CN105112530A - Double digital PCR fluorescent quantitative detection method for transgenic maize BT176 - Google Patents

Double digital PCR fluorescent quantitative detection method for transgenic maize BT176 Download PDF

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CN105112530A
CN105112530A CN201510585882.7A CN201510585882A CN105112530A CN 105112530 A CN105112530 A CN 105112530A CN 201510585882 A CN201510585882 A CN 201510585882A CN 105112530 A CN105112530 A CN 105112530A
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付伟
朱鹏宇
杜智欣
王晨光
魏霜
张亮亮
彭萱子
朱水芳
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Chinese Academy of Inspection and Quarantine CAIQ
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Abstract

The invention provides a double digital PCR fluorescent quantitative detection method for transgenic maize BT176. According to the method, quantitative detection can be directly performed on the transgenic maize BT176 in genomes of samples to be detected, and therefore the standard curve plotting step in quantitative detection of a conventional PCR method and the sample preprocessing step in existing digital PCR detection are omitted; in addition, transgenic ingredients are calculated according to the proportion of the exogenous gene copy number and the reference gene copy number, and therefore the stability of quantitative detection of the transgenic ingredients can be improved by performing double detection on one reaction system. Through the method, absolute quantitative detection on the transgenic maize BT176 can be achieved, the quantitative detection limitation can reach 0.5%, the sensitivity can reach 0.1%, and the requirements of actual detection of all the transgenic ingredients can be met. In addition, the method is easy to operate and high in flexibility and serves as an effective method for absolute quantitative detection of transgenes.

Description

The dual digital pcr fluorescence quantitative detecting method of transgenic corns BT176
Technical field
The present invention relates to technical field of molecular biological detection, specifically, relate to the dual digital pcr fluorescence quantitative detecting method of transgenic corns BT176.
Background technology
From the first transgenic Fructus Lycopersici esculenti in 1996 since U.S.'s successful commercialization, the research recent two decades for genetically modified crops achieves and develops rapidly.According to ISAAA statistic data, by 2014, the cultivated area of global genetically modified crops has reached 1.8 hundred million hectares, improves nearly 100 times, account for 3.4% of world crops total cultivated area than 1996 Annual planting areas.The fast development of genetically modified crops is not only modern agriculture and provides breeding mode more easily, because the potential safety hazard of its unknown causes every country and regional extensive concern yet.For this reason, the mark system of transgene component is set up in various countries and area one after another.In the transgene component mark system that world community man and area formulate, quantitative identifying system occupies larger specific gravity, wherein with European Union and Japan and Korea S's most representativeness.China mainly takes qualitative mark system in transgene component mark, because qualitative mark system exists different from quantitative identifying system in detection technique etc., makes China in crop import and export, easily be subject to the restriction of international standard.For this reason, China needs to greatly develop transgene component quantitative measurement technology and sets up relevant quantitative identifying system.
Digital pcr (Digital-PCR) is a kind of novel absolute quantitation PCR detection method, it is the new technology of a detection and quantitative nucleic acid, its ultimate principle is by micro-example is carried out large multiple dilutions and separatory, until testing molecule number contained in each sample is no more than 1, again all samples is carried out pcr amplification under the same conditions, pcr amplification fluorescent signal is had to be designated as 1, unstressed configuration signal is designated as 0, there is in the reaction member of fluorescent signal the target molecule at least comprising a copy, Poisson's distribution (Poissondistribution) formula is adopted for reaction result, just original copy number or the concentration of sample can be calculated.This technology does not rely on the cycle threshold of amplification curve, without the need to house-keeping gene and typical curve, can realize DNA absolute quantitation yet.This technology is at copy number changes analysis (copynumbervariation, CNV) (Qinetal., 2008), genetic mutation detects (Yungetal., 2009), gene type (Loetal., 2007), gene quantification (Warrenetal., 2010), unicellular genetic expression (Guoetal., 2010) etc. the research of aspect achieves breakthrough development, is that the disease detection such as cancer, tumour provides new diagnostic method at clinicing aspect.In detection GMOs, Corbisier etc. (2010) utilize digital pcr to analyze the ratio of the copy number of foreign gene and reference gene in corn seed DNA, this result take plasmid DNA as coming to the same thing of detecting of reference material with utilizing common fluorescent quantitative PCR technique, demonstrates the reliability of digital pcr.Burns etc. (2010) have evaluated digital pcr detectability (LOD) and quantitative limit (LOQ) in detection GMOs, explore the feasibility of digital pcr in detection GMOs and reaction conditions, show that digital pcr can carry out absolute quantitation to initial template copy number.But utilize the research of digital pcr to detection GMOs to be also in the starting stage.Existing digital pcr detection method all needs through sample pretreatment process, and in reality detects, pretreatment process often causes experimental result to produce deviation.Therefore existing digital pcr detection method is not suitable for directly as the detection method of transgene component.
