CN109825631A - Transgene rape strain Topas19-2 detection method and reagent - Google Patents
Transgene rape strain Topas19-2 detection method and reagent Download PDFInfo
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Abstract
This application discloses a kind of transgene rape strain Topas19-2 detection method and reagents.The detection method of the application, special primer and probe including napus lines Topas19-2 foreign gene and PEP gene is added simultaneously in PCR reaction system, and mineral oil is added, droplet is made in mixing, dual ddPCR is carried out in droplet, after reaction, each droplet amplified signal is read, the copy number of strain foreign gene and PEP is calculated；Wherein, the probe of strain foreign gene and PEP gene uses FAM and HEX to mark respectively.The application detection method, design matching used endogenous gene PEP and strain specificity primed probe, using two-channel method, copy number of the two in rapeseed gene group is quantified respectively, transgene rape strain Topas19-2 absolute content is calculated, it is significant to the research of rape transgene component and the accurate quantitative detection of rape product transgenic ingredient of passing in and out.
Description
Technical field
This application involves transgene rape detection fields, examine more particularly to a kind of transgene rape strain Topas19-2
Survey method and reagent.
Background technique
With the rapid development of biotechnology industry, the research and development speed and cultivated area of global genetically modified crops persistently increase
Add.Transgenic technology is promoted to have become the development strategy having an eye on the future and solve food shortage, ensure that national food is pacified
Full inevitable choice.But GMO bio-safety problem also becomes the hot spot of World Focusing therewith.At present countries in the world for
The various purposes such as economy, health, environmental protection are strengthened management and are detected to genetically modified organism and products thereof one after another.Including China
50 or so countries and regions promulgate in succession and have carried out " transgenic labeling system ", to genetically modified organism and its converted products into
Line identifier and management.In recent years, the foundation with various countries in relation to GMO (GeneticallyModified organism) labeling acts
With constantly improve, in food GMO content lower limit have defined.Many countries not only require to determine GM food
Property detection, it is also necessary to in food GMO content carry out quantitative detection, to identify.The threshold value that it is identified is generally 0.9%-
Between 5% (European Commission, 2003；Notification,2000).Establish the detection technique of transgenic product
The especially accurate quantitative measurement technology of standard is the technology premise for implementing transgenic product mark.Therefore, the essence of transgene component
Quasi- quantitative technique will be as the whole world identifies the perfect of system and develops increasingly important.
Quantitative detecting method most widely used at this stage is real-time fluorescence PCR (qPCR), but stability, accuracy are poor, institute
To need more accurate detection method to be substituted.Digital pcr (dPCR) is the gene to grow up on the basis of fluorescent PCR
Quantitative technique, the absolute copy number for nucleic acid detect.DPCR is by being divided into phase for conventional Fluorescence PCR system equivalent
The micro- reaction system of big quantity being mutually isolated, usual equivalent are divided into 10000 or more micro- reaction systems, keep each template independent
It is randomly assigned into micro- reaction system, while carrying out pcr amplification reaction under identical rated condition, according to the fluorescent value of setting
Judge the testing result of each micro- reaction system.Poisson distribution point is finally carried out with the positive of experiment and the ratio of negative findings
Analysis obtains the template concentrations in Fluorescence PCR system.Compared with traditional qPCR, there are following advantages by dPCR: (1) qPCR
Result judgement relies on the Ct value of amplification curve, is easy to be influenced by the external world；And dPCR result judgement is independent of amplification curve, only
It needs to determine the presence or absence of amplified signal, so that it may determine sample, stability is much stronger than qPCR；(2) qPCR be easy by
The influence of inhibiting factor in matrix；And dPCR can weaken PCR inhibiting factor and tie to reaction by the way that reaction system to be split
The influence of fruit, to improve the stability and accuracy of result judgement.
Currently, digital pcr (dPCR) has been achieved for very big progress in terms of scientific research, examined in clinic
Disconnected, copy number identification, absolute quantitation etc. achieve many scientific achievements.Taly etc. (2013) utilizes droplet type digital pcr
(ddPCR) the KRAS mutation gene of technology detection Patients With Rectal Carcinoma cyclic DNA, finally realizes the multiple sample including wild type
Detection.In the detection research of plant source product, Corbisier etc. (2010) is analyzed in corn seed DNA using digital pcr
The ratio between foreign gene and the copy number of internal standard gene, the result and utilization common fluorescent quantitative PCR technique are mark with Plasmid DNA
The result of quasi- substance detection is identical, it was demonstrated that the reliability of digital pcr.Burns etc. (2010) has evaluated the detection limit of digital pcr
(LOD) and quantitative limit (LOQ), digital pcr is explored in the feasibility and reaction condition of context of detection, article shows digital pcr
Absolute quantitation can be carried out to initial template copy number.Morisset etc. (2013) is detected using droplet type digital pcr (ddPCR)
Transgenic corns MON810 contents, obtain it is consistent with quantitative PCR as a result, the result also indirect proof dPCR technology for
The contribution of transgenosis quantitative detection.In general, in terms of dPCR technology is more applied to medical diagnosis at present, it has also become clinical
Most potential one of the diagnostic techniques of application aspect, and breakthrough progress is achieved in other industries.But in transgenosis
In terms of the research of detection, digital pcr is also in initial phase, especially in all temporary nothing of the standard aspect of China or even world community
It is related to, this greatly constrains application of the digital pcr in actually detected work.
