CN102424863B - 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, the mankind have changed the method for plant conventional breeding, by engineered means, foreign gene are changed in plant materials, thereby cultivate a collection of high yield, transgenic plant kind that resistance 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 on a large scale.However, countries in the world are still very careful to the attitude that turns 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, the detection methods such as ordinary gel PCR.Real-time fluorescence PCR (RT-PCR): refer to add fluorophor in the PCR reaction system, utilize the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, the method for by the fluorescent signal that detects, unknown template being analyzed at last.Yet all there is the deficiency of self in these detection methods, as some deep processed product and the lower product existence of transgenosis content are judged the blind area, namely can't carry out disposable judgement to detected result.
This just need exploitation efficiently, accurately, sensitiveer transgenic detection method.This foreign trade for country, transgenosis safe and be all the most important problem solving of needing for customs, commodity inspection, inspection and quarantining for import/export department, food-processing department.
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 positive 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 cumulative volume 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 described primer sets middle and upper reaches primer sequence is as shown in SEQ ID No.1, and the downstream primer sequence is as shown in SEQ ID No.2.
More preferably, described probe sequence is as shown in SEQ ID No.3.Described probe can utilize various nucleic acid labeling methods known in the art to make probe molecule marker beacon molecule, and described method includes but not limited to, the methods such as PCR, RT-PCR, in-vitro transcription, random primer labelling, nick translation and end metastatic marker.Described beacon molecule can be for selecting suitable fluorescein; Described fluorescein 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 cumulative volume 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 described primer sets middle and upper reaches primer sequence is as shown in SEQ ID No.4, and the downstream primer sequence is as shown in SEQ ID No.5.
More preferably, described probe sequence is as 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, comprise: the optimization of primer concentration and probe concentration and proportioning, by selecting different primers concentration, and reach a conclusion at primers different under same primers as concentration, concentration and probe concentration ratio: in same primers as and concentration and probe concentration than in situation, primer and concentration and probe concentration are higher, and the Δ Rn value of PCR in real time curve is also just relatively higher, 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 less.And change annealing temperature to C
TValue and fluorescent value impact are little.And middle cycle number is more, C
TBe worth less and fluorescent value affected little.
By 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 illustrates that in 100% left and right the ERT-PCR reaction conditions has reached optimization.
Relatively find by ERT-PCR and RT-PCR, it is highly sensitive that RT-PCR has detection method, and it detects lower limit and exceeds 10 times of left and right than conventional RT-PCR.ERT-PCR adopts identical template, the C of ERT-PCR with RT-PCR
TThe value scope is at 12-30, and the C of conventional RT-PCR
TThe value scope and be it is generally acknowledged C between 22-39
TValue just is difficult to judge negative or positive in 40 left and right, analyze from this angle, and ERT-PCR has improved the sensitivity that detects.In addition, we adopt respectively ERT-PCR and RT-PCR respectively transgenic seed (0.5%, 1%, 5%) to be carried out quantitative definite value result to meet the requirements, demonstration ERT-PCR detected result more near actual value, illustrates that the accuracy of ERT-PCR is higher than RT-PCR than RT-PCR.
To sum up, detect with present transgenosis the fluorescence quantitative PCR detection technique that generally uses and compare, it is highly sensitive that ERT-PCR of the present invention has detection method, is about 10 times of RT-PCR sensitivity.The havoc of the DNA molecular that transgenic product lower for transgenosis content or deep processing causes, extract and to contain a large amount of PCR inhibitor in sample and because present applied fluorescent quantitative PCR detection method can't be judged the sample of negative positive gray area and is particularly suitable for, and can use in the detection of multiple transgenic strain, be with a wide range of applications.
Definition and general terms
The present invention will list the content of specializing of determining in detail.The those skilled in the art will identify many similar or be equal to method described herein and material, and these can be applied to go in practice of the present invention.The present invention is limited to absolutely not the description of method and material.
Enhancement type quantitative fluorescent PCR (ERT-PCR): fluorescence quantifying PCR method is improved, and the improvement aspect comprises: annealing temperature, cycle number etc., and greatly improve a kind of detection method of fluorescence quantitative PCR detection sensitivity and accuracy.
Real-time fluorescence PCR (RT-PCR): real-time fluorescence PCR technology, refer to add fluorophor in the PCR reaction system, utilize the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, the method for by the fluorescent signal that detects, unknown template being analyzed at last.
Gray area: can't judge feminine gender and the positive when the CT of quantitative fluorescent PCR value between 35-40, 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, all similarly replace and change apparent to those skilled in the art, and they all are deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change methods and applications as herein described within not breaking away from content of the present invention, spirit and scope or suitably change and combination, realizes and use the technology of the present invention.
In order to make those skilled in the art understand better technical scheme of the present invention, the present invention is described in further detail below in conjunction with specific embodiment.
Agents useful for same consumptive material in 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 extracting method (In house) are available from Haikang Life Sciences Co., Ltd; Taqman genetic expression premixed liquid (Master mix) is available from ABI company.