Summary of the invention
The object of this invention is to provide a kind of sample pre-treatments step that do not need to carry out, for the dual digital pcr fluorescence quantitative detecting method of transgenic corns BT176, the absolute quantitation to transgenic maize BT 176 can be realized.
In order to realize the object of the invention, first the present invention is provided for primer and the probe combinations of the dual digital pcr fluorescent quantitation detection of transgenic corns BT176, and described primer and probe combinations are:
Foreign gene forward primer: 5'-GGCCGTGAACGAGCTGTT-3'
Foreign gene reverse primer: 5'-GGGAAGAAGCCTACATGTTTTCTAA-3'
Foreign gene probe: 5'-FAM-AGCAACCAGATCGGCCGACACC-BHQ1-3'
Reference gene adh1-135 forward primer: 5'-CGTCGTTTCCCATCTCTTCCTCC-3'
Reference gene adh1-135 reverse primer: 5'-CCACTCCGAGACCCTCAGTC-3'
Reference gene adh1-135 probe: 5'-VIC-AATCAGGGCTCATTTTCTCGCTCCTCA-BHQ1-3'.
The present invention also provides the transgenic corns BT176 digital pcr double fluorescent quantitative detection kit containing described primer and probe combinations.
Preferably, described test kit also comprises dNTPs, Taq DNA polymerase, Mg 2+, PCR reaction buffer, at least one in standard positive template etc.
The present invention further provides the dual digital pcr fluorescence quantitative detecting method of transgenic corns BT176, said method comprising the steps of:
1) genomic dna of testing sample is extracted;
2) with the genomic dna extracted for template, the primer described in utilization and probe combinations, carry out digital pcr amplified reaction;
3) pcr amplification product is detected.
Wherein, the reaction system of digital pcr amplified reaction is counted with 4 μ l: genomic dna 1 μ l, the each 0.04 μ l of the forward and reverse primer of 225nM foreign gene, the each 0.04 μ l of the forward and reverse primer of 225nM reference gene, 50nM foreign gene probe 0.02 μ l, 50nM reference gene probe 0.02 μ l, 2 × MasterMix (purchased from Fluidigm company) 2 μ l, 20 × LoadingReagent (purchased from Fluidigm company) 0.4 μ l, ddH 2o complements to 4 μ l.
The program of digital pcr amplified reaction is: 50 DEG C of hot activation 300s; 95 DEG C of denaturation 300s; 95 DEG C of sex change 15s, 60 DEG C of annealing and extension 60s, totally 50 circulations; Fluorescent signal is gathered at 60 DEG C.
Aforesaid method, step 3) in detect pcr amplification product specific as follows: after digital pcr amplified reaction terminates, record the fluorescent signal value of foreign gene and reference gene in each reacting hole, by the copy number of foreign gene and reference gene in each reacting hole of Poisson's distribution formulae discovery, obtained the absolute concentration of transgene component by the ratio of both copy numbers.
