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

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 Rice TT51-1 Nucleic Acid Quantitative Detection Kit

Technical field:

The invention relates to a quantitative detection method and a detection kit for transgenic rice TT51-1 nucleic acid.

Background technique:

The rapid development of commercial planting and application of genetically modified crops has brought challenges to the safety management of genetically modified crops. Component testing of genetically modified products is an important part of the safety management and labeling of genetically modified organisms.

The transgenic rice TT51-1 (BT63) is an insect-resistant rice, and the Ministry of Agriculture has issued a safety certificate for its production and application in Hubei Province in August 2009. There are the following reports about the transgenic detection method of TT51-1:

The article "Characterization of the transgenic riceevent TT51-1 and construction of a reference plasma" published in J Agric Food Chem by Cao Y. et al reported a qualitative detection method for transgenic rice TT51-1. The article "Evaluation of four genes in rice for their suitability as endogenous reference standards in quantitative PCR" published in J Agric Food Chem by Wang C et al. reported that the SPS gene was used as the internal standard gene in the detection of TT51-1. Neither of the above two methods could accurately and quantitatively detect the transgenic rice TT51-1.

In order to ensure the accuracy and reliability of the results of the quantitative detection method of genetically modified crops, it is also necessary to use relevant standards and quality control substances in the detection method. But so far, the content of the precise quantitative detection method for transgenic rice TT51-1 has not been seen yet.

Invention content:

The purpose of the present invention is to provide a method and a detection kit for the accurate quantitative detection of transgenic rice TT51-1 and its products, which have high sensitivity, strong specificity, good accuracy and high precision.

The present invention establishes a method for the accurate and quantitative detection of transgenic rice TT51-1 and its products for the first time. In order to meet the precise and quantitative detection of transgenic rice TT51-1, the amplification effect and amplification efficiency of the standard gene in rice are required to be the same as those of exogenous Gene TT51-1 is the same. The inventor has carried out following work:

①Select the appropriate internal standard gene

After synthesizing primers for five commonly used rice internal standard genes, rice genes GOS9, PLD, SPS, RBE4, and UBQ5, based on the extracted transgenic rice TT51-1, the five internal standard genes and the exogenous gene TT51-1 were respectively synthesized. For PCR amplification, the amplified PCR product was subjected to electrophoresis analysis. It was found that the brightness of the amplified product of RBE4 gene was the closest to that of the exogenous gene TT51-1, and the brightness of the other four internal standard gene products were all dark.

Therefore, the internal standard gene for the detection of transgenic rice TT51-1 was determined to be RBE4.

Optimal conditions for the amplification of two genes were obtained

References The primers and probes of the internal standard gene RBE4 and the exogenous gene TT51-1 were synthesized, and the PCR system was optimized to obtain the best conditions suitable for the amplification of the two genes.

We optimized the proportioning system of the five elements of the detection method for transgenes in the prior art, and obtained the best detection PCR system.

Validate the effectiveness of the detection method

The extracted transgenic rice TT51-1 genomic DNA was serially diluted and used as a template, and the fluorescence quantitative PCR amplification of the internal standard gene RBE4 and the exogenous gene TT51-1 were respectively performed, and the RBE4 amplified by each dilution gradient concentration template was Compared with the Ct value of TT51-1 gene amplification, it was found that:

The Ct ratios of TT51-1 and RBE4 were all between 0.98 and 1, which proved that RBE4, as an internal standard gene, had a good effect in real-time fluorescent quantitative PCR detection.

The detection limit and quantification limit of RBE4 were 5 copies and 15 copies, respectively, and the detection line and quantification limit of TT51-1 were 5 copies and 10 copies, respectively, which proved that the detection sensitivity of the two genes was very high.

According to the detection method established by the above-mentioned present invention, the present invention also provides a quantitative detection kit for transgenic rice TT51-1 nucleic acid. Contains the following ingredients:

The upstream primers, downstream primers, and probes of the internal standard gene RBE4; the upstream primers, downstream primers, and probes of the exogenous gene TT51-1; see Table 1 for details.

Quality control (1% and 5% transgenic rice TT51-1 seed powder standard substance)

Standard: 3-8 plasmid standard substances containing TT51-1 and RBE4 gene fragments at different concentrations;

Although detection accuracy is directly proportional to the number of standards, the cost of detection is also proportional to the number of standards. The inventor has proved through experiments that the preferred number of standard products is 4 to 6

In an example of the present invention, 5 different concentrations of standards (containing 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , and 10 ² copy/ml of TT51-1 and RBE4 gene fragments respectively) were used.

Table 1 Internal standard gene and exogenous gene probe sequences for quantitative detection of transgenic rice TT51-1

Note: In the table, FAM is the luminescent group of the probe, and BHQ1 and MGBNFQ are the quenching groups of the probe.

The operation method of carrying out the quantitative detection of transgenic rice TT51-1 nucleic acid with the kit of the present invention is characterized in that:

1. Accurately weigh quality control products and samples to be tested;

To ensure accurate weighing, a calibrated balance should be used; usually, weigh 100 mg each of the quality control and the sample to be tested.

2. Extract the genomic DNA of the quality control product and the sample to be tested respectively. The purity of the obtained DNA should be A ₂₆₀ 1.8-2.0, and the concentration should be greater than 20ng/μL.

Wherein, extraction DNA can be according to prior art method, for example, can adopt plant genome extraction kit Genomic DNA Purification Kit (Promega, USA), the extraction steps were performed according to the instructions of the product.

Determination of DNA purity can adopt prior art methods or products, such as measuring with PicoGreen kit;

3. The RBE4 internal standard primer pair and probe and the TT51 exogenous primer pair and probe described in Table 1 were dissolved in deionized water to prepare a working solution with a concentration of 10 μM;

4. Each standard product, quality control product DNA and sample DNA to be tested are subjected to fluorescent quantitative PCR amplification respectively, and said standard product is as described in claim 1;

5. Perform fluorescent quantitative PCR detection according to the PCR program settings shown in Table 3. According to the indicated concentration and measured Ct value of each standard, the standard curve of the internal standard gene and exogenous gene, and the DNA of the sample to be tested can be obtained. and the Ct value of the internal standard gene and exogenous gene of the DNA of the quality control product, and calculate the copy number and ratio of the internal standard gene and the exogenous gene of the sample to be tested and the quality control product according to the standard curve, so as to obtain the The precise content of the transgenic rice TT51-1 in samples and quality control products is used to monitor the entire experimental process and ensure the accuracy and reliability of experimental data by using quality control products.

In step 4, as shown in Table 2, prepare the PCR system of internal standard gene and exogenous gene, DNA template is 5 standard products, sample DNA to be tested and quality control product DNA respectively; Fluorescent quantitative PCR reaction procedure is according to Table 3 conduct;

In step 4, each sample is used for three repeated tests;

In step 5, the copy numbers of the exogenous gene and the internal standard gene are calculated respectively according to the equations of the standard curves of the exogenous gene and the internal standard gene, and finally the ratio of the copy number of the exogenous gene/the copy number of the internal standard gene is calculated to obtain a quantitative detection result.

Table 2 internal standard gene and exogenous gene PCR system

The PCR enzyme premix described in the table can be selected from commercially available products, such as Taqman gene expression PCR mastermix (Life Technology Company) and the like.

Table 3 Fluorescent quantitative PCR reaction program

The detection method of the present invention uses a plant genome extraction kit to extract the genomic DNA of transgenic rice TT51-1 or its products as the basis, and the upstream and downstream primers and probes of the rice internal standard gene RBE4 are used to quantitatively detect the Ct value of the internal standard gene, exogenous The upstream and downstream primers and probes of the gene TT51-1 are used to quantitatively detect the Ct value of the exogenous gene, and the standard curve established by serial dilution of the transgenic rice TT51-1 genomic DNA is used to calculate the copy number of the exogenous gene and the internal standard gene, which can be The precise content of the genetically modified components in the transgenic rice TT51-1 or its products can be obtained quickly and accurately.

Experiments have proved that this method can not only qualitatively detect the presence of the transgenic rice TT51-1 component in the sample to be tested, but also quantitatively detect the precise content of the transgenic component (see Example 1).

The present invention has the following innovations and advantages:

1. By screening the commonly used rice internal standard genes, the internal standard gene RBE4 with the best effect suitable for the detection of transgenic rice TT51-1 was found;

2. Through the optimization of the real-time fluorescent quantitative PCR system, the best PCR system suitable for the quantitative detection of transgenic rice TT51-1 or its products was found.

3. The test kit comes with standard materials for making standard curves and quality control products, which can eliminate the difference between samples from interfering with data analysis, and ensure the accuracy and accuracy of quantitative testing to the greatest extent. It breaks through the shortcomings of low precision and large uncertainty in the quantitative detection method of the previous detection of genetically modified plants, and can ensure the accuracy and traceability of the detection results.

4. High sensitivity

At least 5 copies of internal standard genes and exogenous genes can be detected quantitatively, and 15 copies of internal standard genes and 10 copies of exogenous genes can be quantitatively detected.

5. Strong specificity

Other representative insect-resistant rice primers and herbicide-resistant transgenic rice primers at home and abroad were selected (Keqidao No. 1, Keqidao No. 2, Kefeng No. 6, LLRICE601, LLRICE62). Using the genomic DNA of TT51-1 as a template for PCR amplification, it was found that only TT51-1 primers could amplify specific bands.

6. Good repeatability

From the experimental results given in the examples, it can be confirmed that 5 gradiently diluted matrix DNAs were used as samples, and each sample was repeated 6 times, and there was no significant difference between groups within a group (p<0.05).

Description of drawings:

Fig. 1 is the electropherogram of five different candidate internal standard gene products and exogenous gene TT51-1 product;

Among them, lane M is the DNA marker with a maximum molecular weight of 2000bp, lane 1 is the product of exogenous primer TT51-1, and lanes 2-6 are the products of five candidate internal standard primers, respectively GOS, PLD, RBE4, SPS, UBQ5;

Figure 2 and Figure 3 are the standard curves of the exogenous gene TT51-1 and the internal standard gene RBE4 respectively (the abscissa in the figure is the logarithm of the initial copy number (concentration) of the template, and the ordinate is the Ct value);

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

Figure 5 is the standard curve of the exogenous gene TT51-1 of the standard.

Detailed ways

Example 1 Screening of internal standard gene for detection of transgenic rice TT51-1 and optimization of fluorescent quantitative PCR reaction system

1. Materials and methods

1. 100% transgenic rice TT51-1 rice seed powder (from China Institute of Metrology)

2. Synthesize the primers of 5 rice internal standard genes and exogenous gene TT51-1 including GOS9, PLD, SPS, RBE4 and UBQ5 (sequence information comes from the article published by Jeong S-C et al. in Food Control, Volume 18: 1434-1442 "Molecular analysis and quantitative detection of a transgenic rice line expressing abifunctional fusion TPSP" and the article "Evaluation of four genes in rice for their suitability as endogenous reference standards inquantitative PCR ") see Table 4.

3. Using the extracted TT51-1 DNA as a template, use 5 rice internal standard gene primers and exogenous gene TT51-1 primers to carry out PCR reaction respectively. The set system is: TaqMan Universal PCR Master Mix 12.5ul, upstream primer (400nM) 1ul, downstream primer (400nM) 1ul, TT51-1 genomic DNA 5ul, deionized water to make up 25ul system. PCR reaction program: the first step is 95°C for 5 minutes; the second step is 40 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 60 seconds; the third step is 72°C for 10 minutes. PCR products were analyzed by electrophoresis (Figure 1).

4. Synthesize the probes of the selected internal standard gene and exogenous gene (Table 1), and optimize the PCR system, according to the quantitative PCR amplification system: the first step is 95 ° C for 5 minutes; the second step is 45 cycles of 95 ° C for 15 minutes seconds, 60 seconds at 60°C; the third step is 10 minutes at 50°C. The best conditions suitable for the amplification of internal standard genes and exogenous genes were obtained.

5. PCR enzyme master mix (Taqman gene expression PCR mastermix) was purchased from Life Technology Company.

Table 4 Sequence information of internal standard gene and exogenous gene primers for detection of transgenic rice

5. The extracted transgenic rice TT51-1 genomic DNA was serially diluted as a template, and the fluorescent quantitative PCR amplification of the internal standard gene RBE4 and the exogenous gene TT51-1 were respectively performed, and the templates of each dilution gradient concentration were amplified The Ct values of the RBE4 and TT51-1 gene amplifications were compared. Tables 5-7 below are the data for selecting internal standard genes and verifying the stability of the experimental procedure.

Table 5 Ct value of exogenous gene in transgenic rice TT51-1 at different concentrations of template

Table 6 Ct value of internal standard gene in transgenic rice TT51-1 with different concentrations of template

According to the average values of exogenous gene and internal standard gene Ct values in Table 5 and Table 6, the ratio relationship between exogenous and exogenous Ct values at various concentrations of templates of transgenic rice TT51-1 can be calculated respectively, as shown in Table 7:

Table 7 Ratio of exogenous and exogenous Ct values in transgenic rice TT51-1 with different concentrations of templates

template (copy number/ul) Ratio of internal and external Ct values 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

2. Experimental results

1. Screening of standard genes in rice

Using the pure Yang rice TT51-1 genome as a template, five candidate internal standard gene primers and exogenous gene TT51-1 primers were used for qualitative PCR amplification, and the amplification results were compared with the respective exogenous primer amplification results.

It can be seen from the electropherogram that the bands of the five internal standard gene products and the exogenous gene TT51-1 and KMD2 products are clear and single, indicating that their amplification has good specificity.

It can be seen from Figure 1 that the brightness of the bands amplified by the RBE4 internal standard primer is the closest to that of the product amplified by the TT51-1 exogenous primer. It shows that the RBE4 amplification effect of the internal standard gene is the best in the detection of transgenic rice TT51-1.

2. Optimization of fluorescent quantitative PCR reaction system

Adjust the amount of primers, probes and DNA templates used in the PCR amplification system to obtain an optimized reaction system.

RBE4 reaction system: PCR enzyme master mix 12.5ul, upstream primer (200nM) 0.5ul, downstream primer (200nM) 0.5ul, probe (100nM) 0.25ul, TT51-1 genomic DNA 5ul, deionized water to make up 25ul system. TT51-1 reaction system: PCR enzyme master mix 12.5ul, upstream primer (400nM) 1ul, downstream primer (400nM) 1ul, probe (200nM) 0.5ul, TT51-1 genomic DNA 5ul, deionized water to make up 25ul system.

3. Drawing of standard curve and system stability

The extracted transgenic rice genomic DNA was measured with Picogreen, and then serially diluted according to the gradient, and a standard curve was made by real-time fluorescent quantitative PCR to verify whether the internal standard gene and the exogenous gene had a good relationship between the template copy number and the Ct value. Linear relationship, and whether the Ct values of the internal standard gene and the exogenous gene are consistent when the template concentration is the same. Whether its quantitative system is suitable for quantitative detection of samples. According to the relationship between the Ct value obtained by real-time fluorescence quantitative PCR and the corresponding template concentration, the standard curves of the two genes were drawn (Fig. 2, