CN102424863A - Enhanced fluorescence quantitative PCR (polymerase chain reaction) method - Google Patents
Enhanced fluorescence quantitative PCR (polymerase chain reaction) method Download PDFInfo
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Abstract
The invention relates to the field of biotechnologies, and discloses an enhanced fluorescence quantitative PCR (polymerase chain reaction) method for detecting transgenic plants and products thereof. The method disclosed by the invention is high in sensitivity, strong in specificity, simple in operation and wide in sample range, and is especially suitable to be applied to the quantitative detection and qualitative detection of transgenic plants.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of enhancement type fluorescence quantifying PCR method that detects transgenic plant and products thereof.
Background technology
Along with the continuous progress of life science and technology, the mankind have changed the method for plant conventional breeding, through engineered means, foreign gene are changed in the plant materials, thereby cultivate a collection of high yield, transgenic plant kind that resistance property is strong.Played 2010 from the nineties, the cultivated area accumulative total of whole world transgenic plant reaches 1,000,000,000 hectares.Genetically modified crops have become scale.However, countries in the world are still very careful to the attitude of changeing basic plant, and put into effect a lot of policies and regulations, require transgene component is detected, and require the positive person of detected result to need sign.
At present, detection method commonly used has real-time fluorescence PCR, detection methods such as ordinary gel PCR.Real-time fluorescence PCR (RT-PCR): be meant in the PCR reaction system to add fluorophor, utilize the fluorescent signal accumulation whole PCR process of monitoring in real time, the method for through detected fluorescent signal unknown template being analyzed at last.Yet all there is the deficiency of self in these detection methods, as there are the judgement blind area in some deep processed product and the lower product of transgenic content, promptly can't carry out disposable judgement to detected result.
This just need exploitation efficiently, accurately, sensitive transgenic detection method more.This foreign trade, transgenosis safe and all be a most important problem that need to solve for customs, commodity inspection, inspection and quarantining for import/export department, food-processing department for country.
Summary of the invention
The present invention is directed to the lower sample of transgenic plant dna molecular content that prior art can't detect or cause because of the deep processing transgenic product dna molecular havoc sample and use the sample that fluorescent quantitative PCR detection method can't be judged negative male gray area; A kind of enhancement type fluorescence quantifying PCR method is provided; Its sensitivity, accuracy are higher, can be widely used in the detection of transgenic plant and converted products thereof.
A kind of enhancement type fluorescence quantifying PCR method that detects transgenic corns strain BT11 provided by the invention; The reaction system that will comprise the TV 25 μ l of primer sets 0.5 μ M, probe 0.5 μ M, template 1～100ng is carried out the real-time fluorescence quantitative PCR reaction, and reaction conditions is:
50℃，2min；
95℃10min；
95 ℃ of 15s, 45 ℃ of 30s, 72 ℃ of 30s, 10 circulations;
95℃15s，60℃?1min。
As preferably, the testing goal gene is the border sequence of BT11 structure specific gene cry1Ab and adh1-IVS6, and said primer sets middle and upper reaches primer sequence is shown in SEQ ID No.1, and the downstream primer sequence is shown in SEQ ID No.2.
More preferably, said probe sequence is shown in SEQ ID No.3.Said probe can utilize various nucleic acid labeling methods known in the art to make probe molecule marker beacon molecule, and said method includes but not limited to, methods such as PCR, RT-PCR, in-vitro transcription, random primer labelling, nick translation and terminal metastatic marker.Said beacon molecule can be for selecting suitable resorcinolphthalein; Said resorcinolphthalein includes but not limited to, Cy3, Cy5, fluorescein isothiocyanate, texas Red etc.
The present invention also provides a kind of enhancement type fluorescence quantifying PCR method that detects transgenic paddy rice strain TT51-1; The reaction system that will comprise the TV 25 μ l of primer sets 0.5 μ M, probe 0.25 μ M, template 1～100ng is carried out the real-time fluorescence quantitative PCR reaction, and reaction conditions is:
50℃，2min；
95℃10min
95 ℃ of 15s, 45 ℃ of 30s, 72 ℃ of 30s, 10 circulations;
95℃?15s，60℃?1min。
As preferably, the testing goal gene is TT51 foreign gene cry1Ab, and said primer sets middle and upper reaches primer sequence is shown in SEQ ID No.4, and the downstream primer sequence is shown in SEQ ID No.5.