The present invention carries out digital pcr amplified reaction in pcr chip.By each well integrally, adding genetically modified crops digital pcr amplification system and carry out real-time fluorescence amplification, by monitoring the fluorescent signal value in each reacting hole, obtaining the overall amplification curve in all reacting holes and hotspot graph.
The judgement of detected result is carried out in such a way:
1, utilizing digital pcr to carry out in the process increased, the decision method in positive hole is: (in negative reaction hole, template is 1 μ lddH to occur obviously being different from negative reaction hole 2o) amplified signal, is designated as positive amplification hole by this reacting hole; The decision method of positive is: in three parallel group of same sample, at least occurs that has a positive amplification hole, then show to detect in sample gene group containing genetically modified crops composition;
2, after digital pcr amplification terminates, record the bright spot number of foreign gene and reference gene in each amplification wells, by the copy number of foreign gene and reference gene in each hole of Poisson's distribution formulae discovery, obtained the absolute concentration of transgene component by the ratio of both copy numbers;
3, each sample arranges three parallel group, and obtained the relative standard deviation (RSD) of transgene component concentration in three parallel group by statistical study, RSD≤25% shows that quantitative result is meaningful;
4, when being negative findings in testing sample reacting hole, show genetically modified crops composition not detected; When being positive findings in testing sample reaction tubes, show in testing sample containing genetically modified crops composition.
The inventive method directly can carry out detection by quantitative to the transgenic corns BT176 in the genome of detected sample, thus the sample pre-treatments step eliminated in existing digital pcr method, make testing process simple and easy to do, avoid the detrimentally affect of pre-treatment step to detected result accuracy.In addition, in the methods of the invention, because transgene component is calculated by the ratio of copy number of foreign gene and reference gene copy number, therefore carry out in same reaction system the stability that double check can improve transgene component detection by quantitative.
Utilize dual digital pcr fluorescence quantitative detecting method provided by the invention and detection kit can realize detecting the absolute quantitation of transgenic corns BT176, present method detection by quantitative limit can reach 0.5%, sensitivity can reach 0.1%, can meet the needs of the actual detection of transgene component.In addition, present method is simple to operate, and handiness is strong, is the effective ways that a kind of transgenosis absolute quantitation detects.
Accompanying drawing explanation
Fig. 1 is the expanding effect figure that in the embodiment of the present invention 2, transgenic corns BT176 foreign gene primed probe combines from different reference gene primed probe; Wherein, A combines with reference gene adh1-70bp, and B combines with reference gene adh1-135bp, and C combines with reference gene hmga-79bp, and D combines with reference gene zssIIb-88bp.
Fig. 2 is transgenic corns BT176 event-specific detection result in the embodiment of the present invention 2; Wherein, X-coordinate is different Transgenic corn lines, and ordinate zou is utilize dual digital pcr method to increase the copy number of foreign gene obtained.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
Embodiment 1 is for the dual primer of digital pcr fluorescent quantitation detection of transgenic corns BT176 and the design of probe combinations
The primer and probe combinations (SeqIDNo.1-6) that detect for the dual digital pcr fluorescent quantitation of transgenic corns BT176 is as follows devised according to transgenic corns BT176 strain foreign gene insertion sequence and border sequence:
Foreign gene forward primer: 5'-GGCCGTGAACGAGCTGTT-3'
Foreign gene reverse primer: 5'-GGGAAGAAGCCTACATGTTTTCTAA-3'
Foreign gene probe: 5'-FAM-AGCAACCAGATCGGCCGACACC-BHQ1-3'
Reference gene adh1-135 forward primer: 5'-CGTCGTTTCCCATCTCTTCCTCC-3'
Reference gene adh1-135 reverse primer: 5'-CCACTCCGAGACCCTCAGTC-3'
Reference gene adh1-135 probe: 5'-VIC-AATCAGGGCTCATTTTCTCGCTCCTCA-BHQ1-3'.