Embodiment 1: for 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 as follows:
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 |
| Cumulative volume | 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: 60 ℃ of 1min place's collection fluorescence
Be respectively 0.2 μ M, 0.3 μ M, 0.5 μ M by 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.Set up the negative control (primer and probe ratio are 1: 1) take water as template; Template is that the BT11 transgenic corn seed adopts the Promega magnetic bead to extract test kit extraction genome, and concentration is 11.2 μ g/mL.
Experimental result shows, by selecting different primers concentration, and reach a conclusion in same primers as and concentration and probe concentration than in situation at no primer, concentration and probe concentration ratio under same primers as concentration, primer and concentration and probe concentration are higher, the Δ Rn value of PCR in real time curve is also just relatively higher, 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, in view of the specificity of PCR in real time middle probe and reaction has certain relation, determining to select 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: for the ERT-PCR condition optimizing of BT11---cycle number optimization
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, be respectively 5 cycle numbers and 10 cycle numbers compare.
B. primer is selected in this experiment: 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: 60 ℃ of 1min place's collection fluorescence
2)50℃，2min；
95℃ 10min；
95℃ 5min，45℃ 30s，72℃ 1min(10cycles)
95℃ 15s，60℃ 1min(40cycles)
Note: 60 ℃ of 1min place's collection fluorescence
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 that the BT11 transgenic corn seed adopts the Promega magnetic bead to extract test kit extraction genome, and concentration is 11.2 μ g/mL, the results are shown in following table.
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 by 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 under these two kinds of cycle numbers, the difference of Δ Rn value is little.
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 as follows:
Reaction conditions for the ERT-PCR of BT11 is:
50℃，2min；
95℃ 10min
95℃ 15s，45℃ 30s，72℃ 30s(10cycles)；
95℃ 15s，60℃ 1min (40cycles)
Reaction system for 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 |
| Cumulative volume | 25μL |
Use Haikang Food Ex and Food Ex plus Plant Genome to extract the positive gene group that test kit extracts by gradient dilution, carry out gradient dilution (248ng, 24.8ng, 2.48ng, 248pg, 24.8pg), the Criterion 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 only 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, illustrates that the detection method of ERT-PCR is sensitiveer.Result also shows: the C of ERT-PCR result
TValue is than the C of same template concentration RT-PCR
TBe worth little, and C
TValue can't be judged feminine gender and the positive between 35-40, this interval is called gray area.
Embodiment 4: for 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 as follows:
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 |
| Cumulative volume | 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: 60 ℃ of 1min place's collection fluorescence
Be respectively 0.2 μ M, 0.3 μ M, 0.5 μ M by 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.Set up the negative control (primer and probe ratio are 1: 1) take water as template; Template is that the BT63 transgenic paddy rice adopts the Promega magnetic bead to extract test kit extraction genome, and after dilution, concentration is 2ng/ μ L.
Result shows, when the specific primer concentration is 0.2 μ M, the higher Δ R of concentration and probe concentration n value is higher, 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 impact 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 higher Δ Rn of concentration and probe concentration value was higher, 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 3.6 left and right to 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 1.4 left and right to concentration and probe concentration=2: 1.The concentration ratio of primer and probe is little on the impact 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 higher Δ Rn of concentration and probe concentration value was higher, 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 5.3 left and right to 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 2.3 left and right to concentration and probe concentration=2: 1.The concentration ratio of primer and probe is little on the impact 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.
By selecting different primers concentration, and reach a conclusion at primers different under same primers as concentration, concentration and probe concentration ratio: in same primers as and concentration and probe concentration than in situation, primer and concentration and probe concentration are higher, and the Δ R n value of Real timePCR curve is also just relatively higher, 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, in view of the specificity of Real time PCR middle probe and reaction has certain relation and considers from the angle of saving cost, it is 0.5 μ M that primer concentration is selected in this experiment, and concentration and probe concentration is that 0.25 μ M is as final optimum result.
Embodiment 5: for the ERT-PCR condition optimizing of TT51-1 (BT63)---cycle number optimization
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, be respectively: 5 cycle numbers, 10 cycle numbers compare.
B. by experiment on the 20100205th, primer is selected in this experiment: 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: 60 ℃ of 1min place's collection fluorescence
2)50℃，2min；
95℃ 10min；
95℃ 15s，45℃ 30s，72℃ 30s(10cycles)
95℃ 15s，60℃ 1min(40cycles)
Annotate: 60 ℃ of 1min place's collection fluorescence
Respectively primer being carried out cycle number is 5,10 ERT-PCR, NTC is set: the negative control take water as template (primer and probe ratio are 1: 1); 2 repetitions.Template is that the BT63 transgenic paddy rice adopts the Promega magnetic bead to extract test kit extraction genome, and after dilution, concentration is 2ng/ μ L, the results are shown in following table.
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 by cycle number 5cycles and 10cycles can be reached a conclusion, and the CT value of 10cycles is than little 3 left and right of CT value of 5Cycles, thus cycle number be 10 sensitivity than cycle number be 5 exceed 2
3Doubly.And under these two kinds of cycle numbers, the difference of Δ Rn value is little.