More preferably, said probe sequence is shown in SEQ ID No.6.
The present invention is directed to transgenic corns strain BT11, transgenic paddy rice strain TT51-1 filters out suitable primer and probe; And whole detection system is optimized, being comprised: the optimization of primer concentration and probe concentration and proportioning, through selecting different primer concentrations for use; And reach a conclusion at primers different under the same primers as concentration, concentration and probe concentration ratio: in same primers as and concentration and probe concentration than under the situation; Primer and concentration and probe concentration are high more, and the Δ Rn value of PCR in real time curve is also just high relatively more, simultaneously C
TValue also presents respective change.When ratio amplification curve shape in 2: 1 of primer and probe better, good reproducibility, C
TBe worth littler.And change annealing temperature to C
TValue and fluorescent value influence are little.And the intermediary cycle number is many more, then C
TBe worth more little and little to fluorescent value influence.
Through standard substance are carried out gradient dilution, the amplification efficiency of ERT-PCR native gene and foreign gene all reaches requirement, and linear relationship is good, R
2＞0.98.Amplification efficiency explains that the ERT-PCR reaction conditions has reached optimization about 100%.
Find relatively that through ERT-PCR and RT-PCR it is highly sensitive that RT-PCR has detection method, it detects the more common RT-PCR of lower limit and exceeds about 10 times.ERT-PCR adopts identical template, the C of ERT-PCR with RT-PCR
TThe value scope is at 12-30, and the C of common RT-PCR
TThe value scope and it is generally acknowledged C then between 22-39
TValue just is difficult to judge negative or positive about 40, analyzes from this angle, and ERT-PCR has improved the sensitivity that detects.In addition; We adopt ERT-PCR and RT-PCR respectively transgenic seed (0.5%, 1%, 5%) to be carried out quantitative definite value result respectively to meet the requirements; Demonstration ERT-PCR detected result more near actual value, explains that the accuracy of ERT-PCR is higher than RT-PCR than RT-PCR.
To sum up, detect the fluorescence quantitative PCR detection technique that generally uses with present transgenic and compare, it is highly sensitive that ERT-PCR according to the invention has detection method, is about 10 times of RT-PCR sensitivity.The havoc of the dna molecular that transgenic product lower to transgenic content or deep processing causes; Extract and to contain a large amount of PCR suppressor factor in the sample and because present applied fluorescent quantitative PCR detection method can't judge that the sample of negative male gray area is particularly suitable for; And can in the detection of multiple transgenic strain, use, be with a wide range of applications.
Definition and general terms
The present invention will list the content of confirming of specializing in detail.The those skilled in the art will discern many similar or be equal to method described herein and material, and these can be applied to go in the practice of the present invention.The present invention is limited to the description of method and material absolutely not.
Enhancement type quantitative fluorescent PCR (ERT-PCR): fluorescence quantifying PCR method is improved, and the improvement aspect comprises: annealing temperature, cycle number etc., and improve a kind of detection method of fluorescence quantitative PCR detection sensitivity and accuracy greatly.
Real-time fluorescence PCR (RT-PCR): the real-time fluorescence PCR technology, be meant in the PCR reaction system to add fluorophor, utilize the fluorescent signal accumulation whole PCR process of monitoring in real time, the method for through detected fluorescent signal unknown template being analyzed at last.
Gray area: between 35-40, can't judge the feminine gender and the positive when the CT of quantitative fluorescent PCR value, this interval is called gray area.
Embodiment
The invention discloses a kind of enhancement type fluorescence quantifying PCR method that detects transgenic plant and products thereof, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Method of the present invention and application are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is done further detailed description below in conjunction with specific embodiment.
Agents useful for same consumptive material in the experiment:
Material: positive transgenic corn seed BT11, positive transgenic paddy rice seed TT51-1, above sample is provided by Hong Kong Haikang Life Sciences Co., Ltd.The NK603 standard substance (being respectively 0.1%, 0.5%, 1%, 2%, 5%) that IRMM buys
Transgenic plant and deep processed product process for extracting (In house) are available from Haikang Life Sciences Co., Ltd; Taqman genetic expression premixed liquid (Master mix) is available from ABI company.