The foundation of the dual digital pcr quantitative detecting method of embodiment 2 transgenic corns BT176
1.1 experiment material
The transgenic corns BT176 sample used in the present embodiment and parent's non-transgenic sample thereof, Transgenic corn lines NK603, MON810, MIR604, MIR162,3272, MON863 provides by China Inst. of Quarantine Inspection Sciences.Pcr chip purchased from American Fluidigm company, model is 48.770DigitalArrayChip; BioMarkHD high-throughput gene alaysis system (BiomarkHDSystem) is purchased from Fluidigm company.
1.2 experiments genetically modified crops seed sample extracting genome DNA
(1) by also thoroughly air-dry for the grinding of crop seed sample;
(2) Transgenic corn lines is carried out mixing of different ratios with parent's non-transgenic seed sample, and obtain the sample of the different transgenosis concentration of testing by the mixing of spending the night of mixing instrument;
(3) take 100mg sample with analytical balance, add the RNaseA of 400 μ LAP1 damping fluids and 4 μ L, after concuss mixing, water-bath 20min at 65 DEG C, period concussion mixing 2-3 time;
(4) above-mentioned system taken out from water-bath, add 130 μ LP3 damping fluids, mixing is placed in ice bath 5min on ice;
(5) by above-mentioned system from taking out on ice, be placed in the centrifugal 5min of whizzer 14000rpm;
(6) the supernatant liquor rifle head in above-mentioned system is taken out, be placed in QIAshredderMinispincolumn, the centrifugal 2min of 14000rpm;
(7) filtrate of previous step is transferred in new centrifuge tube, adds the AW1 damping fluid of 1.5 times of volumes, fully mix with rifle head;
(8) mixing solutions of previous step is transferred in DNeasyMinispincolumn, each transfer 700 μ L, the system had more shifts several times, DNeasyMinispincolumn is placed in the centrifugal 1min of whizzer 8000rpm, centrifugal complete after abandon filtrate;
(9) be transferred in new 2mL centrifuge tube by the DNeasyMinispincolumn of previous step, add 500 μ LAW2 damping fluids, the centrifugal 1min of 14000rpm, abandons filtrate;
(10) repeating step 8;
(11) DNeasyMinispincolumn obtained in the previous step is put into new centrifuge tube, the centrifugal 2min of 14000rpm, the AW2 damping fluid thoroughly on removing filter membrane, and thoroughly dry filter membrane;
(12) by 50 μ L aseptic deionized water points on filter membrane, fully dissolve genome DNA sample 10min, the centrifugal 1min of 8000rpm collects filtrate.By concentration and the purity of determined by ultraviolet spectrophotometry gained genomic dna, genomic dna is diluted to 50ng/ μ L stand-by.
1.3 digital pcr amplified reactions
(1) blue protection film is taken off from pcr chip, controllinefluid is injected the hole of the upper and lower both sides of chip, be placed in IFCcontrollerMX, perform Prime (167 ×) operation.
(2) each reaction system in digital pcr well is 4 μ L, comprise genomic dna 1 μ l, the each 0.04 μ l of the forward and reverse primer of 225nM foreign gene, the each 0.04 μ l of the forward and reverse primer of 225nM reference gene, 50nM foreign gene probe 0.02 μ l, 50nM reference gene probe 0.02 μ l, 2 × MasterMix (purchased from Fluidigm company) 2 μ l, 20 × LoadingReagent (purchased from Fluidigm company) 0.4 μ l, ddH 2o complements to 4 μ l.Template is replaced with aseptic deionized water in negative control hole.Above-mentioned primer and probe are from the primer of embodiment 1 and probe combinations.
(3) above-mentioned system is added in the well of chip, and in combined hole, add 10 μ L1 × GESampleLoadingReagent, in application of sample process, can not bubble be produced.
