CN106636360A - Microdroplet type digital PCR (polymerase chain reaction) detection method and kit for transgenic rice Bt63 - Google Patents
Microdroplet type digital PCR (polymerase chain reaction) detection method and kit for transgenic rice Bt63 Download PDFInfo
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Abstract
The invention provides a microdroplet type digital PCR (polymerase chain reaction) detection method and a kit for transgenic rice Bt63. The detection method comprises the following steps of providing a specific primer and a probe of a target strain and endogenous gene; extracting DNA (deoxyribonucleic acid) of a sample; performing digital microdroplet PCR proliferation, and analyzing results. The detection method has the advantages that whether the transgenic rice Bt63 is contained in rice or rice products or not can be precisely and quantitatively detected; the site quick and quantitative detection of transgenic detection items at ports is realized, and the application value is higher.
Description
Technical field
The invention belongs to the detection technique of pest-resistant transgenic rice Bt63, more particularly to pest-resistant transgenic rice Bt63 shows
The whether detection method containing transgenic paddy rice Bt63 in quantitative measurement technology, specially rice or rice made products.
Background technology
In recent years, the commercialization fast development of global genetically modified crops, cultivated area is increased by 1,700,000 hectares of 1996
To 1.8 hundred million hectares of 2014, more than 100 times is increased in 19 years.Paddy rice is one of most important cereal crops in the world, at me
Occupy very important status in state's grain-production, transgenic paddy rice Recent Progresses In The Development is also more rapid, there are multiple transgenosis
Paddy rice excellent strain enters the Environment release stage, applies for commercial growth.At the same time, transgenic technology is pacified to environment and food
Full potential risk has caused the extensive concern in the whole world, international trade dispute to happen occasionally.At the beginning of 2012, EU Committee issues
《Decision to taking urgent measure containing transgene component in Chinese exports rice food》, European Union will be to Chinese 25 kinds of metric systems
Product take mandatory detection of GMOs, and take the return of goods according to testing result and destroy measure, and the Chinese authority is necessary
The survey report for submitting to rice made products non-transgenic to prove before to European Union's outlet, indicates whether containing transgene component.2006
Since, 7 countries such as existing France, Germany, Italy and Austria are because genetically modified rice is polluted to China's export rice made products
Take and the measure such as deny entry or remove frame, recall.On the other hand, whether the focus of current public attention is from " being transgenosis "
It is transferred to " containing which transgene component and respective content " to come up, is to meet public right to information, managerial skills and detection skill
The demand of art ability, many countries have launched respectively transgenosis limitation regulation, and such as European Union specifies that the composition of genetically modified crops exceedes
0.9%, it is necessary to be identified；And the current standard of Japan is 5%；The current standard of China is most stringent, to transgenic product
Carry out " zero tolerance ", as long as detect transgene component in the product to be accomplished by being identified, it is contemplated that transgenosis low-level mixes
Etc. objective reality, at present this " zero tolerance " policy is excessively harsh for technical enforcement angle.Therefore, turn from each port of China
Actually setting out for genetic test level, is that national policy department proposes that rational transgenosis threshold value is extremely urgent, is stood in the breach
It is the accurate quantitative detecting method for developing transgene component, realizes the absolute quantitation to transgenic product.
The content of the invention
In view of the active demand of field of food safety detection technique safe to food and product at present, the present invention is based on droplet
Formula digital pcr technology, develops a accurate quantitative detecting method and kit that can be used for pest-resistant transgenic rice Bt63.