Embodiment 1: to ERT-PCR condition optimizing-probe, the primer concentration optimization of BT11
Material: BT11 Genome; The BT11SSIIB primers F, R (10 μ M), SSIIB probe (10 μ M); The Specific primers F, R (10 μ M), Specific probe (10 μ M);
Reagent: ABI 2 * Master Mix, ddH
2O
Instrument: ABI 7500
The testing goal gene is: the border sequence of BT11 structure specific gene cry1Ab and adh1-IVS6.The primer probe sequence is following:
Method and step:
A. experimental design
The application of sample system:
| Composition | Store concentration | Single tube application of sample volume (μ L) |
| ddH 2O | - | 9-A-B |
| 2×Master?Mix | 2× | 10 |
| Primer-F/R | 10μM | A |
| Probe | 10μM | B |
| Template | - | 1.0 |
| TV | 25 |
A=(20 μ L* primer concentration)/10 μ M wherein
B=(20 μ L* concentration and probe concentration)/10 μ M
Template: Genome concentration is 110ng/ μ L
B. amplification condition:
50℃，2min；
95℃?10min
95℃?15s，45℃?30s，72℃?30s(10cycles)；
95℃?15s，60℃?1min(40cycles)
Note: fluorescence is collected at 60 ℃ of 1min places
Be respectively 0.2 μ M, 0.3 μ M, 0.5 μ M through primer concentration, primer: concentration and probe concentration is that 2: 1,1: 1,1: 2 different situations are carried out PCR in real time, and 2 repetitions are done in each experiment.Setting up with water is the negative control (primer and probe ratio are 1: 1) of template; Template is extracted genome for the BT11 transgenic corn seed adopts the Promega magnetic bead to extract test kit, and concentration is 11.2 μ g/mL.
Experimental result shows; Through selecting different primer concentrations for use; And reach a conclusion in same primers as and concentration and probe concentration than under the situation at no primer, concentration and probe concentration ratio under the same primers as concentration; Primer and concentration and probe concentration are high more, and the Δ Rn value of PCR in real time curve is also just high relatively more, and the CT value also presents respective change simultaneously; When primer concentration reaches 0.5 μ M; The SSIIB gene of BT11 and Specific gene PCR in real time curve are all better; And Δ Rn value is all more than 1.5; Seeing that the specificity of PCR in real time middle probe and reaction has certain relation, confirming to select for use primer concentration is 0.5 μ M, and concentration and probe concentration is that 0.5 μ M is as final optimum result.
Embodiment 2: to ERT-PCR condition optimizing---the cycle number optimization of BT11
Material: BT11Genome; BT11 SSIIB primers F, R (10 μ M), SSIIB probe (10 μ M); The Specific primers F, R (10 μ M), Specific probe (10 μ M) is with embodiment 1;
Reagent: ABI 2 * Master Mix, ddH
2O
Instrument: ABI 7500
Method and step:
A. experimental design: this experiment will be selected two different cycle number conditions for use, be respectively 5 cycle numbers and 10 cycle numbers compare.
B. primer is selected in this experiment for use: probe=1: 1, concentration are respectively 0.5 μ M
The application of sample system:
C. amplification condition:
1)50℃，2min；
95℃?10min；
95℃?5min，45℃?30s，72℃?1min(5cycles)
95℃?15s，60℃?1min(40cycles)
Note: fluorescence is collected at 60 ℃ of 1min places
2)50℃，2min；
95℃?10min；
95℃?5min，45℃?30s，72℃?1min(10cycles)
95℃?15s，60℃?1min(40cycles)
Note: fluorescence is collected at 60 ℃ of 1min places
Respectively SSIIB group primer, Specific group primer being carried out cycle number is 5,10 ERT-PCR; 2 repetitions are done in each experiment.Template is extracted genome for the BT11 transgenic corn seed adopts the Promega magnetic bead to extract test kit, and concentration is 11.2 μ g/mL, and the result sees the following form.
Different cycle number ERT-PCR C
TValue relatively
| The sample title | C T(10cycles) | C T(5cycles) |
| specific-1 | 20.1174 | 24.2902 |
| specific-2 | 20.1461 | 24.3405 |
| SSIIB-1 | 19.7149 | 23.6693 |
| SSIIB-2 | 19.6259 | 23.7318 |
Conclusion: the comparison through cycle number 5cycles and 10cycles can be reached a conclusion, C under the 10cycles cycle number
TValue is than the C under the 5Cycles cycle number
TBe worth little nearly 4 circulations, so cycle number is that 10 sensitivity is 5 height than cycle number.And the difference of Δ Rn value is little under these two kinds of cycle numbers.