Studies have shown that is counted according to cultivated area, and the rape in the whole world about 30% is all transgenic product；However international, state
It is interior that the qualitative or quantitative detection method of the specificity of transgene rape is but quite lacked, especially to transgene rape strain
The quantitative detection of Topas19-2, there has been no relevant research or reports.Therefore, it needs to study a kind of effective transgene rape
Strain Topas19-2 detection method.
Summary of the invention
The purpose of the application is to provide a kind of transgene rape strain Topas19-2 detection method and reagent.
To achieve the goals above, the application uses following technical scheme:
The one side of the application discloses the detection method of transgene rape strain Topas19-2 a kind of, including following step
It is rapid:
PCR amplification system preparation steps, including strain Topas19-2 special primer group, strain Topas19-2 are specifically visited
Needle, PEP gene specific primer group, PEP gene specific probe and sample to be tested DNA be added in PCR reaction solution, be made dual
PCR reaction system；
Droplet generation step is included in double PCR reaction system and droplet generation oil is added, after mixing, is transferred into
Droplet generates on instrument, and concussion generates droplet；
PCR reaction and droplet analytical procedure, including droplet is transferred to progress PCR reaction in reaction tube, PCR reaction terminates
Afterwards, the amplified signal of each droplet is read using droplet analyzer；
Quantitative detection step is copied according to the amplified signal of reading using the specific amplified of software analysis strain Topas19-2
Shellfish number accounts for the percentage of interior source reference gene PEP gene specific amplification copy number, indicates transgene rape strain with this
The content of Topas19-2；
The forward primer of strain Topas19-2 special primer group is sequence shown in Seq ID No.1, strain Topas19-2
The reverse primer of special primer group is sequence shown in Seq ID No.2, and strain Topas19-2 specific probe is Seq ID No.3
Shown sequence, the forward primer of PEP gene specific primer group are sequence shown in Seq ID No.4, PEP gene specific primer group
Reverse primer is sequence shown in Seq ID No.5, and PEP gene specific probe is sequence shown in Seq ID No.6；
Seq ID No.1:5 '-GTTGCGGTTCTGTCAGTTCC-3 '
Seq ID No.2:5 '-CGACCGGCGCTGATATATGA-3 '
Seq ID No.3:5 '-TCCCGCGTCATCGGCGG-3 '
Seq ID No.4:5 '-CCCTTGTGAAGCTCGACATC-3 '
Seq ID No.5:5 '-CTTGTCCTCTGACCATTCTTTGT-3 '
Seq ID No.6:5 '-CCGACCGTCACACCGATGTTTTAGA-3 '
Wherein, in the strain Topas19-2 specific probe of sequence shown in Seq ID No.3,5 ' ends have fluorophor,
3 ' ends have quenching group；In the PEP gene specific probe of sequence shown in Seq ID No.6,5 ' ends have fluorophor,
3 ' ends have quenching group.The application is red, and Topas19-2 is by being abbreviated as Topas19/2 again for transgene rape strain.
Preferably, in strain Topas19-2 specific probe, the fluorophor of 5 ' ends is FAM, the quenching group of 3 ' ends
For BHQ-1；In PEP gene specific probe, the fluorophor of 5 ' ends is HEX, and the quenching group of 3 ' ends is BHQ-1.
It should be noted that the detection method of the transgene rape strain Topas19-2 of the application, actually droplet
Formula digital pcr.Its principle is that grease reaction system is prepared into nearly up to ten thousand oil by drop generator in a PCR reaction tube
Bao Shui little droplet, when DNA profiling molecular number to be measured is lower than certain number, droplet contains only the DNA profiling and foot of a molecule
Enough measure for all other component such as dNTP, Taq archaeal dna polymerase and probe and primer etc. needed for Fluorescence PCR.
