CN103614476A - Constant-temperature detection method and detection kit for transgenic ingredient CaMV35S in edible oil - Google Patents
Constant-temperature detection method and detection kit for transgenic ingredient CaMV35S in edible oil Download PDFInfo
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Abstract
The invention discloses a constant-temperature detection method and a detection kit for a transgenic ingredient CaMV35S in edible oil. According to the method, a nucleic acid component can be stably extracted from plant oil, and the transgenic ingredient can be detected within short time by a constant-temperature amplification method. The method comprises the following steps of (1) extracting a DNA (deoxyribonucleic acid) of a sample to be detected; (2) performing constant-temperature amplification reaction on the sample to be detected under a condition that a primer and BstDNA polymerase exist; and (3) judging whether the transgenic ingredient CaMV35S exists in the sample to be detected according to an amplification result. The method disclosed by the invention has the characteristics of quickness, high efficiency, simplicity in operation, high specificity, high sensitivity, simplicity in authentication, suitability for on-site detection and the like, and the transgenic ingredient CaMV35S in the edible oil can be quickly detected.
Description
Technical field
The invention belongs to biomolecule detection technical field, be specifically related to Constant Temperature Detection method and the detection kit of transgene component CaMV35S in a kind of edible oil.
Background technology
Since 20 century 70 genetically modified organisms and transgenic product are developed, transgenic product development is swift and violent.Up to the present, having had the transgenic plant more than surpassing 100 kinds to be planted by commercialization, is wherein soybean, rape and the corn processing raw material as edible oil the most widely.Because these transgenosis oil plant crop oil yields are high, the numerous and confused import genetically engineered soybean of liquefaction cost Di，Ge developing country and rape be as liquefaction raw material.But owing to still lacking at present the security of scientific basis proof genetically modified food, thereby also more and more urgent to the demand of genetically modified food detection technique.In recent years, in the liquefaction raw material of Chinese import, there are a considerable amount of transgenic product, genetically engineered soybean especially, the soybean of Chinese import 100% is almost genetically engineered soybean.Due to had a lot of national regulations in the world genetically modified food be must labelling or import prohibition, therefore, food oils are carried out to the detection of transgene component, seem particularly important.How differentiating whether food oils are genetically modified food, except detecting raw material, is more from finished product edible oil, directly to detect.
Cauliflower mosaic virus CaMV35S promotor is the constitutive promoter the most often using, and it has multiple cis-acting elements, can nearly all etap of various plants species and institute in a organized way in high efficient expression, be widely used in the structure of transfer-gen plant.At present on the market common corn, cotton, soybean, etc. in transgenic plant, approximately have 85%-90% transgenic plant to use this promotor.Although CaMV35S promotor exists a plurality of versions at present, its conserved sequence is relatively stable, is one of best target gene of transgenosis detection.And from food oils, extract the DNA that can be used for detection of nucleic acids, be the key of testing.But the DNA extraction of edible oil is still one of difficult problem both domestic and external, due to treating processess such as process high temperature and high pressures in the edible fat production course of processing, DNA in product is seriously damaged, be degraded to length shorter, fragment not of uniform size, and being subject to the impact of physics, chemistry and the factor such as biological, the quality of DNA also reduces greatly.At present, domestic and international still more stable edible oil DNA extraction method or the test kit of neither one.A kind of method that can stablize extraction nucleic acid from edible oil is set up in this research, and sets up a kind of method of Constant Temperature Detection CaMV35S promotor.
Summary of the invention
One object of the present invention is to provide the Constant Temperature Detection method of transgene component CaMV35S in a kind of rapid detection edible oil.
Another object of the present invention is to provide a kind of LAMP test kit for detection of transgene component CaMV35S in edible oil.
Another object of the present invention is to provide a kind of LAMP primer sets for detection of transgene component CaMV35S in edible oil.
