CN109825630A - A kind of transgene rape strain T45 detection reagent, kit and detection method - Google Patents
A kind of transgene rape strain T45 detection reagent, kit and detection method Download PDFInfo
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Abstract
This application discloses reagent, kit and the detection methods of a kind of transgene rape strain T45 detection.The detection method of the application, including the specific primer and probe of napus lines T45 foreign gene and PEP gene are added in PCR reaction system simultaneously, and mineral oil is added, it is mixed evenly to prepare droplet, dual ddPCR reaction is carried out in droplet, after reaction, the amplified signal of each droplet is read, the copy number of strain T45 foreign gene and PEP is calculated；Wherein, the probe of strain foreign gene and PEP gene uses FAM and HEX fluorescent marker respectively.The detection method of the application, design matching used endogenous gene PEP and strain foreign gene specific primer probe, using two-channel method, the copy number of the two is quantified respectively, transgene rape strain T45 absolute content is calculated, is of great significance 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, more particularly to a kind of examination of transgene rape strain T45 detection
Agent, kit and detection method.
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, as the related GMO in various countries (Genetically Modified organism) labeling acts is built
It stands and constantly improve, have defined to the GMO content lower limit in food.Many countries not only require to carry out GM food
Qualitative detection, it is also necessary to quantitative detection be carried out to the GMO content in food, to identify.Its threshold value identified generally exists
Between 0.9% -5% (European Commission, 2003；Notification,2000).Establish the inspection of transgenic product
The especially accurate quantitative measurement technology of survey technology standard is the technology premise for implementing transgenic product mark.Therefore, transgenosis at
The accurate quantitative technique divided will identify the perfect of system with the whole world and develop 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 T45
Quantitative detection, there has been no relevant research or reports.Therefore, it needs to study a kind of effective transgene rape strain T45 inspection
Survey method.
Summary of the invention
The purpose of the application is to provide reagent, kit and the detection method of a kind of transgene rape strain T45 detection.
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 T45 a kind of, comprising the following steps:
PCR amplification system preparation steps, including strain T45 special primer group, strain T45 specific probe, PEP gene is special
The DNA of different primer sets, PEP gene specific probe and sample to be tested is added in PCR reaction solution, and double PCR reaction system is made；
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 accounted for according to the amplified signal of reading using the specific amplified copy number of software analysis strain T45
The percentage of interior source reference gene PEP gene specific amplification copy number, the content of transgene rape strain T45 is indicated with this；
The forward primer of strain T45 special primer group is sequence shown in Seq ID No.1, strain T45 special primer group
Reverse primer is sequence shown in Seq ID No.2, and strain T45 specific probe is sequence shown in Seq ID No.3, and PEP gene is special
The forward primer of different primer sets is sequence shown in Seq ID No.4, and the reverse primer of PEP gene specific primer group is Seq ID
Sequence shown in No.5, PEP gene specific probe are sequence shown in Seq ID No.6；
Seq ID No.1:5 '-TGCATATGGAATACAGTTGTAAATGAATT-3 '
Seq ID No.2:5 '-TCGTAAGAGACTCTGTATGAACTGTT-3 '
Seq ID No.3:
5’-CCAGTCTTTACGGCGAGTTCTGTTAGGTCCTC-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 T45 specific probe of sequence shown in Seq ID No.3,5 ' ends have fluorophor, 3 ' ends
With quenching group；In the PEP gene specific probe of sequence shown in Seq ID No.6,5 ' ends have fluorophor, 3 ' ends
With quenching group.
Preferably, in strain T45 specific probe, the fluorophor of 5 ' ends is FAM, and the quenching group of 3 ' ends is 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 T45 of the application, actually droplet type are digital
PCR.Its principle is, it is small by drop generator to be prepared into nearly a Water-In-Oils up to ten thousand for grease reaction system in a PCR reaction tube
Droplet, when DNA profiling molecular number to be measured is lower than certain number, droplet contains only the DNA profiling and sufficient amount of molecule
For all other component needed for Fluorescence PCR such as dNTP, Taq archaeal dna polymerase and probe and primer etc..To all
Droplet PCR after reaction, checks each droplet one by one, as long as droplet has fluorescence signal detection, has been regarded as PCR reaction hair
Raw, note has the template to be measured an of molecule to be detected, and counts all positive droplet numbers, so that it may obtain DNA profiling to be measured in sample
Molecular number or copy number.The application uses two-channel method, i.e., respectively in napus lines T45 specific probe and rape
Source gene-specific probe measures endogenous gene and product using different fluorescent markers simultaneously in the same PCR reaction system
It is the copy number of distinguished sequence.Wherein, copy number of the endogenous gene PEP of the application detection in rapeseed gene group is determining
, and copy number of the napus lines T45 specific sequence in rapeseed gene group is also determining；Therefore, by calculating sample
The content of transgene rape strain T45 can be calculated in the ratio of strain specificity sequence and PEP sequence copy numbers in product.