Embodiment 3: detect transgenic strain BT11 specific gene with the method for the invention
The testing goal gene is: the border sequence of BT11 structure specific gene cry1Ab and adh1-IVS6.The primer probe sequence is following:
Reaction conditions to the ERT-PCR of BT11 is:
50℃，2min；
95℃?10min
95℃?15s，45℃?30s，72℃?30s(10cycles)；
95℃?15s，60℃?1min (40cycles)
Reaction system to the ERT-PCR of BT11 sees the following form:
| Composition | Final concentration |
| ddH 2O | |
| 2×Master?Mix | 1× |
| Primer-F/R | 0.5μM |
| Probe | 0.5μM |
| Template | 1～100ng |
| TV | 25μL |
Use Haikang Food Ex and Food Ex plus Plant Genome to extract the positive gene group that test kit extracts through gradient dilution, carry out gradient dilution (248ng, 24.8ng; 2.48ng; 248pg 24.8pg), sets up typical curve; Amplification shows, is limited to 24.8pg under the detection of the method for the invention detection transgenic corn BT 11 special gene sequence.And the detection lower limit of RT-PCR is merely 248pg.This presentation of results: its detection lower limit of detection transgenic corn BT 11 for ERT-PCR used in the present invention exceeds 10 times than use RT-PCR, explains that the detection method of ERT-PCR is sensitive more.The result also shows: ERT-PCR result's C
TValue is than the C of same template concentration RT-PCR
TBe worth little, and C
TValue can't be judged the feminine gender and the positive between 35-40, this interval is called gray area.
Embodiment 4: to ERT-PCR condition optimizing-probe, the primer concentration optimization of TT51-1 (BT63)
Material: BT63Genome; The Specific primers F, R (10 μ M), Specific probe (10 μ M);
Reagent: ABI 2 * Master Mix, ddH2O
Instrument: ABI 7500
The testing goal gene is: TT51 foreign gene cry1Ab.The primer probe sequence is following:
Experimental design
The application of sample system:
| Composition | Store concentration | Single tube application of sample volume (μ L) |
| ddH 2O | - | - |
| 2×Master?Mix | - | 12.5 |
| Primer-F/R | 10μM | A |
| Probe | 10μM | B |
| Template | - | 5.0 |
| TV | 25 |
A=(20 μ l* primer concentration)/10 μ M wherein
B=(20 μ l* concentration and probe concentration)/10 μ M
Template: Genome concentration is 2ng/ μ L
Amplification condition:
50℃，2min；
95℃?10min
95℃?15s，45℃?30s，72℃?30s(10cycles)；
95℃?15s，60℃?1min
Annotate: fluorescence is collected at 60 ℃ of 1min places
Be respectively 0.2 μ M, 0.3 μ M, 0.5 μ M through primer concentration, primer: concentration and probe concentration is that 2: 1,1: 1,1: 2 different situations are carried out PCR in real time, and 2 repetitions are done in each experiment.Setting up with water is the negative control (primer and probe ratio are 1: 1) of template; Template is extracted genome for the BT63 transgenic paddy rice adopts the Promega magnetic bead to extract test kit, and dilution back concentration is 2ng/ μ L.
The result shows that when the specific primer concentration was 0.2 μ M, the high more Δ R of concentration and probe concentration n value was high more, and when primer: concentration and probe concentration is that (primer concentration: 0.2 μ M, concentration and probe concentration: in the time of 0.4 μ M), the Δ R n value of Real time PCR curve reached 2.6 in 1: 2; And when primer: concentration and probe concentration is that (primer concentration: 0.2 μ M, concentration and probe concentration: in the time of 0.1 μ M), the Δ Rn value of Real time PCR curve only reached 0.7 in 2: 1.The concentration ratio of primer and probe is to C
TThe influence of value is little, and when primer: during concentration and probe concentration=2: 1, the CT value is respectively: 21.0,21.5; Primer: concentration and probe concentration=1: 2 o'clock, the CT value is respectively: 19.5,19.5.