The detection method is in a kind of rice or rice made products the whether droplet type digital pcr containing transgenic paddy rice Bt63
Detection method, including：
(1) primed probe is designed：
The endogenous and strain specificity primed probe of design pest-resistant transgenic rice Bt63：
PLD-Forward：TGGTGAGCGTTTTGCAGTC
PLD-Reverse：CTGATCCACTAGCAGGAGGTC
PLD-Probe：FAM-TGTTGTGCTGCCAATGTGGCCT-TAMRA
Bt63-Forward：AGAGACTGGTGATTTCAGCGG
Bt63-Reverse：GCGTCCAGAAGGAAAAGGAAT
Bt63-Probe：FAM-ATCTGCCCCAGCACTCGTCC-BHQ1
(2) testing sample, positive control, the DNA of negative control are extracted：
(3) digital droplet type PCR amplifications：
By step 2) obtained by the DNA of testing sample, the DNA of positive control, the DNA of negative control be utilized respectively step 1)
The primed probe of gained carries out following operation：
1. step 1) obtained by primer probe process water with DEPC and carry out dissolved dilution, primer is diluted to concentration and is
900nM, probe dilution is to concentration 250nM；
2. amplification reaction system is set：
PCR reaction systems
So as to obtain testing sample reaction system, positive control reaction system, negative control reaction system respectively；
3. the testing sample reaction system, positive control reaction system, negative control reaction system carry out respectively following
Operation：
Add droplet to occur (to avoid producing bubble) in the sample well of plate reaction system, occur in oilhole in corresponding droplet
Add 70 μ L droplets that oil occurs, install adhesive tape, be placed in drop generator and form droplet.Afterwards, by 8 roads of the droplet for having occurred
Pipettor is transferred in 0.2mL PCR reaction tubes (slow inhale slow puts), with supporting aluminium foil sealing after, be placed in the enterprising performing PCR of PCR instrument anti-
Should, PCR response parameters are：95℃/10min；94 DEG C/30s, 57 DEG C/30s, 40 circulations；98℃/10min.Ramp rate 2
℃/s。
So as to obtain testing sample reactant liquor, positive control reactant liquor, negative control reactant liquor respectively；
(4) result detection and analysis：
Reactant liquor obtained by step (3) is put into the droplet analyzer of droplet type digital pcr instrument, and in software
Detection pattern is set on QuantaSoft as ABS, the fluorescence signal of FAM is detected.Instrument can automatically analyze fluorescence in each droplet
Signal, then, by QuantaSoft each sample related gene copy Particle density is calculated.
Primer of the present invention can entrust TAKARA company systems standby, HPLC purifying.
In the setting amplification reaction system of the step (3) of the present invention：2 × ddPCR TM Supermix for Probes can
Obtained by commercial mode.
The present invention also provides in a kind of rice or rice made products whether the droplet type digital pcr containing transgenic paddy rice Bt63 is detected
Kit, including：
The endogenous primer and probe of pest-resistant transgenic rice Bt63：
PLD-Forward：TGGTGAGCGTTTTGCAGTC
PLD-Reverse：CTGATCCACTAGCAGGAGGTC
PLD-Probe：FAM-TGTTGTGCTGCCAATGTGGCCT-TAMRA
The specific primer and probe of pest-resistant transgenic rice Bt63 strains：
Bt63-Forward：AGAGACTGGTGATTTCAGCGG
Bt63-Reverse：GCGTCCAGAAGGAAAAGGAAT
Bt63-Probe：FAM-ATCTGCCCCAGCACTCGTCC-BHQ1.
The kit also includes 2 × ddPCR TM Supermix for Probes.
In the present invention, extracting testing sample, positive control, the DNA of negative control can be entered using CTAB Centrifugation method DNAs
Row DNA extract, performance equivalent or more than this method DNA extraction kit (such as QIAGEN DNA extraction kits
Deng), when using commercial reagents box, then operating procedure is carried out with reference to kit specification.
The invention belongs to the field of detection of food safety of nucleic acid is based on, different from traditional nucleic acid amplification, detection sensitivity
Height, can realize accurate quantitative determination, realize the absolute quantitation to transgenic product.And the method also has very strong prediction
Property, in that context it may be convenient to other transformed varieties of detection required for increasing.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.
5 parts of samples used in example 1 below, are detected in advance according to traditional fluorescence quantifying PCR method, tool
Body testing result is as shown in table 1.
The fluorescence quantifying PCR method testing result of table 1
Remarks explanation：
1st, positive control (positive) is Bt63 transgenic paddy rice standard items.
2nd, negative control (negative) is non-transgenic rice.
The whether detection containing transgenic paddy rice Bt63 in 1 meter of embodiment or rice made products
Using the sample 1～5 in table 1 as testing sample, with Bt63 transgenic paddy rice standard items as positive control, with non-turn
Transgenic rice is negative control；Follow the steps below successively：
1) primer is designed：
The corresponding probe primer of design transgenic paddy rice Bt63, using real time fluorescent PCR method in design synthesis
Source gene and strain gene primer probe detected, as a result shows that all primed probes can effectively amplify target gene (table
1), specially：
PLD-Forward：TGGTGAGCGTTTTGCAGTC
PLD-Reverse：CTGATCCACTAGCAGGAGGTC
PLD-Probe：FAM-TGTTGTGCTGCCAATGTGGCCT-TAMRA
Bt63-Forward：AGAGACTGGTGATTTCAGCGG
Bt63-Reverse：GCGTCCAGAAGGAAAAGGAAT
Bt63-Probe：FAM-ATCTGCCCCAGCACTCGTCC-BHQ1
2) pre-treatment of rice to be measured or rice made products, follows the steps below successively：
Carry out DNA extractions using CTAB Centrifugation method DNAs, so as to extract obtain testing sample (i.e. sample 1～5) DNA,
The DNA of positive control, the DNA of negative control.