After reaction to all droplet PCR, each droplet is checked one by one, as long as droplet has fluorescence signal detection, be regarded as PCR
Reaction occurs, and note has the template to be measured an of molecule to be detected, and counts all positive droplet numbers, so that it may obtain to be measured in sample
The molecular number or copy number of DNA profiling.The application uses two-channel method, i.e., visits respectively to napus lines Topas19-2 specificity
Needle and rape endogenous gene specific probe are surveyed in the same PCR reaction system simultaneously using different fluorescent markers
Determine the copy number of endogenous gene and strain distinguished sequence.Wherein, the endogenous gene PEP of the application detection is in rapeseed gene group
Copy number is determining, and copy number of the napus lines Topas19-2 specific sequence in rapeseed gene group is also determining
's；Therefore, transgenic rape can be calculated by calculating the ratio of strain specificity sequence and PEP sequence copy numbers in sample
The content of vegetable system Topas19-2.
In a kind of implementation of the application, specifically, including being calculated separately out using QuantaSoft V1.3.2 software
After the copy number and PEP gene copy number of transgene rape strain Topas19-2, according to transgene rape strain Topas19-2
The ratio of copy number and PEP gene copy number calculates the content of transgene rape strain Topas19-2.
The another side of the application discloses a kind of reagent of transgene rape strain Topas19-2 quantitative detection, the reagent
Including the first primer probe combinations and the second primer combination of probe, the first primer probe combinations are special for strain Topas19-2
Property detection, the second primer combination of probe for PEP gene specific detection；In the first primer probe combinations, forward primer Seq
Sequence shown in ID No.1, reverse primer are sequence shown in Seq ID No.2, and probe is sequence shown in Seq ID No.3, and
5 ' ends of probe have fluorophor, and 3 ' ends have quenching group；In second primer combination of probe, forward primer Seq
Sequence shown in ID No.4, reverse primer are sequence shown in Seq ID No.5, and probe is sequence shown in Seq ID No.6, and
5 ' ends of probe have fluorophor, and 3 ' ends have quenching group.
It should be noted that the reagent of the application, the actually quantitative detection of transgene rape strain Topas19-2
Strain Topas19-2 specific detection primer probe and PEP gene specific detection primer employed in method and probe.
Therefore, in the probe of sequence shown in Seq ID No.3, the fluorophor of 5 ' ends is FAM, and the quenching group of 3 ' ends is BHQ-
1；In the probe of sequence shown in Seq ID No.6, the fluorophor of 5 ' ends is HEX, and the quenching group of 3 ' ends is BHQ-1.
It is appreciated that the primer and probe of the application is designed for droplet type digital pcr, it is of course possible to be prepared separately into corresponding
Strain Topas19-2 detection reagent use.
The another side of the application discloses the reagent of the application in prepare transgenosis rape detection kit or detection device
In application.
The application's discloses a kind of transgene rape detection kit, the examination containing the application in the kit on one side again
Agent.
Preferably, the kit of the application using the application detection method to transgene rape strain Topas19-2 into
Row quantitative detection.
Due to using the technology described above, the beneficial effects of the present application are as follows:
The detection method of the transgene rape strain Topas19-2 of the application, devises matching used endogenous gene PEP
Specific primer probe and strain Topas19-2 specific primer probe, the two match, fixed respectively using two-channel method
Copy number of the two in rapeseed gene group is measured, the content of transgene rape strain Topas19-2 is finally calculated.The application
Detection method can be to measuring samples transgenic napus lines different from the relative quantification of conventional real-time fluorescence PCR
Topas19-2 ingredient carries out accurate quantitative detection, and quantitative detection lower bound is copied up to 4, and qualitative detection lower bound is copied up to 2
Shellfish, research and China to rape transgene component pass in and out rape product transgenic ingredient accurate quantitative detection have weight
Want meaning.
Detailed description of the invention
Fig. 1 is the output result figure in the channel stability test FAM in the embodiment of the present application；
Fig. 2 is the output result figure in the channel stability test HEX in the embodiment of the present application；
Fig. 3 is the output result figure in the channel specific detection FAM in the embodiment of the present application；
Fig. 4 is the output result figure in the channel specific detection HEX in the embodiment of the present application；
Fig. 5 is the fit standard curve graph for the HEX channel data that gradient dilution detects in the embodiment of the present application；
Fig. 6 is the fit standard curve graph for the FAM channel data that gradient dilution detects in the embodiment of the present application.
Specific embodiment
Transgene rape strain Topas19-2 is at present still without corresponding qualitative or quantitative detection method.The application takes the lead in
It studies and proposes the detection method of transgene rape strain Topas19-2 a kind of, the detection method of the application can not only determine
Property detection strain Topas19-2, and accurate absolute quantitation can be carried out to it.