When the specific primer concentration was 0.3 μ M, the high more Δ Rn of concentration and probe concentration value was high more, and when primer: (primer concentration: 0.3 μ M, concentration and probe concentration: in the time of 0.6 μ M), the Δ R n value of Real time PCR curve reaches about 3.6 concentration and probe concentration=1: 2; And when primer: (primer concentration: 0.3 μ M, concentration and probe concentration: in the time of 0.15 μ M), the Δ Rn value of Real time PCR curve only reaches about 1.4 concentration and probe concentration=2: 1.The concentration ratio of primer and probe is little to the influence of CT value, and when primer: during concentration and probe concentration=2: 1, the CT value is respectively: 19.7,20.0; Primer: concentration and probe concentration=1: 2 o'clock, the CT value is: 19.4,19.5.
When the specific primer concentration was 0.5 μ M, the high more Δ Rn of concentration and probe concentration value was high more, and when primer: (primer concentration: 0.5 μ M, concentration and probe concentration: in the time of 1 μ M), the Δ R n value of Real time PCR curve reaches about 5.3 concentration and probe concentration=1: 2; And when primer: (primer concentration: 0.5 μ M, concentration and probe concentration: in the time of 0.25 μ M), the Δ Rn value of Real time PCR curve only reaches about 2.3 concentration and probe concentration=2: 1.The concentration ratio of primer and probe is little to the influence of CT value, and when primer: during concentration and probe concentration=2: 1, the CT value is respectively: 19.6,19.6; Primer: concentration and probe concentration=1: 2 o'clock, the CT value is respectively: 19.4,19.4.When primer concentration was 0.5 μ M, the Δ Rn value of Real time PCR and CT value changed all less.
Through selecting different primer concentrations for use; And reach a conclusion at primers different under the same primers as concentration, concentration and probe concentration ratio: in same primers as and concentration and probe concentration than under the situation; Primer and concentration and probe concentration are high more, and the Δ R n value of Real timePCR curve is also just high relatively more, simultaneously C
TValue also presents respective change; When primer concentration reaches 0.5 μ M; The Specific gene Real time PCR curve of BT63 is all better; And Δ Rn value is all more than 1.5; Seeing that the specificity of Real time PCR middle probe and reaction has certain relation and considers that from the angle of practicing thrift cost it is 0.5 μ M that primer concentration is selected in this experiment for use, concentration and probe concentration is that 0.25 μ M is as final optimum result.
Embodiment 5: to ERT-PCR condition optimizing---the cycle number optimization of TT51-1 (BT63)
Material: BT63Genome; The BT63Specific primers F, R (10 μ M), Specific probe (10 μ M);
Reagent: ABI 2 * Master Mix, ddH
2O
Instrument: ABI 7500
A. experimental design: this experiment will be selected two different cycle number conditions for use, be respectively: 5 cycle numbers, 10 cycle numbers compare.
B. through experiment on the 20100205th, primer is selected in this experiment for use: probe=2: 1, concentration, probe were respectively: 0.5 μ M
The application of sample system:
A=(20 μ l* primer concentration)/10 μ M
B=(20 μ l* concentration and probe concentration)/10 μ M
Template: Genome concentration is 2ng/ μ Lc. amplification condition:
1)50℃，2min；
95℃?10min；
95℃?15s，45℃?30s，72℃?30s(5cycles)
95℃?15s，60℃?1min(40cycles)
Annotate: fluorescence is collected at 60 ℃ of 1min places
2)50℃，2min；
95℃?10min；
95℃?15s，45℃?30s，72℃?30s(10cycles)
95℃?15s，60℃?1min(40cycles)
Annotate: fluorescence is collected at 60 ℃ of 1min places
Respectively primer being carried out cycle number is 5,10 ERT-PCR, NTC is set: the negative control (primer and probe ratio are 1: 1) that with water is template; 2 repetitions.Template is extracted genome for the BT63 transgenic paddy rice adopts the Promega magnetic bead to extract test kit, and dilution back concentration is 2ng/ μ L, and the result sees the following form.
Different cycle number ERT-PCR CT values relatively
| The sample name | C T(10cycles) | C T(5cycles) |
| specific-1 | 19.5689 | 24.3398 |
| specific-2 | 19.6372 | 24.5139 |
Comparison through cycle number 5cycles and 10cycles can be reached a conclusion, and the CT value of 10cycles is little by about 3 than the CT value of 5Cycles, thus cycle number be 10 sensitivity than cycle number be 5 exceed 2
3Doubly.And the difference of Δ Rn value is little under these two kinds of cycle numbers.