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 T45, according to transgene rape strain T45 copy number and PEP base
Because of the ratio of copy number, the content of transgene rape strain T45 is calculated.
The another side of the application discloses a kind of reagent of transgene rape strain T45 quantitative detection, which includes the
One primer combination of probe and the second primer combination of probe, the first primer probe combinations are used for strain T45 specific detection, and second draws
Object probe combinations are detected for PEP gene specific；In the first primer probe combinations, forward primer is shown in Seq ID No.1
Sequence, 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
With fluorophor, 3 ' ends have quenching group；In second primer combination of probe, forward primer is shown in Seq ID No.4
Sequence, 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
With fluorophor, 3 ' ends have quenching group.
It should be noted that the reagent of the application, actually in the quantitative detecting method of transgene rape strain T45
Used strain T45 specific detection primer probe and PEP gene specific detection primer and probe.Therefore, Seq ID
In the probe of sequence shown in No.3, the fluorophor of 5 ' ends is FAM, and the quenching group of 3 ' ends is BHQ-1；Seq ID
In the probe of sequence shown in 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 corresponding strain T45 be prepared separately into
Detection reagent uses.
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 quantifies transgene rape strain T45 using the detection method of the application
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 T45 of the application devises matching used endogenous gene PEP specificity
Primed probe and strain T45 specific primer probe, the two match, and using two-channel method, quantify the two respectively in oil
Copy number in dish genome finally calculates the content of transgene rape strain T45.The detection method of the application, is different from
The relative quantification of conventional real-time fluorescence PCR can carry out measuring samples transgenic napus lines T45 ingredient precisely quantitative
Detection, quantitative detection lower bound are copied up to 7, and qualitative detection lower bound is copied up to 2, to the research of rape transgene component and
The pass in and out accurate quantitative detection of rape product transgenic ingredient of China is of great significance.
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 T45 is at present still without corresponding qualitative or quantitative detection method.The application takes the lead in research simultaneously
The detection method of transgene rape strain T45 a kind of is proposed, the detection method of the application not only can be with qualitative detection strain
T45, 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 T45.Also, in the quantitative detecting method of the application, while devising endogenous
It with reference to two sets of matching used primed probes of gene and strain gene, is expanded by dual digital pcr, with two fluorescence channels point
Not Tong Jifenxi in source reference gene PEP and strain T45 specific sequence copy number, copied according to transgene rape strain T45
The ratio of shellfish number and PEP gene copy number calculates the absolute content of transgene rape strain T45.
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 7 parts of experimental material, respectively transgene rape standard substance Ms1, Ms8, Rf1,
Rf2, Rf3, MON88302 and T45, the above material is both from American Oil chemist association (AOCS).
Reagent and consumptive material: ddPCR Super Mix, ddPCR Droplet Generation Oil, ddPCR Droplet
Reader Oil, Droplet Generator DG8 Cartridge, Droplet Generator DG8 Gasket are purchased from
Bio-Rad company, the U.S.；DNeasyPlantMinikit 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 T45 special primer group and strain T45 specific probe of this example are held according to foreign gene insertion point 5 ' across side
Boundary's sequence design, PEP gene specific primer group and PEP gene specific tip reference SN/T 1204-2016.Primer and probe by
Sangon Biotech's synthesis and modification.See Table 1 for details for the primer and probe sequence of this example.
1 endogenous gene of table and strain T45 specific primer and probe
Wherein, 5 ' end of T45-P probe carries out the modification of FAM fluorophor, and 3 ' ends carry out the modification of BHQ-1 quenching group；
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: the napus lines of 2 × ddPCR Super Mix, 10 μ L, 3.6 μm of ol/L
1 μ L of specific probe T45-P, 1 μ L of napus lines forward primer T45-F, the napus lines of 6 μm of ol/L of 6 μm of ol/L are reversed
1 μ L of primer T45-R, 3.6 μm of ol/L interior 1 μ L of source reference gene probe PEP-P, 6 μm of ol/L in source reference gene forward direction draw
The 4 μ L of DNA profiling of 1 μ L of source reference gene reverse primer PEP-R, 12.5ng/ μ L in 1 μ L of object PEP-F, 6 μm of ol/L.