The detection method of the application and existing transgene rape strain detect in conventional use of general PCR, glimmering in real time
The methods of light PCR, PCR-DHPLC difference；General PCR can only qualitative detection, although real-time fluorescence PCR can be by dilute to gradient
The standard items released are detected, and standard curve is established, and carry out relative quantification by standard curve；But it can not also accomplish absolutely to determine
Amount detection.As transgenic crop is more and more common, the detection method of existing relative quantification is no longer satisfied China mouthful
The use demand of bank detection GMOs.Therefore, the application utilizes advanced digital pcr detection technique, takes the lead in studying and propose
The quantitative detecting method of transgene rape strain Topas19-2.Also, it in the quantitative detecting method of the application, designs simultaneously
Two sets of matching used primed probes of interior source reference gene and strain gene, are expanded by dual digital pcr, with two fluorescence
Channel statisticallys analyze the copy number of interior source reference gene PEP and strain Topas19-2 specific sequence respectively, according to transgenic rape
The ratio of vegetable system Topas19-2 copy number and PEP gene copy number calculates the absolute of transgene rape strain Topas19-2
Content.
The application is described in further detail below by specific embodiments and the drawings.Following embodiment and attached drawing are only right
The application is further described, and should not be construed as the limitation to the application.
Embodiment
One, material and method
1. material to be tested
Experimental material: this example for select 10 parts of experimental material, respectively transgene rape standard substance Ms1, Ms8, Rf1,
Rf2, Rf3, MON88302, T45, OXY235, DP-073496-4 and Topas19/2, the above material is both from American Oil
Scholar association (AOCS).
Reagent and consumptive material: ddPCR Super Mix, ddPCR Droplet Generation Oil, ddPCRDroplet
Reader Oil, Droplet Generator DG8Cartridge, Droplet Generator DG8Gasket are purchased from beauty
Bio-Rad company, state；DNeasy Plant Mini kit plant genome DNA extracts kit is purchased from QIAGEN company, Germany.
Laboratory apparatus: QX100TM Droplet Digital PCR system, including PCR instrument, droplet generate instrument and droplet is read
Four part of instrument and sealer instrument is taken, U.S. Bio-Rad company is purchased from；Centrifuge is purchased from Beckman Coulter Inc., the U.S.；Be vortexed shake
Instrument is swung for the manufacture of IKA company, Germany；Thermostat water bath is purchased from Beijing Liuyi Instrument Factory, Nanodrop 2000c nucleic acid-protein point
Analyzer is purchased from U.S. Thermo Scientific company.
2. primer and probe designs
The strain Topas19-2 special primer group and strain Topas19-2 specific probe of this example are inserted into according to foreign gene
Hold cross-border sequence design, PEP gene specific primer group and PEP gene specific tip reference SN/T 1204-2016 in site 3 '.
Primer and probe is synthesized and is modified by Sangon Biotech (Shanghai) Co., Ltd..The primer and probe sequence of this example is detailed in
Table 1.
1 endogenous gene of table and strain Topas19-2 specific primer and probe
Wherein, 5 ' end of Topas19-2-P probe carries out the modification of FAM fluorophor, and 3 ' ends carry out BHQ-1 quenching group
Modification；5 ' end of PEP-P probe carries out the modification of HEX fluorophor, and 3 ' ends carry out the modification of BHQ-1 quenching group.
3. dual digital pcr reaction system and condition
The extracting genome DNA of the materials to be tested such as this example transgene rape uses DNeasy Plant Mini kit plant
Genome DNA extracting reagent kit, detailed process refer to kit specification.3 parallel PCR are set when each DNA sample detects
Reaction.
DdPCR reaction system totally 20 μ L, comprising: 2 × ddPCR SuperMix, 10 μ L, the napus lines of 3.6 μm of ol/L are special
The rape of 1 μ L of specific probes Topas19-2-P, napus lines forward primer Topas19-2-F1 μ L of 6 μm of ol/L, 6 μm of ol/L
1 μ L of strain reverse primer Topas19-2-R, interior 1 μ L of source reference gene probe PEP-P, the 6 μm of ol/L of 3.6 μm of ol/L are endogenous
With reference to the DNA of 1 μ L of source reference gene reverse primer PEP-R, 12.5ng/ μ L in 1 μ L of gene forward primer PEP-F, 6 μm of ol/L
4 μ L of template.
20 μ L reaction systems of preparation and 70 μ L droplets oily (droplet generation oil) is generated respectively to be added in
Droplet generates in the corresponding cell card slot (Droplet Generator DG8Cartridge), covers rubber mat (Droplet
Generation DG8Gasket) after be put into droplet generate instrument in, by instrument independently shake generation droplet.It will after wait shake
The droplet of the about 40 μ L generated or so is transferred in 96 orifice plates, and carries out sealer to it with heat-sealing film instrument, is finally placed in PCR instrument
Carry out PCR reaction.