(4), after completion of the sample, chip is put back in Controller and perform Load (167 ×) operation.The dust on chip is removed after Load.
(5) chip is put into BiomarkHDSystem, adopt the mono-fluorescence channel of FAM-MGB, following response procedures is set: 50 DEG C of hot activation 300s; 95 DEG C of denaturation 300s; 95 DEG C of sex change 15s, 60 DEG C of annealing and extension 60s, totally 50 circulations; Fluorescent signal is gathered at 60 DEG C.
(6) there is the amplified signal being obviously different from negative reaction hole, this reacting hole is designated as positive amplification hole; The decision method of positive is: in three parallel group of same group, at least occurs that a group is for positive amplification hole, then show to detect in genome containing genetically modified crops composition.Digital pcr amplification curve diagram and hotspot graph are as shown in Figure 1B.
1.4 data analysis
After digital pcr has increased, the fluorescent signal that all samples amplification produces is by BiomarkHDSystem system acquisition.Through system automatic analysis, the reacting hole of all Ct values between 25-45 is considered to positive amplification hole.
By the actual copy number of Poisson's distribution formulae discovery foreign gene and reference gene, obtained the absolute concentration of transgene component by the ratio of both copy numbers.
N (reference gene)=-ln [(N 0-X)/N 0] × N 0
N'(foreign gene)=-ln [(N 0-Y)/N 0] × N 0
In above-mentioned formula, N and N' is respectively the actual copy number of reference gene and foreign gene, N 0for total reaction hole count, X and Y is respectively the positive amplification hole count of reference gene and foreign gene.
Absolute concentration=N'/N × 100% of transgene component
1.5 digital pcr amplifications
Likely produce the suppression between primer due to double PCR and cause amplification efficiency unstable, therefore first the present embodiment has carried out the combination expanding effect of BT176 transformation event foreign gene primer probe (SeqIDNo.1-3) and different reference gene primed probe.Different primed probe combinations is in table 1.
As shown in Figure 1, as seen from Figure 1, the foreign transforming event primer probe of BT176 and reference gene adh1-135 (i.e. adh1-135bp) primed probe combined effect are best for amplification.
The transgenic sample that the present embodiment mixes to obtain different relative content by carrying out different ratio with non-transgenic corn and BT176 strain sample.By the detection of this experimental system, with theoretical transgene component mass content for X-coordinate, actual detection transgene copy number content (i.e. the absolute concentration of transgene component) is ordinate zou Criterion curve.The parameter of this typical curve is as shown in table 2.Can be found out by linear regression coeffficient, there is good linear relationship in this detection system, this linearity range can meet the needs of all detection of GMOs between the transgene component concentration of 0.5%-100%.
1.6 dual digital pcrs have better stability compared to substance digital pcr
In order to verify that dual digital pcr detects the stability detected relative to substance digital pcr, the substance digital pcr that the present embodiment has carried out BT176 strain respectively detects and the detection of dual digital pcr.Wherein, substance digital pcr only uses the forward and reverse primer of foreign gene and probe in detecting) (SeqIDNo.1-3).
Detected result is as shown in table 3.As can be seen from Table 3, for substance digital pcr detects, dual digital pcr detection system has better organizes internal stability, can better for the Quantitative measurement of transgene component.
The determination of detection by quantitative limit in 1.7 present method
According to the respective specified of European Union, the detection by quantitative of quantivative approach limit is defined as the lowermost turn gene element concentration of relative deviation (RSD) within 25% in group.Detected result is as shown in table 4, and when transgene component mass content is 0.5% time, RSD value in group can control within 25% by this detection system, and therefore the detection by quantitative of present method is limited to gm content 0.5%.
1.8 dual digital pcr reaction system specificity verification
In the present embodiment, specificity is defined as and can not detects Positive fluorescence signal in other transgenic strain.Choose common Transgenic corn lines NK603, MON810, MIR604, MIR162,3272, MON863 is experiment material.Adopt dual digital pcr detection method, verify the specificity of this detection system.Shown in detected result Fig. 2.