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 DG8 Cartridge), covers rubber mat (Droplet
Generation DG8 Gasket) after be put into droplet generate instrument in, by instrument independently shake generation droplet.After shaking
The droplet of the about 40 μ L by generation 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 carries 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 QuantaSoft V1.3.2
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 T45, according to the copy number of the available interior source reference gene PEP of HEX fluorescence channel.Root
The absolute of transgene rape strain T45 can be calculated according to strain T45 copy number and the ratio of interior source reference gene PEP copy number
Content.
4. stability experiment
Working concentration using 2.5ng/ μ L transgene rape strain T45 genomic DNA as template DNA, transgenic
Component content is 100%.Reaction system is prepared according to " 3. dual digital pcr reaction systems and condition " and carries out PCR reaction.Surely
Qualitative experiment is arranged 3 in parallel, it is desirable that relative standard deviation (the Relative Standard of 3 repetition experimental results
Deviation, RSD) it is no more than 25%.
5. specificity experiments
Selecting transgene rape strain Ms1, Ms8, Rf1, Rf2, Rf3, MON88302 and T45 is experimental material.With this 7 kinds
Transgenic sample genomic DNA prepares ddPCR reaction system according to " 3. dual digital pcr reaction systems and condition " for template,
And PCR reaction is carried out, wherein T45 is as positive control.
6. quantitative linearity scope measures
This example by transgene rape strain T45 genomic DNA respectively with pure water be diluted to 12.5ng/ μ L, 2.5ng/ μ L,
0.5ng/ μ L, 0.1ng/ μ L, 0.02ng/ μ L, 0.004ng/ μ L, 0.0008ng/ μ L totally 7 working concentrations.It is above-mentioned each dilute with 4 μ L
DNA is released as template " 3. dual digital pcr reaction systems and condition " according to the present example and carries out ddPCR reaction, each concentration weight
Multiple 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 Of Quantitation, LOQ) is defined as detection knot
Minimum amount of samples or samples copy number of the fruit RSD value no more than 25%, as take the lower limit of " measurement of 6. quantitative linearity scopes "
DNA sample carry out ten parallel reactions, RSD value≤25% of testing result.
Detection lower bound (Limit Of Detection, LOD) is defined as the minimum copy number of sample that can be detected or sample
Dosage, 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 T45 base
Because of a group progress digital pcr experiment, experimental setup 3 parallel.The hotspot graph that digital pcr obtains is as depicted in figs. 1 and 2, experiment knot
The data analysis of fruit is as shown in table 2.Wherein, Fig. 1 is the output result figure in the channel FAM, the i.e. testing result of strain T45；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, C07, E07, F07 are respectively
Three parallel tests.Fig. 1 and Fig. 2's the results show that having apparent fluorescence between digital pcr negative point obtained and positobe focus
Signal difference is away from trailing phenomenon is not serious, can be by the way that unified threshold value limit progress negative reaction point and positive reaction point is arranged
It distinguishes.
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
88.1,85.7 and 86.9, average detected copy number is 86.9, RSD 1.38%, in acceptable range, that is, be less than etc.
In 25%；It is respectively 87.1,86 and 90 that foreign gene expands duplicate actually detected copy number three times, and average detected copy number is
87.7, RSD 2.36%, in acceptable range, that is, it is less than or equal to 25%.As it can be seen that used in the detection method of this example
The primed probe amplification of reference gene and foreign gene have good stability, and the positive sample stablize it is available.
2 strain T45 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 T45；Fig. 4 is the defeated of the channel HEX
Result figure out, i.e., the testing result of interior source reference gene PEP；In Fig. 3 and Fig. 4, A09, B09, C09, D09, E09, F09 are sequentially
The testing result of Ms1, Ms8, Rf1, Rf2, Rf3, MON88302, G09 are the testing result of T45.
Fig. 3 and Fig. 4's the result shows that, the strain T45 specific primer and probe of this example are to other several transgene rapes
Strain does not expand, and PEP gene has amplification in other transgene rape strains and strain T45.Illustrate the T45 of this example
Specific primer and probe have good specificity, can be used in the specific detection of transgene rape strain T45.