Reaction condition are as follows: 95 DEG C of initial denaturation 10min；It is recycled subsequently into 40: 95 DEG C of denaturation 15s, 56 DEG C of annealing 1min；
98 DEG C after circulation terminates, 10min make enzyme heat inactivation, 4 DEG C are standby.
After amplification, 96 orifice plates are placed in droplet analyzer and read signal, and is soft using QuantaSoftV1.3.2
Part analyzes experimental data.Sample transgenic component content is accounted for interior with sample gene group DNA transgenic distinguished sequence copy number
The percentage of source gene copy number indicates, i.e., the content of transgene component is characterized for copy number percentage composition.
Specifically, opening droplet fluorescence detector application software, the 96 hole reaction plates of PCR after reaction are inserted directly into
In fluorescence detector, equipment detects the fluorescence of all droplets in every PCR reaction tube, i.e. PCR response situation automatically, last soft
Part directly gives DNA sequence dna copy number to be measured according to amber pine distribution law.This example according to FAM fluorescence channel specifically, can obtain
To the copy number of transgene rape strain Topas19-2, according to copying for the available interior source reference gene PEP of HEX fluorescence channel
Shellfish number.Transgene rape can be calculated according to the ratio of strain Topas19-2 copy number and interior source reference gene PEP copy number
The absolute content of strain Topas19-2.
4. stability experiment
Using 2.5ng/ μ L transgene rape strain Topas19/2 genomic DNA as the working concentration of template DNA, wherein
Gm content is 100%.Reaction system is prepared according to " 3. dual digital pcr reaction systems and condition " and carries out PCR
Reaction.Stability experiment is arranged 3 in parallel, it is desirable that the relative standard deviation (Relative of 3 repetition experimental results
Standard Deviation, RSD) it is no more than 25%.
5. specificity experiments
Select transgene rape strain MS1, MS8, RF1, RF2, RF3, MON88302, T45, OXY235, DP-073496-4
It is experimental material with Topas19/2.It is template according to " 3. dual digital pcrs react using this 10 kinds of transgenic sample genomic DNAs
System and condition " prepares ddPCR reaction system, and carries out PCR reaction, and wherein Topas19/2 is as positive control.
6. quantitative linearity scope measures
Transgene rape strain Topas19/2 genomic DNA is diluted to 2.5ng/ μ L, 0.5ng/ with pure water respectively by this example
μ L, 0.1ng/ μ L, 0.02ng/ μ L, 0.01ng/ μ L, 0.004ng/ μ L, 0.0008ng/ μ L, 0.00016ng/ μ L totally 8 work
Concentration.It is carried out using the above-mentioned each dilution DNA of 4 μ L as template " 3. dual digital pcr reaction systems and condition " according to the present example
DdPCR reaction, each concentration repeat 3 reactions, calculate the RSD value of 3 repetition experimental results of each concentration samples.
7. the measurement of quantitative detection limit and detection limit
In the detection method of this example, quantitative detection limit (Limit OfQuantitation, LOQ) is defined as testing result
Minimum amount of samples or samples copy number of the RSD value no more than 25%, as take the lower limit of " measurement of 6. quantitative linearity scopes "
DNA sample carries out ten parallel reactions, RSD value≤25% of testing result.
Detection lower bound (Limit OfDetection, LOD) is defined as the minimum copy number of sample that can be detected or sample is used
It measures, in as ten parallel reactions, the sum of copy numbers of all positive detections are not less than 9.
8. precision test measures
Precision refers under conditions of repeatability, with identical method, identical test item, and in same laboratory,
By same personnel in very short time interval, the standard deviation for the testing result that identical instrument and equipment obtains, this example 6 are used
A parallel digital pcr reaction result RSD value≤25%.
9. accuracy test measures
Be consistent degree of the definition of accuracy between testing result and the group public affairs definite value.
The experiment of this step take LOQ value, 5 times of LOQ value, 3 groups of positive samples of 25 times of LOQ value carry out accuracy verifying,
Three parallel digital pcr experiments, standard error≤25% are carried out to above 3 groups of DNA samples.
Two, result and analysis
1. positive sample expands stability result
Reaction system according to the present example carries out the preparation of digital pcr system, by 2.5ng/ μ L transgene rape strain
Topas19-2 genome carries out digital pcr experiment, and experimental setup 3 parallel.Hotspot graph such as Fig. 1 and Fig. 2 that digital pcr obtains
Shown, the data analysis of experimental result is as shown in table 2.Wherein, Fig. 1 is the output result figure in the channel FAM, i.e. strain Topas19-
2 testing result；Fig. 2 is the output result figure in the channel HEX, i.e., the testing result of interior source reference gene PEP；In Fig. 1 and Fig. 2,
D06, E06, F06 are respectively three parallel tests.Fig. 1 and Fig. 2's the results show that digital pcr negative point obtained with it is positive
There is apparent fluorescence signal gap between point, trailing phenomenon is not serious, can be negative anti-by the way that unified threshold value limit progress is arranged
The differentiation with positive reaction point should be put.