Embodiment 6: to ERT-PCR condition optimizing-temperature optimization of TT51-1 (BT63)
Material: BT63Genome; The BT63Specific primers F, R (10 μ M), Specific probe (10 μ M);
Reagent: ABI 2 * Master Mix, ddH
2O
Instrument: ABI 7500
Method and step:
A. experimental design: this experiment will be selected two different temperature conditions for use, be respectively: 45 degree, 50 degree compare.
B. through experiment on the 20100205th, primer is selected in this experiment for use: probe=2: 1, concentration were respectively 0.5 μ M
The application of sample system:
A=(20 μ L* primer concentration)/10 μ M
B=(20 μ L* concentration and probe concentration)/10 μ M
Template: Genome concentration is 2ng/ μ L
C. amplification condition:
1)50℃，2min；
95℃?10min；
95℃?15s，45℃?30s，72℃?30s(10cycles)
95℃?15s，60℃?1min(40cycles)
Annotate: fluorescence is collected at 60 ℃ of 1min places
2)50℃，2min；
95℃10min；
95℃?15s，50℃?30s，72℃?30s(10cycles)
95℃?15s，60℃?1min(40cycles)
Annotate: fluorescence is collected at 60 ℃ of 1min places
When temperature is 45 ℃, 50 ℃, carry out ERT-PCR respectively, NTC is set: the negative control (primer and probe ratio are 1: 1) that with water is template; 2 repetitions.Template is extracted genome for the BT63 transgenic paddy rice adopts the Promega magnetic bead to extract test kit, and concentration is 2ng/ μ L, and the result sees the following form:
ERT-PCR CT value relatively under the differing temps
Through temperature is that the comparison of 50 ℃ and 45 ℃ can be reached a conclusion, and the CT value of 50 ℃ CT value than 45 ℃ is little by about 0.2, and difference is little.And the difference of Δ Rn value is also little under these two kinds of temperature.
Embodiment 7: detect transgenic strain TT51-1 specific gene with the method for the invention
The testing goal gene is: TT51 foreign gene cry1Ab.The primer probe sequence is following:
Use Haikang Food Ex and Food Ex plus Plant Genome to extract the positive gene group that test kit extracts, be made into following 25 μ L reaction systems, carry out pcr amplification reaction:
Reaction conditions to the ERT-PCR of TT51-1 is:
50℃，2min；
95℃10min
95℃?15s，45℃?30s，72℃?30s(10cycles)；
95℃?15s，60℃?1min(40cycles)
Reaction system to the ERT-PCR of TT51-1 sees the following form.
| Composition | Final concentration |
| ddH 2O | |
| 2×Master?Mix | 1× |
| Primer-F/R | 0.5μM |
| Probe | 0.25μM |
| Template | 1～100ng |
| TV | 25μL |
Use Haikang Food Ex and Food Ex plus Plant Genome to extract the positive gene group that test kit extracts through gradient dilution, carry out gradient dilution (625ng, 125ng; 25ng, 5ng, 1ng; 200pg, 40pg, 8pg); Set up typical curve, the amplification curve result shows, is limited to 40pg under the detection of transgenic paddy rice TT51-1 special gene sequence.And the C RT-PCR 40pg the time
T＞35.Be merely 200pg so detect lower limit, not as ERT-PCR method sensitivity.From experimental result picture, it can also be seen that ERT-PCR result's C
TValue is than the C of same template concentration RT-PCR
TBe worth little, and C
TValue can't be judged the feminine gender and the positive between 35-40, this interval is called gray area.
Embodiment 8: the accuracy that the method for the invention detects
Reaction conditions to the ERT-PCR of transgenic corns NK603 is:
50℃，2min；
95℃10min
95℃?15s，45℃?30s，72℃?30s(10cycles)；
95℃?15s，60℃?1min(40cycles)
Reaction system to the ERT-PCR of NK603 sees the following form.
When detecting, adopt NK603 Haikang, Beijing company to detect the primer probe of usefulness; Adopt above amplification system; Use the standard substance (being respectively 0.1%, 0.5%, 1%, 2%, 5%) of IRMM to set up typical curve respectively; Three blind appearance of ABC are carried out definite value, and to detect the accuracy of ERT-PCR, the result sees the following form.
The result shows that there is not significant difference (P＞0.05) in two groups of results.But because A1, B1, the true value of three groups of blind appearance of C1 (Hong Kong Haikang Life Sciences Co., Ltd provides) is respectively 0.5%, 1%, 5%, and the ERT-PCR detected result more near actual value, explains that the accuracy of ERT-PCR is higher than RT-PCR than RT-PCR.
