CN102952861A - Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize - Google Patents
Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize Download PDFInfo
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Abstract
The invention discloses a multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and a detection method for genetically modified maize. The primer has strong specificity and can be used for PCR amplification and DHPLC analysis. The detection method is simple and convenient to operate, good in expansion performance and high in sensitivity, and multi-target detection of the genetically modified maize is realized. The DHPLC is used to analyze PCR amplified products, fragment size resolution can reach multiple bases, and resolution ratio is high. The multiplex PCR-DHPLC detection primer and the detection method for genetically modified maize have the advantages that the detection method is simple, convenient, effective, reliable, high in throughput and especially suitable for departments of port inspection and quarantine and the like.
Description
Technical field
The present invention relates to a kind of detection of transgenic product, particularly relate to a kind of transgenic corns multiplex PCR-DHPLC and detect primer and detection method.
Background technology
At present the detection method of transgenic corns mainly adopted multiplex PCR, the detection methods such as multiple real time fluorescence PCR, PCR-gene chip.Traditional multiple conventional PCR detection method has certain limitation in the platform extension method, along with the increase of target to be checked, need to re-start optimization to consumption and the ratio of the every cover primer in the system, and takes into account the factors such as amplification efficiency, and workload is larger; On the other hand, the method for the gel electrophoresis analysis amplified production that usually adopts, it is not high to distinguish efficient, and detected result is undesirable.More multiple conventional PCR detection method has superiority although multiple real time fluorescence PCR is at aspects such as detection sensitivities, but because the restriction of present instrument self and fluorescence dye development, non-interfering 4 fluorescence channels only can be provided simultaneously, also limit this technology and detected the flux expansion.Conventional many cover PCR-gene chip detection methods need many cover primers to increase, complex operation step, and be not suitable for large-scale high throughput testing.Sex change high-efficient liquid phase chromatogram technology (Denaturing High-performance Liquid Chromatography, DHPLC) be a kind of method for nucleic acid analysis of simple, quick, non-gel, have the advantages such as extendability is strong, good resolution, sensitivity height.The method is analytic sample under 50 ℃ of conditions, and the wash-out at sample peak only determines elution order by the quantity of base pair, and the acetonitrile concentration that has served as post improves, and nucleic acid fragment can be according to molecular weight order from small to large by wash-out out.Relatively determine molecular size range by elution peak and the marker that obtains, determine whether and contain the target detect gene.At present, still there is not the PCR of transgenic corns in conjunction with the detection technique (PCR-DHPLC) of DHPLC.
Summary of the invention
The purpose of this invention is to provide a kind of primer for transgenic corns multiplex PCR-DHPLC detection.
The transgenic corns multiplex PCR that another object of the present invention provides that a kind of scalability based on above-mentioned primer is good, sensitivity and resolving power are high-DHPLC detection method.
For achieving the above object, the present invention has adopted following technical scheme:
The invention discloses the primer that detects for transgenic corns multiplex PCR-DHPLC, described primer comprise primer to 1, primer to 2, primer to 3, primer to 4, primer to 5, primer to 6 and primer at least one pair of primer in 7; Primer contains sequence shown in the Seq ID No.1 to 1 upstream primer, and downstream primer contains sequence shown in the Seq ID No.2; Primer contains sequence shown in the Seq ID No.3 to 2 upstream primer, and downstream primer contains sequence shown in the Seq ID No.4; Primer contains sequence shown in the Seq ID No.5 to 3 upstream primer, and downstream primer contains sequence shown in the Seq ID No.6; Primer contains sequence shown in the Seq ID No.7 to 4 upstream primer, and downstream primer contains sequence shown in the Seq ID No.8; Primer contains sequence shown in the Seq ID No.9 to 5 upstream primer, and downstream primer contains sequence shown in the Seq ID No.10; Primer contains sequence shown in the Seq ID No.11 to 6 upstream primer, and downstream primer contains sequence shown in the Seq ID No.12; Primer contains sequence shown in the Seq ID No.13 to 7 upstream primer, and downstream primer contains sequence shown in the Seq ID No.14.
It is pointed out that above-mentioned Seq ID No.1 to Seq ID No.14 is and the distinguished sequence of the detection target sequence complementary pairing of transgenic corns strain, has very strong specific recognition.