3) digital droplet type PCR amplifications：
By step 2) obtained by testing sample DNA (that is, the DNA of sample 1～5), the DNA of positive control, negative control
DNA be utilized respectively step 1) primer sets carry out following operation：
The dissolved dilution of primer：
Primer DEPC processes water and is diluted to concentration into 900nM；
Probe DEPC processes water and is diluted to concentration into 250nM.
Amplification reaction system is set, 2 are shown in Table：
Table 2PCR reaction systems
So as to obtain testing sample reaction system, positive control reaction system, negative control reaction system respectively；
3. the testing sample reaction system, positive control reaction system, negative control reaction system carry out respectively following
Operation：
Add droplet to occur (to avoid producing bubble) in the sample well of plate reaction system, occur in oilhole in corresponding droplet
Add 70 μ L droplets that oil occurs, install adhesive tape, be placed in drop generator and form droplet.Afterwards, by 8 roads of the droplet for having occurred
Pipettor is transferred in 0.2mL PCR reaction tubes (slow inhale slow puts), with supporting aluminium foil sealing after, be placed in the enterprising performing PCR of PCR instrument anti-
Should, PCR response parameters are：95℃/10min；94 DEG C/30s, 57 DEG C/30s, 40 circulations；98℃/10min.Ramp rate 2
℃/s。
So as to obtain testing sample reactant liquor, positive control reactant liquor, negative control reactant liquor respectively；
4) interpretation of result：
By step 3) obtained by reactant liquor be put into the droplet analyzer of droplet type digital pcr instrument, and in software
Detection pattern is set on QuantaSoft as ABS, the fluorescence signal of FAM is detected.Instrument can automatically analyze fluorescence in each droplet
Signal, then, by QuantaSoft each sample related gene copy Particle density is calculated.Concrete outcome is as shown in table 3.
The droplet type digital pcr testing result of table 3
| Testing result | |
| Positive control (positive) | Detection |
| Negative control (negative) | Do not detect |
| Genetically modified rice Bt63 (1) | Detection |
| Genetically modified rice section peak 6 (2) | Do not detect |
| Genetically modified rice Kemingdao (3) | Do not detect |
| Genetically modified rice LLRICE601 (4) | Do not detect |
| Ground rice (5) | Do not detect |
Embodiment 2
With transgenic paddy rice Bt63 samples that non-transgenic rice and content are 0.1%, 1%, 10% as template, 3 are carried out
Secondary repetition quantitative test, verifies the quantitative sensitivity of digital pcr detection method.
When non-transgenic rice quantitative result is 0%, 10% genetically modified rice Bt63 sample detections result is 9.94%,
1% genetically modified rice Bt63 sample detections result is 1.01%, and 0.1% genetically modified rice Bt63 sample detection results are
0.114%, show ddPCR methods that this research sets up to the quantitative determination sensitivity of genetically modified rice Bt63 up to 0.1%, inspection
Survey the results are shown in Table 4.
The different content Bt63 paddy rice sample amounts testing results of table 4
| Theoretical value (%) | Repeat 1 | Repeat 2 | Repeat 3 | Mean value (%) |
| 10 | 9.95 | 9.89 | 9.97 | 9.94 |
| 1 | 0.975 | 1.018 | 1.023 | 1.01 |
| 0.10 | 0.112 | 0.109 | 0.121 | 0.114 |
| 0 | 0 | 0 | 0 | / |
Claims (9)
1. the droplet type digital pcr detection method of transgenic paddy rice Bt63, comprises the following steps：
(1) primer and probe are obtained, the primer and probe include：
The endogenous primer and probe of pest-resistant transgenic rice Bt63：
PLD-Forward：TGGTGAGCGTTTTGCAGTC
PLD-Reverse：CTGATCCACTAGCAGGAGGTC
PLD-Probe：FAM-TGTTGTGCTGCCAATGTGGCCT-TAMRA
The specific primer and probe of pest-resistant transgenic rice Bt63 strains：
Bt63-Forward：AGAGACTGGTGATTTCAGCGG
Bt63-Reverse：GCGTCCAGAAGGAAAAGGAAT
Bt63-Probe：FAM-ATCTGCCCCAGCACTCGTCC-BHQ1；
(2) testing sample, positive control, the DNA of negative control are extracted；
(3) step (2) gained DNA is carried out into digital droplet type PCR amplifications, obtains reactant liquor；
(4) step (3) reactant liquor is detected and is analyzed.