3. range of linearity measurement result
This example carries out transgene rape strain T45 genomic DNA linearity test using 7 working concentrations, as a result such as 3 institute of table
Show, the data fit standard curve in the range of linearity is as shown in Figure 5 and Figure 6.Wherein, Fig. 5 is the fit standard of HEX channel data
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, i.e. strain
The fit standard curve graph of T45 gene.
The range of linearity of 3 rape T45 digital pcr method 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 T45 genome adds 4 μ L into reaction system；Copy number refers to the copy of unit volume in reaction system
Number, i.e., the every microlitre copy number containing strain T45 or PEP.Fig. 5, Fig. 6 and table 3 the results show that including the detection method of this example
Source reference gene copy number is located in the section 6.2-13930, that is, the amount for corresponding to genomic DNA is the section of 0.016ng to 50ng
It is interior, it can show good linear, across at least four orders of magnitude, meet the requirements, the r of fit standard curve2It is 0.99, symbol
The requirement more than or equal to 0.98 is closed, the RSD value of all concentration groups meets between 4.11% to 22.58% less than 25%
Requirement；This example detection method corresponds to the amount of genomic DNA in the section that copy number of foreign gene is located at 6.8-13880
In section for 0.016ng to 50ng, good linear, the r of fit standard curve is showed2It is 0.99, all concentration groups
RSD value meets the requirements between 5.19% to 23.18%.Therefore, copy of the detection method of this example in interior source reference gene
Numerical digit is located at 6.8-13880 in 6.2-13930, the copy number of foreign gene, has good quantitation capabilities.
4. the quantitative detection of standard limits and detection limit result
The DNA sample of the lower limit of the range of linearity in " 3. range of linearity measurement result " is taken to carry out the verifying of LOQ.By the DNA
Ten parallel digital pcr verifyings are carried out, the reference gene of each parallel unit volume and the copy number of foreign gene are calculated,
Calculate average value and RSD value.Specifically, this example takes the genome DNA sample of 0.004ng/ μ L to be tested, in reaction system
Genomic DNA is 0.016ng, and the results are shown in Table 4.Table 4 the results show that this group of DNA 10 parallel testing results in,
Foreign gene RSD is respectively 10.33% and 10.88%, meets the requirement less than 25%.This illustrates that the detection method of this example can be with
The sample for being 6.8 to copy number of foreign gene carries out stable quantitative detection.
It, will when this example takes the minimum DNA concentration group that can be expanded in " 3. range of linearity measurement result " to carry out the verifying of LOD
The DNA carries out 10 parallel digital pcr verifyings, and result is unable to satisfy the sum of all positive detection copy numbers and is not less than 9
It is required that stable detection cannot be carried out under respective copies number.
Therefore, this example further takes the half of LOQ value to carry out 10 parallel tests, specifically, taking 0.002ng/ μ L's
Genome DNA sample is tested, and the genomic DNA in reaction system is 0.008ng, and the results are shown in Table 5.The result of table 5
It shows, in ten parallel tests, the sum of copy numbers of all positive detections are 10, meet wanting not less than 9 for LOD Quality Control
It asks.This explanation, the sample that the detection method of this example can be 1.34 to copy number of foreign gene carry out stable detection.
The LOQ of 4 rape T45 digital pcr method of table is verified
The LOD of 5 rape T45 digital pcr method 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 0.04ng/ μ L to 0.004ng/ μ L
It is tested, the results are shown in Table 6.
The precision measurement result of 6 rape T45 digital pcr method 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 10.05% and 10.03% 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 0.004ng/ μ L, the genome DNA sample of 0.02ng/ μ L and 0.1ng/ μ L into
Row test, the results are shown in Table 7.
The accuracy determination result of 7 rape T45 digital pcr method of table
In table 7, amount of DNA indicates that the amount of DNA contained in reaction system, such as 0.08ng, actually 0.02ng/ μ L turn
Vector for transgenic rape strain T45 genome adds 4 μ L and obtains into reaction system；" copy number (external source/internal reference) " refers to that detection obtains
Strain T45 copy number and PEP in source reference gene copy number, the unit of two copy numbers is all " a/μ L "；Copy number
The ratio of the copy number of source reference gene in percentage, that is, strain T45 copy number ratio PEP；Theoretical copy number percentage, that is, manage
By the ratio of the copy number of source reference gene in the copy number ratio PEP of upper strain T45, the DNA sample that actually this example uses is
100% transgene rape strain T45 genomic DNA, therefore, the copy number of the strain T45 detected should be with the endogenous ginseng of PEP
Examine the copy number of gene be it is the same, i.e., theoretical copy number percentage is 100%；Average copy number percentage i.e. parallel examination three times
The average value for the copy number percentage tested.
