-time fluorescence PCR or digital pcr amplifing reagent；

It is furthermore preferred that the digital pcr amplifing reagent includes American AB I Life Technology company The chip of 2 × MasterMix of reagent and Fluidigm company, the U.S. in Universal Master Mix II kit Assay Loading Reagent and 20 × GE Sample Loading Reagent.

8. a kind of PCR method for detecting transgene component, characterized by the following steps:

S1, the genomic DNA for acquiring sample to be tested；

S2, using the DNA of step S1 as template, use described in the composition any in claim 3-5 or claim 7 try Agent box carries out PCR amplification；

S3, according to step S2's as a result, judging in sample to be tested whether containing foreign gene or to determine outer described in sample to be tested The copy number of source gene.

9. method according to claim 8, which is characterized in that the PCR amplification is digital pcr amplification,

Preferably, the upstream primer, downstream primer and probe of same target gene are expanded or detected in the digital pcr amplified reaction Molar ratio be (5-1): (5-1): 1, it is furthermore preferred that 4.5:4.5:1；

It is furthermore preferred that expanding or detecting the upstream primer, downstream primer and spy of same target gene in the digital pcr amplified reaction The concentration of needle is respectively 2.25 μM, 2.25 μM and 0.5 μM.

10. method as claimed in claim 9, which is characterized in that the program of the digital pcr amplification are as follows: 50 DEG C of hot activations 300s；95 DEG C of initial denaturation 120s；50 circulations: 95 DEG C of denaturation 15s, 60 DEG C of annealing and extension 60s；Believe in 60 DEG C of acquisition fluorescence Number.