2. detection method according to claim 1, is characterized in that, the digital droplet type pcr amplification reaction system includes：
The μ l of 2 × ddPCR TM Supermix for Probe 10, upstream primer and each 1.8 μ l of downstream primer, the μ l of probe 0.5, concentration
For the μ l of 10～50ng/ μ l DNA profilings 2, moisturizing to reaction system is 20 μ l；Obtain testing sample reaction system, positive control reaction
System, negative control reaction system.
3. detection method according to claim 1, is characterized in that, step (3) also include the primer obtained by step (1) and
Probe DEPC processes water and carries out dissolved dilution, and it is 900nM that primer is diluted to concentration, and probe dilution is to concentration 250nM.
4. detection method according to claim 2, is characterized in that, the testing sample reaction system, positive control reaction
System, negative control reaction system carry out respectively following operation：Droplet is added to occur in the sample well of plate, in phase reaction system
Answer droplet to occur to add 70 μ L droplets that oil occurs in oilhole, install adhesive tape, be placed in drop generator and form droplet；Afterwards, will
8 road pipettors of the droplet for having occurred are transferred in 0.2mL PCR reaction tubes, with supporting aluminium foil sealing after, be placed in PCR instrument enterprising
Performing PCR reacts, and PCR response parameters are：95℃/10min；94 DEG C/30s, 57 DEG C/30s, 40 circulations；98℃/10min.Ramp
2 DEG C/s of rate, so as to obtain testing sample reactant liquor, positive control reactant liquor, negative control reactant liquor respectively.
5. detection method according to claim 1, is characterized in that, the step (4) is included the reaction obtained by step (3)
Liquid is put into the droplet analyzer of droplet type digital pcr instrument, and detection pattern is set on software QuantaSoft as ABS, inspection
The fluorescence signal of FAM is surveyed, instrument can automatically analyze fluorescence signal in each droplet, then, by QuantaSoft each sample be calculated
Related gene copies Particle density.
6., according to the detection method described in claim 1, it is characterized in that, in the step (2) from CTAB Centrifugation method DNAs,
Or DNA extraction kit carries out the extraction of DNA.
7. according to the detection method described in claim 1, it is characterized in that, with rice or rice made products as testing sample, with transgenosis
Rice Bt63 is positive control, and the transgenic rice with non-transgenic rice or non-Bt63 is as negative control.
8. the droplet type digital pcr detection kit of transgenic paddy rice Bt63, is characterized in that, including：
The endogenous primer and probe of pest-resistant transgenic rice Bt63：
PLD-Forward：TGGTGAGCGTTTTGCAGTC
PLD-Reverse：CTGATCCACTAGCAGGAGGTC
PLD-Probe：FAM-TGTTGTGCTGCCAATGTGGCCT-TAMRA
The specific primer and probe of pest-resistant transgenic rice Bt63 strains：
Bt63-Forward：AGAGACTGGTGATTTCAGCGG
Bt63-Reverse：GCGTCCAGAAGGAAAAGGAAT
Bt63-Probe：FAM-ATCTGCCCCAGCACTCGTCC-BHQ1.
9. detection kit according to claim 8, is characterized in that, also including 2 × ddPCR TM Supermix for
Probes。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106868137A (en) * | 2017-03-01 | 2017-06-20 | 浙江省农业科学院 | Transgenic rice multiple digital pcr quantitative detecting method |
| CN107083327A (en) * | 2017-06-29 | 2017-08-22 | 浙江大学 | Determine kit, method and the application of the two of the gene copy numbers of DEFA1 3 |
| CN113430252A (en) * | 2021-06-17 | 2021-09-24 | 中国农业科学院油料作物研究所 | Method for identifying trace amount HSA transgenic rice pollen genotype in air |
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| CN107083327A (en) * | 2017-06-29 | 2017-08-22 | 浙江大学 | Determine kit, method and the application of the two of the gene copy numbers of DEFA1 3 |
| CN113430252A (en) * | 2021-06-17 | 2021-09-24 | 中国农业科学院油料作物研究所 | Method for identifying trace amount HSA transgenic rice pollen genotype in air |
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