Reaction final result is as shown in table 2, and interior source reference gene expands duplicate actually detected copy number three times and is respectively
1018,1172 and 1164, average detected copy number is 1118, RSD 7.75%, in acceptable range, that is, be less than etc.
In 25%；It is respectively 1162,1042 and 1084 that foreign gene expands duplicate actually detected copy number three times, average detected copy
Number is 1096, RSD 5.55%, in acceptable range, that is, is less than or equal to 25%.As it can be seen that in the detection method of this example
The primed probe amplification of reference gene and foreign gene used has good stability, and the positive sample is stable available.
2 strain Topas19-2 Detection of Stability result of table
2. specific detection result
This example carries out specificity experiments, experimental result using interior external source gene primer probe of the digital pcr method to synthesis
As shown in Figure 3 and Figure 4.Fig. 3 is the output result figure in the channel FAM, the i.e. testing result of strain Topas19-2；Fig. 4 is logical for HEX
The output result figure in road, i.e., the testing result of interior source reference gene PEP；In Fig. 3 and Fig. 4, A05, B05, C05, D05, E05,
F05, G05, H05, A06 are sequentially the inspection of Ms1, Ms8, Rf1, Rf2, Rf3, MON88302, T45, OXY235 and DP-073496-4
It surveys as a result, B06 is Topas19/2, C06 is water control.
Fig. 3 and Fig. 4's the result shows that, the strain Topas19-2 specific primer and probe of this example are to other 9 kinds of transgenosis
Napus lines do not expand, and PEP gene has amplification in other 9 kinds of transgene rape strains and strain Topas19-2,
And it is not expanded in water control.The Topas19-2 specific primer and probe for illustrating this example have good specificity, can
Specific detection for transgene rape strain Topas19-2.
3. range of linearity measurement result
This example carries out transgene rape strain Topas19-2 genomic DNA linearity test using 8 working concentrations, as a result
As shown in table 3, the data fit standard curve in the range of linearity is as shown in Figure 5 and Figure 6.Wherein, Fig. 5 is HEX channel data
Fit standard curve graph, i.e., the fit standard curve of interior source reference gene；Fig. 6 is the fit standard curve graph of FAM channel data,
That is the fit standard curve graph of strain Topas19-2 gene.
The range of linearity of -2 digital pcr method of 3 rape Topas-19 of table is verified
In table 3, amount of DNA indicates that the amount of DNA contained in reaction system, such as 10ng, actually 2.5ng/ μ L turn base
It is obtained because napus lines Topas19-2 genome adds 4 μ L into reaction system；Copy number refers to unit volume in reaction system
Copy number, i.e., the every microlitre copy number containing strain Topas19-2 or PEP.Fig. 5, Fig. 6 and table 3 the results show that this example
Detection method is located in the section 5-1006 in interior source reference gene copy number, that is, correspond to genomic DNA amount be 0.04ng extremely
It in the section of 10ng, can show good linear, across at least three orders of magnitude, meet the requirements, fit standard curve
r2It is 0.99, meets the requirement more than or equal to 0.98, the RSD value of all concentration groups is between 1.9% to 23.93%, symbol
Close the requirement less than 25%；This example detection method corresponds to genome in the section that copy number of foreign gene is located at 4-1199
The amount of DNA is to show good linear, the r of fit standard curve in the section of 0.04ng to 10ng2It is 0.99, Suo Younong
The RSD value of degree group meets the requirements between 5.25% to 24.2%.Therefore, the detection method of this example is in interior source reference gene
Copy number be located at 5-1006, the copy number of foreign gene is located at 4-1199, have good quantitation capabilities.
4. the quantitative detection of standard limits and detection limit result
The DNA sample of " 3. range of linearity measurement result " range of linearity lower limit is taken to carry out the verifying of LOQ.The DNA is carried out
Ten parallel digital pcr verifyings, calculate the reference gene of each parallel unit volume and the copy number of foreign gene, calculate
Average value and RSD value.Specifically, this example takes the genome DNA sample of 0.01ng/ μ L to be tested, the gene in reaction system
Group DNA is 0.04ng, and the results are shown in Table 4.Table 4 the results show that 10 inside and outside sources of parallel testing result of this group of DNA
Gene RSD is respectively 24.95% and 23.26%, meets the requirement less than 25%.This illustrates that the detection method of this example can be external
The sample that source gene copy number is 3.44 carries out stable quantitative detection.