The technical solution used in the present invention is:
In edible oil, a Constant Temperature Detection method of transgene component CaMV35S, comprises the steps:
1) according to CaMV35S promoter gene sequences Design specificity constant-temperature amplification, detect primer;
2) extract sample DNA to be checked;
3) sample DNA to be checked above-mentioned primer,
bstunder the condition that DNA polysaccharase exists, carry out isothermal amplification reactions;
4) according to amplification, judge in sample to be checked, whether there is CaMV35S transgene component.
The sequence of described constant-temperature amplification detection primer is as follows respectively:
35S- F3：GGTGGCTCCTACAAATGC（SEQ ID NO:1），
35S- B3：GTCTTGCGAAGGATAGTGG（SEQ ID NO:2），
35S-FIP：GTCCATCTTTGGGACCACTGTCCATCATTGCGATAAAGGAAAGG（SEQ ID NO:3），
35S-BIP： CACGAGGAGCATCGTGGAAACGTCAGTGGAGATATCACATC（SEQ ID NO:4），
35S-FLP：AGAGGCATCTTCAACGATGG（SEQ ID NO:5），
35S-BLP：AGAAGACGTTCCAACCACG（SEQ ID NO:6）。
As preferably, step 2) concrete steps of extracting sample DNA to be checked are:
1) get certain sample oil, add the normal hexane of 1～3 times of volume and the TE of 0.1～0.3 times of volume, under 20～25 ℃ of envrionment conditionss, mix;
2), after stratification, remove upper oil phase;
3) the TE water of lower floor is transferred to centrifuge tube; Add successively 1～2 μ g salmon sperm dna, the Virahol of 0.5～0.7 times of volume precooling, the sodium acetate of 3 mol/L of 0.1～0.2 times of volume; Put upside down back and forth gently to mix and be placed on-20 ℃ of following precipitation DNA;
4) precipitation is centrifugal after spending the night, and abandons supernatant liquor, by 65～75% washing with alcohol precipitation of precooling, transfers to centrifuge tube, centrifugal;
5) empty dry DNA, adds TE dissolving DNA, is placed in-20 ℃ of following preservations.
As preferably, the system of isothermal amplification reactions is described in step 3): 25 μ L LAMP reaction systems contain 1.6 μ mol/L 35S-FIP, 1.6 μ mol/L 35S-BIP, 0.2 μ mol/L 35S-F3,0.2 μ mol/L 35S-B3,0.8 μ mol/L 35S-FLP, 0.8 μ mol/L 35S-BLP, 1 mol/L trimethyl-glycine, 8 mmol/L MgSO4,8 U Bst archaeal dna polymerases, 1.6 mmol/L dNTP, 2.5 μ L 10 * Thermo pol Buffer and DNA profiling to be measured 2 μ L.
As preferably, the program of isothermal amplification reactions is described in step 3): 60～65 ℃ of isothermal reaction 45～60min.
As preferably, step 4) can be according to constant temperature fluorescent method judgement detected result, in reaction system, add 2-20 μ M SYTO-9, use fluorescent PCR instrument (as ABI7500) or other constant-temperature fluorescence detector (Genie II) to detect, according to amplification curve judged result, there is the positive of " s " type amplification curve, negative without " s " type amplification curve.
For detection of a test kit of transgene component CaMV35S in edible oil, it comprises that 6 specificity constant-temperature amplifications according to CaMV35S promoter gene sequences Design detect primer.
The sequence of described constant-temperature amplification detection primer is as follows respectively:
35S- F3：GGTGGCTCCTACAAATGC（SEQ ID NO:1），
35S- B3：GTCTTGCGAAGGATAGTGG（SEQ ID NO:2），
35S-FIP：GTCCATCTTTGGGACCACTGTCCATCATTGCGATAAAGGAAAGG（SEQ ID NO:3），
35S-BIP： CACGAGGAGCATCGTGGAAACGTCAGTGGAGATATCACATC（SEQ ID NO:4），
35S-FLP：AGAGGCATCTTCAACGATGG（SEQ ID NO:5），
35S-BLP：AGAAGACGTTCCAACCACG（SEQ ID NO:6）。
In described test kit, also comprise following component:
bstarchaeal dna polymerase, MgSO
4, trimethyl-glycine, dNTP, 10 * Thermo pol Buffer, SYTO-9, positive control and negative control, described positive control is the recombinant plasmid that contains CaMV35S promoter gene sequence, described negative control product are ddH
2o.