In sum: through standard substance are carried out gradient dilution, the amplification efficiency of ERT-PCR native gene and foreign gene all reaches requirement, and linear relationship is good, R
2＞0.98.Amplification efficiency is explained that the ERT-PCR reaction conditions optimizes, and can be applied to the detection of transgenic plant and products thereof about 100%.
Find relatively that through ERT-PCR and RT-PCR the more common RT-PCR of detection lower limit of ERT-PCR exceeds about 10 times.ERT-PCR adopts identical template, the C of ERT-PCR with RT-PCR
TThe value scope is at 12-30, and the C of common RT-PCR
TThe value scope and it is generally acknowledged C then between 22-39
TValue just is difficult to judge negative or positive about 40, so analyze from this angle, ERT-PCR has improved the sensitivity that detects.In addition, we adopt ERT-PCR and RT-PCR respectively transgenic seed (0.5%, 1%, 5%) to be carried out quantitative definite value result respectively to meet the requirements, and do not have significant difference with RT-PCR definite value result.
The above results shows, detects the fluorescence quantitative PCR detection technique that generally uses with present transgenic and compares.It is highly sensitive that ERT-PCR has detection method, is about 10 times of RT-PCR sensitivity.The havoc of the dna molecular that transgenic product lower to transgenic content or deep processing causes; The DNA that extracts contains a large amount of PCR suppressor factor and because present applied fluorescent quantitative PCR detection method can't judge that the sample of negative male gray area is particularly suitable for, and can in the detection of multiple transgenic strain, use.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (6)
1. enhancement type fluorescence quantifying PCR method that detects transgenic corns strain BT11; It is characterized in that; The reaction system that will comprise the TV 25 μ l of primer sets 0.5 μ M, probe 0.5 μ M, template 1-100ng is carried out the real-time fluorescence quantitative PCR reaction, and reaction conditions is:
50℃，2min；
95℃10min；
95 ℃ of 15s, 45 ℃ of 30s, 72 ℃ of 30s, 10 circulations;
95 ℃ of 15s, 60 ℃ of 1min, 40 circulations.
2. enhancement type fluorescence quantifying PCR method according to claim 1 is characterized in that, said primer sets middle and upper reaches primer sequence is shown in SEQ ID No.1, and the downstream primer sequence is shown in SEQ ID No.2.
3. enhancement type fluorescence quantifying PCR method according to claim 1 and 2 is characterized in that, said probe sequence is shown in SEQ ID No.3.
4. enhancement type fluorescence quantifying PCR method that detects transgenic paddy rice strain TT51-1; It is characterized in that; The reaction system that will comprise the TV 25 μ l of primer sets 0.5 μ M, probe 0.25 μ M, template 100ng is carried out the real-time fluorescence quantitative PCR reaction, and reaction conditions is:
50℃，2min；
95℃?10min
95 ℃ of 15s, 45 ℃ of 30s, 72 ℃ of 30s, 10 circulations;
95 ℃ of 15s, 60 ℃ of 1min, 40 circulations.
5. enhancement type fluorescence quantifying PCR method according to claim 4 is characterized in that, said primer sets middle and upper reaches primer sequence is shown in SEQ ID No.4, and the downstream primer sequence is shown in SEQ ID No.5.
6. according to claim 4 or 5 described enhancement type fluorescence quantifying PCR methods, it is characterized in that said probe sequence is shown in SEQ ID No.6.
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| WO2004097021A1 (en) * | 2003-05-02 | 2004-11-11 | Hong Kong Dna Chips Limited | Nucleic acid detection |
| CN1623002A (en) * | 2000-10-26 | 2005-06-01 | 独立行政法人食品综合研究所 | Methods of quantitative detection of genetic recombinants and standard molecules for the methods |
| CN101812512A (en) * | 2009-12-23 | 2010-08-25 | 中华人民共和国珠海出入境检验检疫局 | Compound gene chip for detecting transgenic foods |
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| WO2004097021A1 (en) * | 2003-05-02 | 2004-11-11 | Hong Kong Dna Chips Limited | Nucleic acid detection |
| CN101812512A (en) * | 2009-12-23 | 2010-08-25 | 中华人民共和国珠海出入境检验检疫局 | Compound gene chip for detecting transgenic foods |
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| LOK-TING LAU 等: "Nucleic acid sequence-based amplification methods to detect avian influenza virus", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
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