Table 7 the results show that for 0.004ng/ μ L, 0.02ng/ μ L, 0.1ng/ μ L concentration DNA standard sample, this
The average value of the copy number percentage detected under the reaction system of example is respectively 105%, 101% and 103%, DNA standard sample
Product theory copy number percentage is 100%, and the relative error that the DNA standard sample of three kinds of concentration is calculated is respectively
5.00%, 1.00% and 3.00%, three groups of deviations meet the requirements, i.e., less than 25%.Therefore the detection method of this example can compare
It is accurate that absolute quantitation detection is carried out to transgene rape strain T45.
The application establishes the specific quantification detection side for the temporary unratified transgene rape strain T45 in China for the first time
Method.The double ddPCR quantitative detecting method stability that the application establishes is good, high specificity, quantification range is wide, quantitative detection is sensitive
Degree is high, accuracy is high, can satisfy at present and in the future to the accurate quantitative actual needs of transgene rape strain T45, the application
Detection method be highly suitable for passing in and out the quantitative detection of the T45 of strain containing transgene rape in rape product.
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>a kind of transgene rape strain T45 detection reagent, kit and detection method
<130> 19I28034
<160> 6
<170> PatentIn version 3.3
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<211> 29
<212> DNA
<213>artificial sequence
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tgcatatgga atacagttgt aaatgaatt 29
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tcgtaagaga ctctgtatga actgtt 26
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<213>artificial sequence
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ccagtcttta cggcgagttc tgttaggtcc tc 32
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cccttgtgaa gctcgacatc 20
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cttgtcctct gaccattctt tgt 23
<210> 6
<211> 25
<212> DNA
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ccgaccgtca caccgatgtt ttaga 25
Claims (7)
1. a kind of detection method of transgene rape strain T45, it is characterised in that: include the following steps,
PCR amplification system preparation steps, including strain T45 special primer group, strain T45 specific probe, PEP gene specific are drawn
The DNA of object group, PEP gene specific probe and sample to be tested is added in PCR reaction solution, and double PCR reaction system is made；
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 is accounted for endogenous according to the amplified signal of reading using the specific amplified copy number of software analysis strain T45
With reference to the percentage of gene PEP gene specific amplification copy number, the content of transgene rape strain T45 is indicated with this；
The forward primer of the strain T45 special primer group is sequence shown in Seq ID No.1, strain T45 special primer group
Reverse primer is sequence shown in Seq ID No.2, and the strain T45 specific probe is sequence shown in Seq ID No.3, described
The forward primer of PEP gene specific primer group is sequence shown in Seq ID No.4, the reverse primer of PEP gene specific primer group
For sequence shown in Seq ID No.5, the PEP gene specific probe is sequence shown in Seq ID No.6；
Seq ID No.1:5 '-TGCATATGGAATACAGTTGTAAATGAATT-3 '
Seq ID No.2:5 '-TCGTAAGAGACTCTGTATGAACTGTT-3 '
Seq ID No.3:
5’-CCAGTCTTTACGGCGAGTTCTGTTAGGTCCTC-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 T45 specific probe of sequence shown in Seq ID No.3,5 ' ends have fluorophor, and 3 ' ends have
Quenching group；In the PEP gene specific probe of sequence shown in Seq ID No.6,5 ' ends have fluorophor, and 3 ' ends have
Quenching group.
2. detection method according to claim 1, it is characterised in that: in the strain T45 specific probe, 5 ' ends it is glimmering
Light group is FAM, and the quenching group of 3 ' ends is BHQ-1；In the PEP gene specific probe, the fluorophor of 5 ' ends is
HEX, the quenching group of 3 ' ends are BHQ-1.
3. a kind of reagent of transgene rape strain T45 quantitative detection, it is characterised in that: the reagent includes the first primer probe
Combination and the second primer combination of probe, the first primer probe combinations are used for transgene rape strain T45 specific detection, institute
The second primer combination of probe is stated to detect 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 T45.
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