1.9 dual digital pcr reaction system sensitivity are determined
In the present embodiment, the detectability of detection system is defined as and stable detection can goes out the transgenosis content group of transgene component.The detected result of this detection system is in table 4.For transgenic maize BT 176, mass content be 0.1% experimental group can stablize and produce positive amplification signal, therefore the detection of this detection system is limited to 0.1%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
The determination of table 4BT176 strain detection by quantitative limit
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Claims (8)

1., for primer and the probe combinations of the dual digital pcr fluorescent quantitation detection of transgenic corns BT176, it is characterized in that, described primer and probe combinations are:
Foreign gene forward primer: 5'-GGCCGTGAACGAGCTGTT-3'
Foreign gene reverse primer: 5'-GGGAAGAAGCCTACATGTTTTCTAA-3'
Foreign gene probe: 5'-FAM-AGCAACCAGATCGGCCGACACC-BHQ1-3'
Reference gene adh1-135 forward primer: 5'-CGTCGTTTCCCATCTCTTCCTCC-3'
Reference gene adh1-135 reverse primer: 5'-CCACTCCGAGACCCTCAGTC-3'
Reference gene adh1-135 probe: 5'-VIC-AATCAGGGCTCATTTTCTCGCTCCTCA-BHQ1-3'.
2. contain the dual digital pcr fluorescence quantitative detection kit of transgenic corns BT176 of primer and probe combinations described in claim 1.
3. test kit according to claim 2, is characterized in that, described test kit also comprises dNTPs, Taq DNA polymerase, Mg 2+, PCR reaction buffer, at least one in standard positive template.
4. the dual digital pcr fluorescence quantitative detecting method of transgenic corns BT176, is characterized in that, said method comprising the steps of:
1) genomic dna of testing sample is extracted;
2) with the genomic dna extracted for template, utilize the primer described in claim 1 and probe combinations, carry out digital pcr amplified reaction;
3) pcr amplification product is detected.
5. method according to claim 4, it is characterized in that, step 2) in the reaction system of digital pcr amplified reaction count with 4 μ l: genomic dna 1 μ l, the each 0.04 μ l of the forward and reverse primer of 225nM foreign gene, each 0.04 μ l of the forward and reverse primer of 225nM reference gene, 50nM foreign gene probe 0.02 μ l, 50nM reference gene probe 0.02 μ l, 2 × MasterMix2 μ l, 20 × LoadingReagent0.4 μ l, ddH 2o complements to 4 μ l.
6. method according to claim 4, is characterized in that, step 2) in carry out digital pcr amplified reaction program be: 50 DEG C of hot activation 300s; 95 DEG C of denaturation 300s; 95 DEG C of sex change 15s, 60 DEG C of annealing and extension 60s, totally 50 circulations; Fluorescent signal is gathered at 60 DEG C.
7. method according to claim 4, is characterized in that, in pcr chip, carry out digital pcr amplified reaction.
8. the method according to any one of claim 4-7, it is characterized in that, step 3) in detect pcr amplification product specific as follows: after digital pcr amplified reaction terminates, record the fluorescent signal value of foreign gene and reference gene in each reacting hole, by the copy number of foreign gene and reference gene in each reacting hole of Poisson's distribution formulae discovery, obtained the absolute concentration of transgene component by the ratio of both copy numbers.
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CN105695595A (en) * 2016-03-22 2016-06-22 曹际娟 Transgenic maize BT176 nucleic acid standard sample and preparation method thereof
CN106755519A (en) * 2017-02-16 2017-05-31 浙江省农业科学院 Based on homozygous and heterozygous transgenic corn dual anti-12 5 the method for digital pcr identification and application
CN108410956A (en) * 2018-03-13 2018-08-17 中国农业大学 A kind of universal primer list fluorescent molecular detection technique and kit

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