Further, described primer to 3, primer to 4, primer to 5 and primer to 6,5 ' end of its upstream primer also comprises sequence shown in the Seq ID No.15,5 ' end of its downstream primer also comprises sequence shown in the Seq ID No.16;
Seq?ID?No.15：5’-CGTGGCCTCGCGATCTGACT-3’
Seq?ID?No.16：5’-CTCAGCGGCGGAGCTACAGA-3’。
It is to be noted, above-mentioned Seq ID No.15 and Seq ID No.16 are irrelevant one section sequences with detecting target sequence of adding at 5 ' end of upstream primer and downstream primer respectively, this sequence can be the sequence of other species far with detecting the target sequence homology, also can be one section sequence that generates at random.This sequence can be used for the size of regulation and control pcr amplification product, so that the further analysis of pcr amplification product.
Among the present invention, preferred, described primer has sequence shown in the Seq ID No.17 to 3 upstream primer, and downstream primer has sequence shown in the Seq ID No.18; Primer has sequence shown in the Seq ID No.19 to 4 upstream primer, and downstream primer has sequence shown in the Seq ID No.20; Primer has sequence shown in the Seq ID No.21 to 5 upstream primer, and downstream primer has sequence shown in the Seq ID No.22; Primer has sequence shown in the Seq ID No.23 to 6 upstream primer, and downstream primer has sequence shown in the Seq ID No.24.
Need to prove, described Seq ID No.17 to Seq ID No.24 sequence is to add regulating and controlling sequence at its 5 ' end respectively by above-mentioned Seq ID No.5 to Seq ID No.12 sequence to form, although above-mentionedly illustrated that regulating and controlling sequence can be the sequence that generates at random shown in Seq ID No.15 and the Seq ID No.16, but, be not that arbitrary sequence can be added, in the present invention, need to consider that regulating and controlling sequence and the unified of physico-chemical property of sequence shown in Seq ID No.5 to the Seq ID No.12 coordinate, and need to consider to add sequence shown in Seq ID No.17 to the Seq ID No.24 behind the regulating and controlling sequence as the overall performance of detection primer.
Among the present invention, preferred, described primer is to 1 primer for detection transgenic corns strain NK603, primer is to 2 primers for detection transgenic corns strain MON863, primer is to 3 primers for detection transgenic corns strain MON810, primer is to 4 primers for detection transgenic corns strain T25, primer is to 5 primers for detection transgenic corns strain MON88017, primer is to 6 primers for detection transgenic corns strain MIR604, and primer is to 7 primers for detection transgenic corns strain 59122.
The invention also discloses a kind of for transgenic corns multiplex PCR-DHPLC detection method, described detection method comprises the above-mentioned primer of employing, carry out pcr amplification take maize dna as template, and pcr amplification product carried out dhplc analysis (Denaturing High-performance Liquid Chromatography, HPLC).
Further, described detection method also comprises take marker as reference standard carries out dhplc analysis, and the dhplc analysis result of pcr amplification product and the dhplc analysis result of marker are compared.
Preferred above-mentioned detection method comprises the steps:
(A) adopt above-mentioned primer, take maize dna as template, carry out pcr amplification;
(B) analyze as sample carries out DHPLC take the pcr amplification product of step (A), carry out simultaneously DHPLC take marker as reference standard and analyze;
(C) the DHPLC analytical results of sample in the step (B) and the DHPLC analytical results of marker are compared, determine the molecular size range of sample, thereby judge whether to contain the target sequence of detection.
Because adopt above technical scheme, beneficial effect of the present invention is:
Transgenic corns Multiple detection primer of the present invention has stronger specificity, can be used in follow-up multiplex PCR amplification and DHPLC and analyzes.The present invention is directed to traditional undesirable problem of electrophoretic analysis pcr amplification result, PCR is combined with DHPLC, provide a kind of easy and simple to handle, scalability good, sensitivity and the high transgenic corns multiple detection method of resolving power, realized the multiplex detection of transgenic corns.Utilize DHPLC that pcr amplification product is analyzed, can reach several bases to different big or small PCR product fragment differentiation rates, even can distinguish the fragment of 1 base difference in size.Primer of the present invention and detection method are particularly suitable for department's uses such as Check and Examination of Port quarantine for transgenic corns strain BT11 detection provides a kind of simple, convenient, effective, reliable high-flux detection method.
Description of drawings
Figure is the partial results that DHPLC analyzes in the embodiment of the invention, and wherein the elution peak of 1 transgenic corns strain MON863,2 is that the elution peak, 3 of transgenic corns strain NK603 is the elution peak of transgenic corns strain 59122 for the elution peak of transgenic corns strain MIR604,7 for the elution peak of transgenic corns strain MON88017,6 for the elution peak of transgenic corns strain T25,5 for the elution peak of transgenic corns strain MON810,4; Curve A is the DHPLC elution curve of the 7 heavy pcr amplification products that carry out take the non-transgenic corn as template as the Auele Specific Primer of 7 strains of the DHPLC elution curve, curve B of the 7 heavy pcr amplification products that carry out take the DNA hybrid template of 7 strains of the Auele Specific Primer of 7 strains, curve M is marker, and each peak value of marker is followed successively by 80bp, 102bp, 174bp, 257bp, 267bp, 296bp, 434bp, 458bp, 587bp from left to right.
Embodiment
The present invention has announced and has been used for the primer that transgenic corns multiplex PCR-DHPLC detects, and this primer designs for the distinguished sequence of transgenic corns strain.Comprise primer to 1, primer to 2, primer to 3, primer to 4, primer to 5, primer to 6 and primer in 7 at least one.Further, also primer to 3, primer to 4 primers to 5 and primer to 6 upstream and downstream added respectively one section with detect the regulating and controlling sequence that target sequence is irrelevant or homology is far, be used for realizing the regulation and control to the clip size of amplified production.It is pointed out that the adding of regulating and controlling sequence just in order to obtain more excellent detection effect, therefore, the present invention is preferred, and primer is added regulating and controlling sequence to 3-6, has respectively sequence shown in Seq ID No.17 to the Seq ID No.24.
The invention also discloses a kind ofly for transgenic corns multiplex PCR-DHPLC detection method, described detection method comprises and adopts described primer, carries out pcr amplification take maize dna as template, and pcr amplification product is carried out dhplc analysis.Further, described detection method also comprises carries out dhplc analysis take marker as reference standard, the dhplc analysis result of pcr amplification product and the dhplc analysis result of marker are compared, judge the size of pcr amplification product according to comparison result, thereby judge whether to contain the target sequence of detection.Among the present invention, primer is 141bp to the DHPLC elution peak of 1 amplified production, primer is 164bp to the DHPLC elution peak of 2 amplified productions, primer is 220bp to the DHPLC elution peak of 3 amplified productions, primer is 241bp to the DHPLC elution peak of 4 amplified productions, primer is 267bp to the DHPLC elution peak of 5 amplified productions, and primer is 283bp to the DHPLC elution peak of 6 amplified productions, and primer is 196bp to the DHPLC elution peak of 7 amplified productions.
Also by reference to the accompanying drawings the present invention is described in further detail below by concrete experimental example.Following experimental example only is further detailed the present invention, should not be construed as limitation of the present invention.
Embodiment 1 DNA extraction
Adopt the CTAB method to extract sample DNA, specific as follows:
A) take by weighing sample 5g, in mortar, add the powder that liquid nitrogen grinding to sample is size about 0.5mm;
B) take by weighing the sample that 300mg grinds, change over to rapidly in the 2mL centrifuge tube, add the CTAB extracting solution 700 μ L of 65 ℃ of preheatings, mixing is put into 65 ℃ of water-bath water-bath 30min;
C) add 5 μ L RNase (10mg/mL), 37 ℃ of water-bath 30min;
D) add the saturated phenol of equal-volume Tris, abundant mixing, the centrifugal 15min of 12000r/min;
E) get supernatant, add isopyknic chloroform/primary isoamyl alcohol (24: 1) mixing, the centrifugal 15min of 12000r/min;
F) get supernatant, add isopyknic chloroform/primary isoamyl alcohol (24: 1) mixing, the centrifugal 15min of 12000r/min;
G) Virahol of adding equal-volume precooling is jiggled, and places-20 ℃ of refrigerators to leave standstill 30min, the centrifugal 15min of 12000r/min;
H) abandon supernatant, add 70% ethanol, 500 μ L, the centrifugal 3min of 12000r/min removes supernatant, repeats 2 times;
I) obtain the DNA precipitation, carry out drying with freeze drier, add 50 μ L～100 μ L TE or aseptic deionized waters, fully after the dissolving, purity and the concentration of measuring DNA are placed in-20 ℃ of refrigerators preserves.
Embodiment 2 DNA concentration determinations
The sample DNA that extracts is carried out concentration and purity testing; Adopt ultraviolet spectrophotometer to measure 260nm and 280nm place absorption value, calculate respectively purity and the concentration of nucleic acid, calculation formula is as follows:
DNA purity=OD260/OD280
DNA concentration=50 * OD260mg/mL
The purity ratio of DNA is between 1.7～1.9, and concentration is greater than 10ng/ μ L.
Embodiment 3 pcr amplifications
According to transgenic corns strain MON863, NK603, MON810, T25, MON88017, MIR604, the binding site of 59122 design specific detection primers, and at MON810, T25, MON88017,5 ' end of the specific detection primer binding site of MIR604 strain adds regulating and controlling sequence, the synthetic detection primer (table 1) that contains regulating and controlling sequence, synthetic primer is to 1 up/down trip primer specificity binding site, primer is to 2 up/down trip primer specificity binding site, primer is swum primer to the primer of 7 up/down trip primer specificity binding site and interpolation regulating and controlling sequence to 3 up/down, add the primer of regulating and controlling sequence to 4 up/down trip primer, add the primer of regulating and controlling sequence to 5 up/down trip primer, the primer of interpolation regulating and controlling sequence carries out pcr amplification to 6 up/down trip primer as primer, the setting of PCR sample comprises, the biased sample of above-mentioned 7 transgenic corns strain DNA and the DNA of non-transgenic corn.
Table 1 transgenic corns detects primer binding site and primer
PCR reaction system cumulative volume is 50 μ L, each composition is respectively: multi-PRC reaction mixed solution Multiplex PCR Mix (TaKaRa) 25 μ L, each 1 μ L of 10 μ mol/L primers, DNA 2 μ L, 5U/ μ L Taq enzyme 0.25 μ L, supplying with the sterilization distilled water is 50 μ L.
PCR reaction conditions: 94 ℃ of sex change 1min; Then enter 35 circulations, 94 ℃ of 30s, 57 ℃ of 1min, 72 ℃ of 1min; 72 ℃ are extended 10min after the loop ends.
Embodiment 4 DHPLC detect
The PCR product is placed on the automatic sampling platform of DHPLC; Open DHPLC control software Navigator, in detection method, select Multiplex, i.e. multi-disc piecewise analysis, setting simultaneously upper limit of detection is 600bp, the lower 70bp that is limited to; Move two blank, i.e. empty needle, balance chromatographic column; Move a marker, be used for reference to contrast, the nucleic acid fragment of test sample size; Successively sample to be tested is analyzed, partial results is seen Fig. 1.
The result judges: observe the elution peak that DHPLC obtains, by comparing with marker and WAVE 4500 automatic analysis are determined the dna fragmentation size of elution peak position, judge accordingly.
Elution peak 141bp is primer to 1 amplified production, elution peak 164bp is primer to 2 amplified production, elution peak 220bp is primer to 3 amplified production, elution peak 241bp is primer to 4 amplified production, elution peak 267bp is primer to 5 amplified production, elution peak 283bp is primer to 6 amplified production, and elution peak 196bp is primer to 7 amplified production.To mixing the sample of transgenic corns strain DNA, its detected result demonstration contains above-mentioned 7 elution peaks; But not the transgenic corns sample is without elution peak.
Above content is the further description of the present invention being done in conjunction with concrete embodiment, can not assert that implementation of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (6)
1. be used for the primer that transgenic corns multiplex PCR-DHPLC detects, it is characterized in that: described primer comprise primer to 1, primer to 2, primer to 3, primer to 4, primer to 5, primer to 6 and primer at least one pair of primer in 7;
Primer contains sequence shown in the Seq ID No.1 to 1 upstream primer, and downstream primer contains sequence shown in the Seq ID No.2;
Primer contains sequence shown in the Seq ID No.3 to 2 upstream primer, and downstream primer contains sequence shown in the Seq ID No.4;
Primer contains sequence shown in the Seq ID No.5 to 3 upstream primer, and downstream primer contains sequence shown in the Seq ID No.6;
Primer contains sequence shown in the Seq ID No.7 to 4 upstream primer, and downstream primer contains sequence shown in the Seq ID No.8;
Primer contains sequence shown in the Seq ID No.9 to 5 upstream primer, and downstream primer contains sequence shown in the Seq ID No.10;
Primer contains sequence shown in the Seq ID No.11 to 6 upstream primer, and downstream primer contains sequence shown in the Seq ID No.12;
Primer contains sequence shown in the Seq ID No.13 to 7 upstream primer, and downstream primer contains sequence shown in the Seq ID No.14;
Seq?ID?No.1：5’-TTATTTTGGACTATCCCGACTCTC-3’
Seq?ID?No.2：5’-CTCAGCGGCGGAGCTACAGAGAGATAACAGGATCCACTCAAACAC-3’
Seq?ID?No.3：5’-AACTATTGACCCTACTTGTTCGGAT-3’
Seq?ID?No.4：5’-TCGGCAGAGGCATCTTGAAT-3’
Seq?ID?No.5：5’-CGTCAACGTGCCCGGTACT-3’
Seq?ID?No.6：5’-AAAGGACCTGACTGCTCGCA-3’
Seq?ID?No.7：5’-GGCAGCTACGACATGATACTCCT-3’
Seq?ID?No.8：5’-CCGAGGAGGTTTCCGGATATTA-3’
Seq?ID?No.9：5’-TTTTCTGTACTTGTGTAATCGGCTAA-3’
Seq?ID?No.10：5’-AGTTGACCATCCAAACCCGA-3’
Seq?ID?No.11：5’-TCTGCGCACGCAATTCAAC-3’
Seq?ID?No.12：5’-CGTTGTGGTCTCCATCCTCTTAC-3’
Seq?ID?No.13：5’-AAGCGAACGATTCAGATGGC-3’
Seq?ID?No.14：5’-CGTTTCCCGCCTTCAGTTTA-3’。
2. primer according to claim 1, it is characterized in that: described primer to 3, primer to 4, primer to 5 and primer to 6,5 ' end of its upstream primer also comprises sequence shown in the Seq ID No.15, and 5 ' end of its downstream primer also comprises sequence shown in the Seq ID No.16;
Seq?ID?No.15：5’-CGTGGCCTCGCGATCTGACT-3’
Seq?ID?No.16：5’-CTCAGCGGCGGAGCTACAGA-3’。
3. primer according to claim 2 is characterized in that:
Described primer has sequence shown in the Seq ID No.17 to 3 upstream primer, and downstream primer has sequence shown in the Seq ID No.18;
Primer has sequence shown in the Seq ID No.19 to 4 upstream primer, and downstream primer has sequence shown in the Seq ID No.20;
Primer has sequence shown in the Seq ID No.21 to 5 upstream primer, and downstream primer has sequence shown in the Seq ID No.22;
Primer has sequence shown in the Seq ID No.23 to 6 upstream primer, and downstream primer has sequence shown in the Seq ID No.24;
Seq?ID?No.17：5’-CGTGGCCTCGCGATCTGACTCGTCAACGTGCCCGGTACT-3’
Seq?ID?No.18：5’-CTCAGCGGCGGAGCTACAGAAAAGGACCTGACTGCTCGCA-3’
Seq?ID?No.19：5’-CGTGGCCTCGCGATCTGACTGGCAGCTACGACATGATACTCCT-3’
Seq?ID?No.20：5’-CTCAGCGGCGGAGCTACAGACCGAGGAGGTTTCCGGATATTA-3’
Seq?ID?No.21：5’-CGTGGCCTCGCGATCTGACTTTTTCTGTACTTGTGTAATCGGCTAA-3’
Seq?ID?No.22：5’-CTCAGCGGCGGAGCTACAGAAGTTGACCATCCAAACCCGA-3’
Seq?ID?No.23：5’-CGTGGCCTCGCGATCTGACTTCTGCGCACGCAATTCAAC-3’
Seq?ID?No.24：5’-CTCAGCGGCGGAGCTACAGACGTTGTGGTCTCCATCCTCTTAC-3’。
4. each described primer according to claim 1-3 is characterized in that:
Described primer is the primer that detects transgenic corns strain NK603 to 1,
Primer is the primers that detect transgenic corns strain MON863 to 2,
Primer is the primers that detect transgenic corns strain MON810 to 3,
Primer is the primers that detect transgenic corns strain T25 to 4,
Primer is the primers that detect transgenic corns strain MON88017 to 5,
Primer is the primers that detect transgenic corns strain MIR604 to 6,
Primer is to 7 primers for detection transgenic corns strain 59122.
5. one kind is used for transgenic corns multiplex PCR-DHPLC detection method, it is characterized in that: described detection method comprises each described primer of employing claim 1-4, carry out pcr amplification take maize dna as template, and pcr amplification product is carried out dhplc analysis.
6. detection method according to claim 5, it is characterized in that: described detection method also comprises take marker as reference standard carries out dhplc analysis, and the dhplc analysis result of pcr amplification product and the dhplc analysis result of marker are compared.
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| CN104195260A (en) * | 2014-09-22 | 2014-12-10 | 天津出入境检验检疫局动植物与食品检测中心 | Novel genetically modified corn strain MIR604 detection kit and detection method of kit |
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| CN104195260A (en) * | 2014-09-22 | 2014-12-10 | 天津出入境检验检疫局动植物与食品检测中心 | Novel genetically modified corn strain MIR604 detection kit and detection method of kit |
| CN104195260B (en) * | 2014-09-22 | 2015-12-30 | 天津出入境检验检疫局动植物与食品检测中心 | A kind of new Transgenic corn lines MIR604 detection kit and detection method thereof |
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