Embodiment 6: for 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 condition, be respectively: 45 degree, 50 degree compare.
B. by experiment on the 20100205th, primer is selected in this experiment: 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: 60 ℃ of 1min place's collection fluorescence
2)50℃，2min；
95℃10min；
95℃ 15s，50℃ 30s，72℃ 30s(10cycles)
95℃ 15s，60℃ 1min(40cycles)
Annotate: 60 ℃ of 1min place's collection fluorescence
Carry out ERT-PCR respectively when temperature is 45 ℃, 50 ℃, NTC is set: the negative control take water as template (primer and probe ratio are 1: 1); 2 repetitions.Template is that the BT63 transgenic paddy rice adopts the Promega magnetic bead to extract test kit extraction genome, and concentration is 2ng/ μ L, the results are shown in following table:
Under differing temps, ERT-PCR CT value relatively
Can reach a conclusion by the comparison that temperature is 50 ℃ and 45 ℃, the CT value of the CT value of 50 ℃ than 45 ℃ little 0.2 left and right, difference is little.And at these two kinds of temperature, the difference of Δ Rn value is also little.
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 as follows:
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 for 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 for 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 |
| Cumulative volume | 25μL |
Use Haikang Food Ex and Food Ex plus Plant Genome to extract the positive gene group that test kit extracts by gradient dilution, carry out gradient dilution (625ng, 125ng, 25ng, 5ng, 1ng, 200pg, 40pg, 8pg), the Criterion 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 when RT-PCR 40pg
T＞35.So detecting lower limit is only 200pg, not as ERT-PCR method sensitivity.It can also be seen that the C of ERT-PCR result from experimental result picture
TValue is than the C of same template concentration RT-PCR
TBe worth little, and C
TValue can't be judged 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 for 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 for the ERT-PCR of NK603 sees the following form.
When detecting, adopt NK603 Haikang, Beijing company to detect the primer probe of use, adopt above amplification system, use respectively standard substance (being respectively 0.1%, 0.5%, 1%, 2%, 5%) the Criterion curve of IRMM, three blind samples of ABC are carried out definite value, to detect the accuracy of ERT-PCR, the results are shown in following table.
The result demonstration, 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 samples of C1 (Hong Kong Haikang Life Sciences Co., Ltd provides) be respectively 0.5%, 1%, 5%, ERT-PCR detected result than RT-PCR more near actual value, illustrate that the accuracy of ERT-PCR is higher than RT-PCR.
In sum: by 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 in 100% left and right, illustrates that the ERT-PCR reaction conditions optimizes, and can be applied to the detection of transgenic plant and products thereof.
Relatively find by ERT-PCR and RT-PCR, the detection lower limit of ERT-PCR exceeds 10 times of left and right than conventional RT-PCR.ERT-PCR adopts identical template, the C of ERT-PCR with RT-PCR
TThe value scope is at 12-30, and the C of conventional RT-PCR
TThe value scope and be it is generally acknowledged C between 22-39
TValue just is difficult to judge negative or positive in 40 left and right, so analyze from this angle, ERT-PCR has improved the sensitivity that detects.In addition, we adopt respectively ERT-PCR and RT-PCR respectively transgenic seed (0.5%, 1%, 5%) to be carried out quantitative definite value result to meet the requirements, and do not have significant difference with RT-PCR definite value result.
The above results shows, detects with present transgenosis the fluorescence quantitative PCR detection technique that generally uses 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 for transgenosis content or deep processing causes, the DNA that extracts contains a large amount of PCR inhibitor and because present applied fluorescent quantitative PCR detection method can't be judged the sample of negative positive gray area and is particularly suitable for, and can use in the detection of multiple transgenic strain.
The above is only the preferred embodiment 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 improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
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 cumulative volume 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°C，2min；
95°C 10min；
95 ° of C 15s, 45 ° of C 30s, 72 ° of C 30s, 10 circulations;
95 ° of C 15s, 60 ° of C 1min, 40 circulations;
Described primer sets middle and upper reaches primer sequence is as shown in SEQ ID No.1, and the downstream primer sequence is as shown in SEQ ID No.2; Described probe sequence is as shown in SEQ ID No.3.
2. 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 cumulative volume 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°C，2min；
95°C 10min
95 ° of C 15s, 45 ° of C 30s, 72 ° of C 30s, 10 circulations;
95 ° of C 15s, 60 ° of C 1min, 40 circulations;
Described primer sets middle and upper reaches primer sequence is as shown in SEQ ID No.4, and the downstream primer sequence is as shown in SEQ ID No.5; Described probe sequence is as shown in SEQ ID No.6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| 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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| 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 |
| 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 |
Non-Patent Citations (2)
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| Lok-Ting Lau 等.Nucleic acid sequence-based amplification methods to detect avian influenza virus.《Biochemical and Biophysical Research Communications》.2004,第313卷第336-342页. |
| Nucleic acid sequence-based amplification methods to detect avian influenza virus;Lok-Ting Lau 等;《Biochemical and Biophysical Research Communications》;20041231;第313卷;第336-342页 * |
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