CN103361409A

CN103361409A - Nucleic acid quantitative detection kit for transgenic rice TT51-1

Info

Publication number: CN103361409A
Application number: CN2012101038115A
Authority: CN
Inventors: 王晶; 隋志伟; 李亮; 余笑波; 臧超; 董莲华
Original assignee: National Institute of Metrology
Current assignee: National Institute of Metrology
Priority date: 2012-04-10
Filing date: 2012-04-10
Publication date: 2013-10-23

Abstract

The invention relates to a nucleic acid quantitative detection method and a detection kit for transgenic rice TT51-1. According to the method and the kit disclosed by the invention, the best endogenous reference gene RBE4 which has an optimal effect and is suitable for the detection of the transgenic rice is screened out; through optimization of a real-time fluorescent quantitative PCR (polymerase chain reaction) system, a PCR system for optimal quantitative detection of the transgenic rice TT51-1 or a product thereof is found. According to the method, the precise content of the transgenic component in the transgenic rice TT51-1 or the product thereof can be rapidly and correctly obtained.

Description

Transgenic paddy rice TT51-1 nucleic acid quantitative determination reagent kit

Technical field:

The present invention relates to a kind of transgenic paddy rice TT51-1 nucleic acid quantification detection method and detection kit.

Background technology:

Genetically modified crops in commercialization plantation and use develop rapidly in, brought challenge for the security control of genetically modified crops.The transgenic product composition detection is the important content of genetically modified organism security control and sign.

Transgenic paddy rice TT51-1 (BT63) is a kind of insect-proof rice, and the Ministry of Agriculture has provided its production application safety certificate in Hubei Province in August, 2009.The transgenic detection method of relevant TT51-1 has following report:

The qualitative checking method to transgenic paddy rice TT51-1 reported in the article " Characterization of the transgenic rice event TT51-1 and construction of a reference plasmid " that Cao Y. etc. is published on the J Agric Food Chem magazine.Article " the Evaluation of four genes in rice for their suitability as endogenous reference standards in quantitative PCR " report that Wang C etc. is published on the J Agric Food Chem magazine is used for the internal standard gene that TT51-1 detects with the SPS gene.More than two kinds of methods all can not carry out accurate quantification to transgenic paddy rice TT51-1 and detect.

For the result that guarantees the genetically modified crops quantitative detecting method accurately and reliably, also need in detection method, use relevant criterion product and quality control product.But up to the present also not yet see for the content of the precise and quantitative detection method of transgenic paddy rice TT51-1.

Summary of the invention:

The purpose of this invention is to provide method and detection kit that a kind of accurate quantification detects transgenic paddy rice TT51-1 and goods thereof, and highly sensitive, high specificity, accuracy are good, tolerance range is high.

The present invention has set up the method that a kind of accurate quantification detects transgenic paddy rice TT51-1 and goods thereof first, the accurate quantification that detects in order to satisfy transgenic paddy rice TT51-1 requires the expanding effect of paddy rice internal standard gene all identical with foreign gene TT51-1 with amplification efficiency.The inventor has carried out following work:

1. select suitable internal standard gene

After having synthesized respectively five kinds of paddy rice internal standard trans-genetic hybrid rice gene GOS9 commonly used, PLD, SPS, RBE4, UBQ5 primer, take the transgenic paddy rice TT51-1 that extracts as the basis, carry out respectively the pcr amplification of five internal standard genes and foreign gene TT51-1, PCR product with after the amplification carries out electrophoretic analysis.Found that the amplified production brightness of RBE4 gene near foreign gene TT51-1, all the other four internal standard gene products brightness are all dark.

Therefore, determined that the internal standard gene that transgenic paddy rice TT51-1 detects is RBE4.

Obtain being fit to the top condition of two gene amplifications

Primer and the probe of the synthetic internal standard gene RBE4 of reference and foreign gene TT51-1 carry out the PCR system optimization, obtain being fit to the top condition of two gene amplifications.

We have drawn best detection PCR system to being optimized on the proportioning system about five elements of this detection method of transgenosis in the prior art.

Checking detection side legal effect

The transgenic paddy rice TT51-1 genomic dna that extracts is carried out behind the serial dilution as template, carry out respectively the fluorescent quantitative PCR of internal standard gene RBE4 and foreign gene TT51-1, the RBE4 that increases of template of each dilution gradient concentration and the Ct value of TT51-1 gene amplification are compared, found that:

The Ct value ratio of TT51-1 and RBE4 all between 0.98 to 1, proves that RBE4 has good effect as internal standard gene in real-time fluorescence quantitative PCR detects.

The detectability of RBE4 and quantitative limit are respectively 5 copies and 15 copies, and the detection line of TT51-1 and quantitative limit are respectively 5 copies and 10 copies, prove that the detection sensitivity of two genes is all very high.

According to the detection method that the invention described above is set up, the present invention also provides a kind of transgenic paddy rice TT51-1 nucleic acid quantitative determination reagent kit, except the due reagent of the quantitative fluorescent PCR of routine, PCR enzyme premixed liquid, also contains following composition in the described test kit:

Upstream primer, downstream primer, the probe of internal standard gene RBE4; The upstream primer of foreign gene TT51-1, downstream primer, probe; Specifically see table 1 for details.

Quality control product (being the transgenic paddy rice TT51-1 seed powder reference material of content 1% and 5%)

Standard substance: be the TT51-1 that contains different concns and the plasmid molecule reference material of RBE4 gene fragment, quantity is 3～8;

Although accuracy of detection is directly proportional with standard substance quantity, testing cost also is directly proportional with standard substance quantity.The inventor the experiment proved that the standard substance preferred amount is 4～6

In example of the present invention, used the standard substance of 5 kinds of different concns (to contain respectively 10 ⁶, 10 ⁵, 10 ⁴, 10 ³, 10 ²The TT51-1 of copy/ml and RBE4 gene fragment).

Table 1 transgenic paddy rice TT51-1 detection by quantitative internal standard gene and foreign gene probe sequence

Annotate: FAM is the luminophore of probe in the table, and BHQ1 and MGBNFQ are the quenching group of probe.

Carry out the working method that transgenic paddy rice TT51-1 nucleic acid quantification detects with test kit of the present invention, it is characterized by:

1, accurately takes by weighing quality control product and testing sample;

Accurate for guaranteeing weighing, should use the balance through calibration; Usually, take by weighing each 100mg of quality control product and testing sample.

2, extract respectively the genomic dna of quality control product and testing sample, gained DNA purity should be A ₂₆₀1.8～2.0, and concentration is greater than 20ng/ μ L.

Wherein, extracting DNA can be by art methods, such as, can adopt Plant Genome to extract test kit Genomic DNA Purification Kit (U.S. Pu Luomaige company), extraction step operates according to the specification sheets of this product.

Measure DNA purity and can adopt art methods or product, as measuring with the PicoGreen test kit;

3, with the described RBE4 interior label primer of table 1 to probe and TT51 external source primer to being mixed with the working fluid that concentration is 10 μ M with probe with deionized water dissolving;

4, each standard substance, quality control product DNA and testing sample DNA are carried out respectively fluorescent quantitative PCR, described standard substance as claimed in claim 1;

5, carry out fluorescence quantitative PCR detection according to the programming of PCR shown in the table 3, can draw the typical curve of internal standard gene and foreign gene according to concentration shown in each standard substance and the Ct value that records, and the Ct value of the internal standard gene of testing sample DNA and quality control product DNA and foreign gene, calculate the internal standard gene of testing sample and quality control product and copy number and the ratio thereof of foreign gene according to typical curve, thereby draw the accurate content of transgenic paddy rice TT51-1 in testing sample and the quality control product, by using quality control product to monitor whole experiment flow and guaranteeing the accurate and reliable of experimental data.

In the step 4, according to shown in the table 2, the PCR system of preparation internal standard gene and foreign gene, dna profiling is respectively 5 standard substance, testing sample DNA and quality control product DNA; The quantitative fluorescent PCR response procedures is undertaken by table 3;

In the step 4, each sample is done three revision tests;

In the step 5, calculate respectively the copy number of foreign gene and internal standard gene according to the equation of the typical curve of foreign gene and internal standard gene, calculate at last the ratio of copy number of foreign gene/internal standard gene copy number, draw the detection by quantitative result.

Table 2 internal standard gene and foreign gene PCR system

The enzyme of PCR described in table premixed liquid can be selected commercially available product, such as Taqman gene expression PCR mastermix (Life Technology company) etc.

Table 3 quantitative fluorescent PCR response procedures

The genomic dna that detection method of the present invention is extracted test kit extraction transgenic paddy rice TT51-1 or its goods with Plant Genome is the basis, paddy rice internal standard gene RBE4 upstream and downstream primer and probe are used for the Ct value of detection by quantitative internal standard gene, foreign gene TT51-1 upstream and downstream primer and probe are used for detection by quantitative foreign gene Ct value, and carry out the typical curve that serial dilution sets up by transgenic paddy rice TT51-1 genomic dna and calculate respectively foreign gene and internal standard gene copy number, can be quick, obtain exactly the accurate content of transgene component in transgenic paddy rice TT51-1 or its goods.

But through the not only whether existence of transgenic paddy rice TT51-1 composition in the qualitative detection testing sample of experiment confirm present method, can also detection by quantitative go out the accurate content (seeing embodiment 1) of transgene component.

The present invention has following innovation and advantage:

1, by existing paddy rice internal standard gene commonly used is screened, the internal standard gene RBE4 that has therefrom found the best suitable transgenic paddy rice TT51-1 of effect to detect;

2, by the optimization to the real-time fluorescence quantitative PCR system, found the PCR system of suitable transgenic paddy rice TT51-1 or its goods detection by quantitative the best.

3, be accompanied with the reference material of production standard curve and quality control product in the detection kit, can eliminate the difference interfering data analysis between sample, guarantee to the full extent detection by quantitative tolerance range and accuracy.Break through the in the past low and large shortcoming of uncertainty of tolerance range of detection detection by quantitative on method of transgenic plant, can guarantee accuracy and the tractability of detected result.

4, highly sensitive

Minimum internal standard gene and the foreign gene that can surely detect 5 copies, and the internal standard gene of energy detection by quantitative to 15 copy and the foreign genes of 10 copies.

5, high specificity

Other representative insect-resistance paddy rice primers of home and overseas and the transgenic paddy rice primer (No. 1, Kemingdao, No. 2, Kemingdao, rich No. 6 of section, LLRICE601, LLRICE62) of anti-weeding have been chosen.Genomic dna with TT51-1 carries out pcr amplification as template, found that to only have the TT51-1 primer can amplify specific band.

6, good reproducibility

The experimental result susceptible of proof that provides from embodiment, take the matrix DNA of 5 gradient dilutions as sample, each sample carries out respectively repeated experiments 6 times, and group difference is without significance (p＜0.05) in the group.

Description of drawings:

Fig. 1 is the electrophorogram of five kinds of different candidate's internal standard gene products and foreign gene TT51-1 product;

Wherein, swimming lane M is that maximum molecular weight is the dna marker thing of 2000bp, and swimming lane 1 is external source primer TT51-1 product, and swimming lane 2-6 is five kinds of candidate's interior label primer products, respectively is GOS, PLD, RBE4, SPS, UBQ5;

Fig. 2 and Fig. 3 are respectively the typical curves (wherein the X-coordinate among the figure is the logarithm of the initial copy number of template (concentration), and ordinate zou is the Ct value) of foreign gene TT51-1 and internal standard gene RBE4;

Fig. 4 is the internal standard gene RBE4 typical curve of standard substance;

Fig. 5 is the foreign gene TT51-1 typical curve of standard substance.

Embodiment

Embodiment 1 transgenic paddy rice TT51-1 detects with the screening of internal standard gene and the optimization of quantitative fluorescent PCR reaction system

One, materials and methods

1.100% transgenic paddy rice TT51-1 rice paddy seed powder (from China National Measuring Science Research Inst.)

2. synthesize GOS9, PLD, SPS, RBE4 and the UBQ5 primer of totally 5 kinds of paddy rice internal standard genes and foreign gene TT51-1 (sequence information derives from Jeong S-C etc. and is published in Food Control magazine 18 volumes: the article of 1434-1442 page or leaf " Molecular analysis and quantitative detection of a transgenic rice line expressing a bifunctional fusion TPSP " and Wang C etc. are published in the article " Evaluation of four genes in rice for their suitability as endogenous reference standards in quantitative PCR " of J.Agric.Food.Chem.58 volume 11543-11547 page or leaf) see Table 4.

3. take the TT51-1DNA that extracts as template, use respectively 5 paddy rice internal standard gene primers and foreign gene TT51-1 primer to carry out the PCR reaction, the setting system is: TaqMan Universal PCR Master Mix 12.5ul, upstream primer (400nM) 1ul, downstream primer (400nM) 1ul, TT51-1 genomic dna 5ul, deionized water is supplied the 25ul system.PCR response procedures: 95 ℃ of the first steps 5 minutes; 95 ℃ of 40 of second steps circulations 15 seconds, 55 ℃ 30 seconds, 72 ℃ 60 seconds; The 3rd the step 72 ℃ 10 minutes.The PCR product carries out electrophoretic analysis (Fig. 1).

4. the synthetic internal standard gene of selecting and the probe (table 1) of foreign gene, the performing PCR system optimization of going forward side by side is according to the quantitative pcr amplification system: 95 ℃ of the first steps 5 minutes; 95 ℃ of 45 of second steps circulations 15 seconds, 60 ℃ 60 seconds; The 3rd the step 50 ℃ 10 minutes.Obtain being fit to the top condition of internal standard gene and foreign gene amplification.

5.PCR enzyme premixed liquid (Taqman gene expression PCR mastermix) is available from Life Technology company.

Table 4 transgenic paddy rice detects with internal standard gene and foreign gene primer sequence information

5. the transgenic paddy rice TT51-1 genomic dna that extracts is carried out behind the serial dilution as template, carry out respectively the fluorescent quantitative PCR of internal standard gene RBE4 and foreign gene TT51-1, the RBE4 that increases of template of each dilution gradient concentration and the Ct value of TT51-1 gene amplification are compared.Following table 5～7th, the data of selection internal standard gene and confirmatory experiment flow process stability.

Foreign gene Ct value during table 5 transgenic paddy rice TT51-1 different concns template

Internal standard gene C t value during table 6 transgenic paddy rice TT51-1 different concns template

According to the foreign gene of table 5, table 6 and the mean value of internal standard gene C t value, interior external source Ct value ratio relation in the time of can calculating respectively the various different concns template of transgenic paddy rice TT51-1 sees Table 7:

External source Ct value ratio in during table 7 transgenic paddy rice TT51-1 different concns template

Template (copy number/ul)	Interior external source Ct value ratio
		54398	0.99
27199	1.00
		5439.8	0.99
1087.96	0.99
		217.39	0.99
108.69	0.99
		43.48	0.98
21.74	0.98
		4.35	0.98

Two, experimental result

1. the screening of paddy rice internal standard gene

Take pure yang edema rice TT51-1 genome as template, carry out qualitative PCR amplification with five kinds of candidate's internal standard gene primers and foreign gene TT51-1 primer, its amplification compares with separately external source primer amplification result respectively.

Can find out that from electrophorogram five internal standard gene products and foreign gene TT51-1 and KMD2 product band are clear single, illustrate that their amplification all has good specificity.

As can be seen from Figure 1, the band brightness of the band brightness of RBE4 interior label primer amplified production and TT51-1 external source primer extension product is the most approaching.The RBE4 expanding effect that shows internal standard gene in the detection of transgenic paddy rice TT51-1 is best.

2. the optimization of quantitative fluorescent PCR reaction system

Adjust primer, probe and dna profiling usage quantity in the pcr amplification system, obtain optimized reaction system.

The reaction system of RBE4: PCR enzyme premixed liquid 12.5ul, upstream primer (200nM) 0.5ul, downstream primer (200nM) 0.5ul, probe (100nM) 0.25ul, TT51-1 genomic dna 5ul, deionized water is supplied the 25ul system.The reaction system of TT51-1: PCR enzyme premixed liquid 12.5ul, upstream primer (400nM) 1ul, downstream primer (400nM) 1ul, probe (200nM) 0.5ul, TT51-1 genomic dna 5ul, deionized water is supplied the 25ul system.

3. the drafting of typical curve and system stability

Extract good transgenic paddy rice genomic dna and measure its concentration with Picogreen, then carry out serial dilution according to gradient, do typical curve by real-time fluorescence quantitative PCR, verify whether internal standard gene and foreign gene have good linear relationship between template copy number and Ct value, and whether the internal standard gene is consistent with foreign gene Ct value when template concentrations is identical.Whether its quantitative system is appropriate to the detection by quantitative of sample.The Ct value that obtains according to real-time fluorescence quantitative PCR and the relation of corresponding templates concentration are drawn the typical curve (Fig. 2, Fig. 3) of two genes.

Experimental result shows: the internal standard gene RBE4 of transgenic paddy rice TT51-1 and foreign gene TT51-1 amplification curve effect are fine, and the R2 of its internal standard gene RBE4 typical curve is 0.9976, slope is-3.33, and its amplification efficiency is 99%;

Foreign gene amplification curve effect is also fine, and the R2 of typical curve is 0.9982, and slope is-3.24, and its amplification efficiency is 104%.

The result shows: the expanding effect of internal standard gene and foreign gene and having good stability is suitable for the detection by quantitative of transgenic paddy rice TT51-1.

The detection by quantitative of embodiment 2 paddy rice powder transfer gene TT51-1 compositions

One, materials and methods

1. testing sample is the transgenic paddy rice TT51-1 rice paddy seed powder reference material (China National Measuring Science Research Inst.'s development) of massfraction 2% content

2. with balance accurate weighing testing sample and quality control product (the transgenic paddy rice TT51-1 rice paddy seed powder reference material of massfraction 1% and 5% content)

3. use Plant Genome to extract test kit

Genomic DNA Purification Kit (U.S. Pu Luomaige company) extracts the genomic dna of testing sample and quality control product, and measures respectively purity and the concentration of the DNA that puies forward with ultraviolet spectrophotometer and Picogreen test kit.

4. standard substance (are contained and have an

appointment

10 ⁶, 10 ⁵, 10 ⁴, 10 ³, 10 ²The plasmid molecule reference material of the TT51-1 of copy/ml and RBE4 gene fragment) and testing sample DNA and quality control product DNA as template, carry out the preparation of PCR system according to table 1, then carry out respectively the fluorescent quantitative PCR of internal standard gene RBE4 and foreign gene TT51-1.

Two, experimental result

1. the Ct value of the internal standard gene of standard substance and testing sample and foreign gene sees Table 8 and table 9.

Table 8 standard substance are as the Ct value of internal standard gene and the foreign gene of typical curve

The Ct value of table 9 testing sample genomic dna behind pcr amplification

2. the detection by quantitative result of testing sample

By typical curve that standard substance are done testing sample is carried out quantitatively.The result shows that the concentration of testing sample can be measured the foreign gene content ratio in the target sample preferably in the linearity range of typical curve, the results are shown in Figure 4 and Fig. 5, and wherein the X-coordinate among the figure is the logarithm of template initial concentration, and ordinate zou is the Ct value.By table 7 and Fig. 4 as can be known, according to formula one: y=-3.4663x+28.287, the internal standard mrna concentration that can calculate testing sample is 19130copy/ul.By table 7 and Fig. 5 as can be known, according to formula two: y=-3.3934x+27.611, the foreign gene concentration that can calculate testing sample is 393copy/ul.By formula three: testing sample foreign gene content ratio=(foreign gene concentration/internal standard mrna concentration) * 100% as can be known, testing sample foreign gene content ratio is 2.05%.

Claims

1. a transgenic paddy rice TT51-1 nucleic acid quantitative determination reagent kit comprises quantitative fluorescent PCR reagent, PCR enzyme premixed liquid in the test kit, it is characterized in that also containing following composition:

Upstream primer, downstream primer, the probe of internal standard gene RBE4, sequence are respectively SEQ ID NO:1～SEQ ID NO:3

The upstream primer of foreign gene TT51-1, downstream primer, probe, sequence are respectively SEQ ID NO:4～SEQ ID NO:6;

Quality control product is the transgenic paddy rice TT51-1 seed powder reference material of content 1% and 5%;

Standard substance are the TT51-1 that contains different concns and the plasmid molecule reference material of RBE4 gene fragment, and quantity is 3～8.

2. test kit claimed in claim 1, the quantity of described standard substance is 4～6.

3. test kit claimed in claim 1, the quantity of described standard substance is 5, the concentration of TT51-1 and RBE4 gene fragment is respectively 10 ⁶, 10 ⁵, 10 ⁴, 10 ³, 10 ²Copy/ml.

4. transgenic paddy rice TT51-1 nucleic acid quantification detection method is characterized by:

1) accurately take by weighing quality control product and testing sample, described quality control product as claimed in claim 1;

2) extract respectively the genomic dna of quality control product and testing sample, gained DNA purity should be A ₂₆₀1.8～2.0, and concentration is greater than 20ng/ μ L;

3) respectively upstream primer, downstream primer, probe, the upstream primer of foreign gene TT51-1, downstream primer, the probe of internal standard gene RBE4 is mixed with the working fluid that concentration is 10 μ M with deionized water dissolving;

4) each standard substance, quality control product DNA and testing sample DNA are carried out respectively fluorescent quantitative PCR, described standard substance as claimed in claim 1;

5) carry out fluorescence quantitative PCR detection, according to concentration shown in the difference of standard substance and the Ct value drafting internal standard gene that records and the typical curve of foreign gene, by this typical curve testing sample is carried out quantitatively.

5. detection method claimed in claim 4, the quantity of described standard substance is 5, the concentration of TT51-1 and RBE4 gene fragment is 10 ⁶, 10 ⁵, 10 ⁴, 10 ³, 10 ²Copy/ml.

6. detection method claimed in claim 4, PCR reaction system used in the described fluorescent quantitative PCR is as follows:

The reaction system of RBE4: PCR enzyme premixed liquid 12.5ul, upstream primer (200nM) 0.5ul, downstream primer (200nM) 0.5ul, probe (100nM) 0.25ul, TT51-1 genomic dna 5ul, deionized water is supplied the 25ul system;

The reaction system of TT51-1: PCR enzyme premixed liquid 12.5ul, upstream primer (400nM) 1ul, downstream primer (400nM) 1ul, probe (200nM) 0.5ul, TT51-1 genomic dna 5ul, deionized water is supplied the 25ul system.

7. detection method claimed in claim 4, response procedures is in the described fluorescent quantitative PCR: 94 ℃ of the first steps 5 minutes; 94 ℃ of 45 of second steps circulations 15 seconds, 60 ℃ 60 seconds.

8. detection method claimed in claim 4, describedly by this typical curve testing sample is carried out quantitatively, it is the copy number that calculates respectively foreign gene and internal standard gene according to the equation of the typical curve of foreign gene and internal standard gene, calculate at last copy number of foreign gene/internal standard gene copy number, draw the detection by quantitative result.

9.RBE4 in transgenic paddy rice TT51-1 detection by quantitative as the application of internal standard gene.