The minimum DNA concentration group that can be expanded in " 3. range of linearity measurement result " is taken to carry out the verifying of LOD, by the DNA
Carry out 10 parallel digital pcr verifyings.This example specifically takes the genome DNA sample of 0.004ng/ μ L to be tested, reactant
Genomic DNA in system is 0.016ng, and the results are shown in Table 5.Table 5 the results show that in ten parallel tests, all positives
The sum of copy number of detection is 17.6, meets the requirement for being not less than 9 of LOD Quality Control.This explanation, the detection method of this example can
With the sample progress stable detection for being 1.76 to copy number of foreign gene.
The LOQ of -2 digital pcr method of 4 rape Topas-19 of table is verified
The LOD of -2 digital pcr method of 5 rape Topas-19 of table is verified
5. precision test measurement result
This example takes the DNA sample of 10 times of concentration of foreign gene LOQ critical value and LOQ critical value to carry out precision verifying, often
A sample does 6 in parallel.Specifically, the genome DNA sample of genome DNA sample and 0.1ng/ μ L to 0.01ng/ μ L into
Row test, the results are shown in Table 6.
The precision measurement result of -2 digital pcr method of 6 rape Topas-19 of table
Table 6 the results show that the testing result of comprehensive analysis LOQ critical point and 10 times of critical point of DNA sample, two groups dense
RSD value is respectively 23.09% and 15.11% in obtained group of the DNA sample of degree, meets entire detection method for precision
Requirement, i.e., RSD is less than 25%.
6. accuracy test measurement result
This example take LOQ value, 5 times of LOQ value, 3 groups of positives of 25 times of LOQ value carry out accuracy verifying.Specifically, this example pair
The genome DNA sample of the genome DNA sample of 0.01ng/ μ L, the genome DNA sample of 0.05ng/ μ L and 0.25ng/ μ L into
Row test, the results are shown in Table 7.
The accuracy determination result of -2 digital pcr method of 7 rape Topas-19 of table
In table 7, amount of DNA indicates that the amount of DNA contained in reaction system, such as 0.04ng, actually 0.01ng/ μ L turn
Vector for transgenic rape strain Topas19-2 genome adds 4 μ L and obtains into reaction system；" copy number (external source/internal reference) " refers to inspection
The copy number of source reference gene in the copy number and PEP of the strain Topas19-2 obtained is surveyed, the unit of two copy numbers is all
" a/μ L "；The ratio of the copy number of source reference gene in copy number percentage, that is, strain Topas19-2 copy number ratio PEP；Reason
By copy number percentage, i.e., the ratio of the copy number of source reference gene in the theoretically copy number ratio PEP of strain Topas19-2,
The DNA sample that actually this example uses is therefore 100% transgene rape strain Topas19-2 genomic DNA detects
Strain Topas19-2 copy number should be with the copy number of source reference gene in PEP as, i.e., theoretical copy number percentage
Than being 100%；Average copy number percentage is the average value of the copy number percentage of parallel test three times.
Table 7 the results show that for 0.01ng/ μ L, 0.05ng/ μ L, 0.25ng/ μ L concentration DNA standard sample, this
The average value of the copy number percentage detected under the reaction system of example is respectively 95.65%, 114.97% and 104.50%,
DNA standard sample theory copy number percentage is 100%, and the relative error point of the DNA standard sample of three kinds of concentration is calculated
Not Wei 4.35%, 14.97% and 4.50%, three groups of deviations meet the requirements, i.e., less than 25%.Therefore the detection method of this example can
More accurately to carry out absolute quantitation detection to transgene rape strain Topas19-2.
The application establishes the specific quantification for the temporary unratified transgene rape strain Topas19-2 in China for the first time
Detection method.The double ddPCR quantitative detecting method stability that the application establishes is good, high specificity, quantification range is wide, quantitatively examines
It surveys that high sensitivity, accuracy are high, can satisfy at present and in the future to the accurate quantitative reality of transgene rape strain Topas19-2
It needs, the detection method of the application is highly suitable for passing in and out the determining of the Topas19-2 of strain containing transgene rape in rape product
Amount detection.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen
Specific implementation please is only limited to these instructions.For those of ordinary skill in the art to which this application belongs, it is not taking off
Under the premise of from the application design, a number of simple deductions or replacements can also be made.
SEQUENCE LISTING
<110>Food Inspection & Quarantine Technology Center of Shenzhen Entry-Exit Inspection
<120>transgene rape strain Topas19-2 detection method and reagent
<130> 19I28035
<160> 6
<170> PatentIn version 3.3
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<211> 20
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cgaccggcgc tgatatatga 20
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tcccgcgtca tcggcgg 17
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cccttgtgaa gctcgacatc 20
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cttgtcctct gaccattctt tgt 23
<210> 6
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<212> DNA
<213>artificial sequence
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ccgaccgtca caccgatgtt ttaga 25
Claims (7)
1. a kind of detection method of transgene rape strain Topas19-2, it is characterised in that: include the following steps,
PCR amplification system preparation steps, including by strain Topas19-2 special primer group, strain Topas19-2 specific probe,
The DNA of PEP gene specific primer group, PEP gene specific probe and sample to be tested is added in PCR reaction solution, and double PCR is made
Reaction system；
Droplet generation step is included in double PCR reaction system and droplet generation oil is added, after mixing, is transferred into droplet
It generates on instrument, concussion generates droplet；
PCR reaction and droplet analytical procedure, including droplet is transferred in reaction tube progress PCR reaction, PCR after reaction,
The amplified signal of each droplet is read using droplet analyzer；
Quantitative detection step, according to the amplified signal of reading, using the specific amplified copy number of software analysis strain Topas19-2
The percentage for accounting for interior source reference gene PEP gene specific amplification copy number, indicates transgene rape strain Topas19-2's with this
Content；
The forward primer of the strain Topas19-2 special primer group is sequence shown in Seq ID No.1, strain Topas19-2
The reverse primer of special primer group is sequence shown in Seq ID No.2, and the strain Topas19-2 specific probe is Seq ID
Sequence shown in No.3, the forward primer of the PEP gene specific primer group are sequence shown in Seq ID No.4, PEP gene specific
The reverse primer of primer sets is sequence shown in Seq ID No.5, and the PEP gene specific probe is sequence shown in Seq ID No.6
Column；
Seq ID No.1:5 '-GTTGCGGTTCTGTCAGTTCC-3 '
Seq ID No.2:5 '-CGACCGGCGCTGATATATGA-3 '
Seq ID No.3:5 '-TCCCGCGTCATCGGCGG-3 '
Seq ID No.4:5 '-CCCTTGTGAAGCTCGACATC-3 '
Seq ID No.5:5 '-CTTGTCCTCTGACCATTCTTTGT-3 '
Seq ID No.6:5 '-CCGACCGTCACACCGATGTTTTAGA-3 '
Wherein, in the strain Topas19-2 specific probe of sequence shown in Seq ID No.3,5 ' ends have fluorophor, 3 ' ends
End has quenching group；In the PEP gene specific probe of sequence shown in Seq ID No.6,5 ' ends have fluorophor, 3 ' ends
End has quenching group.
2. detection method according to claim 1, it is characterised in that: in the strain Topas19-2 specific probe, 5 ' ends
The fluorophor at end is FAM, and the quenching group of 3 ' ends is BHQ-1；In the PEP gene specific probe, the fluorescence of 5 ' ends
Group is HEX, and the quenching group of 3 ' ends is BHQ-1.
3. a kind of reagent of transgene rape strain Topas19-2 quantitative detection, it is characterised in that: the reagent draws including first
Object probe combinations and the second primer combination of probe, the first primer probe combinations are used for transgene rape strain Topas19-2
Specific detection, second primer combination of probe are detected for PEP gene specific；
In the first primer probe combinations, forward primer is sequence shown in Seq ID No.1, and reverse primer is Seq ID
Sequence shown in No.2, probe is sequence shown in Seq ID No.3, and 5 ' ends of probe have fluorophor, 3 ' ends tool
There is quenching group；
In second primer combination of probe, forward primer is sequence shown in Seq ID No.4, and reverse primer is Seq ID
Sequence shown in No.5, probe is sequence shown in Seq ID No.6, and 5 ' ends of probe have fluorophor, 3 ' ends tool
There is quenching group.
4. reagent according to claim 3, it is characterised in that: in the probe of sequence shown in Seq ID No.3,5 ' ends
Fluorophor is FAM, and the quenching group of 3 ' ends is BHQ-1；In the probe of sequence shown in Seq ID No.6,5 ' ends it is glimmering
Light group is HEX, and the quenching group of 3 ' ends is BHQ-1.
5. application of the reagent according to claim 3 or 4 in prepare transgenosis rape detection kit or detection device.
6. a kind of transgene rape detection kit, it is characterised in that: containing described in claim 3 or 4 in the kit
Reagent.
7. transgene rape detection kit according to claim 6, it is characterised in that: the kit is wanted using right
Detection method described in asking 1 or 2 carries out quantitative detection to transgene rape strain Topas19-2.
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