For detection of a primer sets of transgene component CaMV35S in edible oil, its sequence is as follows respectively:
35S- F3：GGTGGCTCCTACAAATGC（SEQ ID NO:1），
35S- B3：GTCTTGCGAAGGATAGTGG（SEQ ID NO:2），
35S-FIP：GTCCATCTTTGGGACCACTGTCCATCATTGCGATAAAGGAAAGG（SEQ ID NO:3），
35S-BIP： CACGAGGAGCATCGTGGAAACGTCAGTGGAGATATCACATC（SEQ ID NO:4），
35S-FLP：AGAGGCATCTTCAACGATGG（SEQ ID NO:5），
35S-BLP：AGAAGACGTTCCAACCACG（SEQ ID NO:6）。
The invention has the beneficial effects as follows: in edible oil, whether containing transgenic rape is the difficult point detecting at present, wherein transgenosis nucleic acid component is to detect comparatively reliably target, but the series of problems such as the detection of nucleic acids rate that is faced with oily amplifying nucleic acid extraction difficulty height and low copy is low, the present invention has announced method and the test kit of CaMV35S promotor in a kind of detection edible oil, there is following characteristics and advantage: 1. set up a kind of method of extracting DNA in oil of stablizing, by adding normal hexane to carry out emulsification in extracting method and adding salmon sperm dna to improve the yield of oily amplifying nucleic acid, 2. adopt isothermal amplification technology, detection efficiency is high, at 63～65 ℃, nucleic acid is carried out to amplified reaction, in 45～60min, complete detection, 3. specificity is high, according to 8 isolated areas on 6 primer energy specific recognition target-gene sequences of CaMV35S promoter gene sequences Design, there is higher specificity, 4. highly sensitive, use a kind of strand displacement activity that has
bstarchaeal dna polymerase, under constant temperature, continual amplification target sequence, the minimum nucleic acid that 1 copy detected.The method has rapidly and efficiently, easy and simple to handle, high specific, highly sensitive, evaluation are easy, be applicable to the features such as Site Detection, the transgene component CaMV35S promotor in can rapid detection edible oil.
figure of description
Fig. 1 is the amplification figure of LAMP optimum response system of the present invention to positive and negative sample;
Fig. 2 is the specific detection result figure of the inventive method;
Fig. 3 is that the method for the reason embodiment of the present invention 1 is extracted the result figure that carries out LAMP reaction after vegetables oil DNA;
Fig. 4 is for adopting conventional commercially available reagent box to extract the result figure that carries out LAMP reaction after vegetables oil DNA.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited to this.
the extraction of DNA in embodiment 1 edible oil
The extracting method of the edible vegetable oil genomic dna that the present invention researches and develops, step is as follows:
1) get 100 mL oil, join in 500 mL triangular flasks; Then add 200 mL normal hexanes and 20 mL TE, under 20～25 ℃ of envrionment conditionss, 120 rpm vibrations mix 3 h;
2) finally the TE water of lower floor (approximately 15 mL) is transferred to 50 mL centrifuge tubes, add successively 1-2 μ g salmon sperm dna (sigma company), the Virahol of 0.6 times of volume precooling, the sodium acetate of 3 mol/L of 1/10 volume; Put upside down back and forth gently to mix and be placed on-20 ℃ of precipitation DNA;
3) precipitation spend the night after centrifugal (12000 * g, 10 min, 4 ℃); Carefully abandon supernatant liquor;
4) 70% of use precooling washing with alcohol precipitation, transfers to 1.5 mL centrifuge tubes, centrifugal (12000 * g, 10 min, 4 ℃);
5) sky is done DNA, adds the TE dissolving DNA of 50 μ L; DNA is placed in-20 ℃ of preservations.
According to CaMV35S promoter gene sequence (GenBank:GU734659.1), design respectively 35S-F3,35S-B3,35S-FIP and tetra-primers of 35S-BIP, designed two ring primers of 35S-FLP and 35S-BLP to shorten detection time simultaneously.Amplification target sequence size is 210 bp.Article 6, primer sequence is as follows respectively:
35S- F3：GGTGGCTCCTACAAATGC（SEQ ID NO:1），
35S- B3：GTCTTGCGAAGGATAGTGG（SEQ ID NO:2），
35S-FIP：GTCCATCTTTGGGACCACTGTCCATCATTGCGATAAAGGAAAGG（SEQ ID NO:3），
35S-BIP： CACGAGGAGCATCGTGGAAACGTCAGTGGAGATATCACATC（SEQ ID NO:4），
35S-FLP：AGAGGCATCTTCAACGATGG（SEQ ID NO:5），
35S-BLP：AGAAGACGTTCCAACCACG（SEQ ID NO:6）。
the optimization of embodiment 3 LAMP detection system
The inventive method is from Mg
2+concentration, trimethyl-glycine concentration, dNTPs concentration, FIP/BIP primer concentration and 5 variable parameters of temperature of reaction, be optimized LAMP reaction conditions by the following method:
mg
2+concentration optimization: 4 Mg are set
2+concentration gradient is followed successively by 4mM, 6mM, 8mM, 10mM;
trimethyl-glycine concentration optimization: 4 trimethyl-glycine concentration gradients are set and are followed successively by 0.8M, 1.0M, 1.2M, 1.6M;
When reaction is carried out, adopt the appearance time of response curve as the index of evaluating, above-mentioned parameter is optimized to research, the final definite LAMP reaction conditions of this research is:
25 μ L LAMP reaction systems contain 1.6 μ mol/L FIP, 1.6 μ mol/L BIP, 0.2 μ mol/L F3,0.2 μ mol/L B3,0.8 μ mol/L FLP, 0.8 μ mol/L BLP, 1 mol/L trimethyl-glycine, 8 mmol/L MgSO
4, 8 U
bstarchaeal dna polymerase, 1.6 mmol/L dNTP, 2.5 μ L 10 * Thermo pol Buffer and DNA profiling 2 μ L.Reaction conditions: 63 ℃, react 1 h.
By above-mentioned optimization postcondition, detect, reaction result is shown in Fig. 1, and experimental result shows: use the optimum response system of setting up can obviously distinguish yin and yang attribute result, 60min is interior without non-specific amplification.
embodiment 4 specificity experiments
Adopt CTAB method to take off and state sample DNA, adopt the best LAMP detection system of embodiment 3 to detect.
Detected result is shown in Fig. 2.Experimental result shows: the sample copy patented method that contains CaMV35S gene No. 1-5 all can detect, No. 6-14 is not all negative containing the transgenosis sample detection result of CaMV35S gene, 15-22 non-transgenic sample detection result is all negative, shows this detection method high specificity.
Genetically engineered soybean oil containing CaMV35S gene element and non-transgenic soybean oil are mixed into CaMV35S gene element content by weight and are respectively 10%, 5%, 1%, 0.5%, 0.1%, 0.05% positive analog sample, adopts respectively the DNA extraction method of the embodiment of the present invention 1 and conventional business DNA extraction test kit to extract respectively, then adopts the LAMP detection system of embodiment 3 to detect.
The extracting method of the edible vegetable oil genomic dna that the present invention researches and develops, step is as follows:
DNA extraction method detected result of the present invention is shown in Fig. 3, conventional business DNA extraction test kit detected result is shown in Fig. 4, experimental result shows: adopt extracting method minimum detectability of the present invention can reach 0.5%, adopt conventional business DNA extraction test kit minimum detectability can reach 5%, the nucleic acid better effects if that method for extracting nucleic acid of the present invention extracts is described, illustrate that extracting method of the present invention mates use with detection kit, minimum detectability can arrive 0.5% simultaneously.
Above embodiment is only for introducing preferred case of the present invention, and to those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention, all should be regarded as a part of the present invention.
Di Ao bio tech ltd, <110> Guangzhou
Constant Temperature Detection method and the detection kit of transgene component CaMV35S in <120> edible oil
<130>
<160> 6
<170> PatentIn version 3.5
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ggtggctcct acaaatgc 18
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Claims (10)
1. a Constant Temperature Detection method of transgene component CaMV35S in edible oil, comprises the steps:
1) according to CaMV35S promoter gene sequences Design specificity constant-temperature amplification, detect primer;
2) extract sample DNA to be checked;
3) sample DNA to be checked above-mentioned primer,
bstunder the condition that DNA polysaccharase exists, carry out isothermal amplification reactions;
4) according to amplification, judge in sample to be checked, whether there is CaMV35S transgene component.
2. method according to claim 1, is characterized in that, the sequence of described constant-temperature amplification detection primer is as follows respectively:
35S- F3：GGTGGCTCCTACAAATGC（SEQ ID NO:1），
35S- B3：GTCTTGCGAAGGATAGTGG（SEQ ID NO:2），
35S-FIP：GTCCATCTTTGGGACCACTGTCCATCATTGCGATAAAGGAAAGG（SEQ ID NO:3），
35S-BIP： CACGAGGAGCATCGTGGAAACGTCAGTGGAGATATCACATC（SEQ ID NO:4），
35S-FLP：AGAGGCATCTTCAACGATGG（SEQ ID NO:5），
35S-BLP：AGAAGACGTTCCAACCACG（SEQ ID NO:6）。
3. method according to claim 1, is characterized in that step 2) concrete steps of extracting sample DNA to be checked are:
1) get certain sample oil, add the normal hexane of 1～3 times of volume and the TE of 0.1～0.3 times of volume, under 20～25 ℃ of envrionment conditionss, mix;
2), after stratification, remove upper oil phase;
3) the TE water of lower floor is transferred to centrifuge tube; Add successively 1～2 μ g salmon sperm dna, the Virahol of 0.5～0.7 times of volume precooling, the sodium acetate of 3 mol/L of 0.1～0.2 times of volume; Put upside down back and forth gently to mix and be placed on-20 ℃ of following precipitation DNA;
4) precipitation is centrifugal after spending the night, and abandons supernatant liquor, by 65～75% washing with alcohol precipitation of precooling, transfers to centrifuge tube, centrifugal;
5) empty dry DNA, adds TE dissolving DNA, is placed in-20 ℃ of following preservations.
4. method according to claim 1, it is characterized in that, the system of isothermal amplification reactions is described in step 3): 25 μ L LAMP reaction systems contain 1.6 μ mol/L 35S-FIP, 1.6 μ mol/L 35S-BIP, 0.2 μ mol/L 35S-F3,0.2 μ mol/L 35S-B3,0.8 μ mol/L 35S-FLP, 0.8 μ mol/L 35S-BLP, 1 mol/L trimethyl-glycine, 8 mmol/L MgSO4,8 U Bst archaeal dna polymerases, 1.6 mmol/L dNTP, 2.5 μ L 10 * Thermo pol Buffer and DNA profiling to be measured 2 μ L.
5. method according to claim 1, is characterized in that, the program of isothermal amplification reactions is described in step 3): 60～65 ℃ of isothermal reaction 45～60min.
6. according to the method described in claim 1～5 any one, it is characterized in that, step 4) can be according to constant temperature fluorescent method judgement detected result, in reaction system, add 2-20 μ M SYTO-9, use fluorescent PCR instrument (as ABI7500) or other constant-temperature fluorescence detector (Genie II) to detect, according to amplification curve judged result, there is the positive of " s " type amplification curve, negative without " s " type amplification curve.
7. for detection of a test kit of transgene component CaMV35S in edible oil, it comprises that 6 specificity constant-temperature amplifications according to CaMV35S promoter gene sequences Design detect primer.
8. test kit according to claim 1, is characterized in that, the sequence of described constant-temperature amplification detection primer is as follows respectively:
35S- F3：GGTGGCTCCTACAAATGC（SEQ ID NO:1），
35S- B3：GTCTTGCGAAGGATAGTGG（SEQ ID NO:2），
35S-FIP：GTCCATCTTTGGGACCACTGTCCATCATTGCGATAAAGGAAAGG（SEQ ID NO:3），
35S-BIP： CACGAGGAGCATCGTGGAAACGTCAGTGGAGATATCACATC（SEQ ID NO:4），
35S-FLP：AGAGGCATCTTCAACGATGG（SEQ ID NO:5），
35S-BLP：AGAAGACGTTCCAACCACG（SEQ ID NO:6）。
9. according to the test kit described in claim 7 or 8, it is characterized in that, in described test kit, also comprise following component:
bstarchaeal dna polymerase, MgSO
4, trimethyl-glycine, dNTP, 10 * Thermo pol Buffer, SYTO-9, positive control and negative control, described positive control is the recombinant plasmid that contains CaMV35S promoter gene sequence, described negative control product are ddH
2o.
10. for detection of a primer sets of transgene component CaMV35S in edible oil, its sequence is as follows respectively:
35S- F3：GGTGGCTCCTACAAATGC（SEQ ID NO:1），
35S- B3：GTCTTGCGAAGGATAGTGG（SEQ ID NO:2），
35S-FIP：GTCCATCTTTGGGACCACTGTCCATCATTGCGATAAAGGAAAGG（SEQ ID NO:3），
35S-BIP： CACGAGGAGCATCGTGGAAACGTCAGTGGAGATATCACATC（SEQ ID NO:4），
35S-FLP：AGAGGCATCTTCAACGATGG（SEQ ID NO:5），
35S-BLP：AGAAGACGTTCCAACCACG（SEQ ID NO:6）。
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| CN106480181A (en) * | 2016-09-30 | 2017-03-08 | 许昌学院 | For detecting LAMP primer group and its method for transgene component CaMV35S |
| CN107794295A (en) * | 2017-11-09 | 2018-03-13 | 湖南工程学院 | A kind of blood coagulation enzyme assay method and kit that ring mediated isothermal amplification is opened based on double Aptamer interlayer structures |
| CN107794295B (en) * | 2017-11-09 | 2020-07-31 | 湖南工程学院 | Thrombin detection method and kit based on double Aptamer sandwich structure open loop-mediated isothermal amplification |
| CN111020053A (en) * | 2019-12-24 | 2020-04-17 | 广州迪澳生物科技有限公司 | Transgenic CAMV35S constant-temperature fluorescence detection primer group capable of avoiding false negative and kit thereof |
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| CN103614476B (en) | 2016-01-06 |
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Inventor after: Tan Guiliang Inventor after: Shi Lei Inventor after: Tang Dayun Inventor after: Zhang Huang Inventor after: Liu Meixian Inventor after: Fan Yaosen Inventor after: Luo Kangjie Inventor after: Ye Lei Inventor before: Shi Lei Inventor before: Tang Dayun Inventor before: Zhang Huang Inventor before: Liu Meixian Inventor before: Fan Yaosen Inventor before: Luo Kangjie Inventor before: Ye Lei |
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Effective date of registration: 20160119 Address after: 510663 Guangdong City, Guangzhou province high tech Industrial Development Zone, Science City Road, No. 182, C2 District, unit thirteenth, unit 1303 Patentee after: Guangzhou Deaou Biotechnology Co., Ltd. Patentee after: Tan Guiliang Address before: 510663 Guangdong City, Guangzhou province high tech Industrial Development Zone, Science City Road, No. 182, C2 District, unit thirteenth, unit 1303 Patentee before: Guangzhou Deaou Biotechnology Co., Ltd. |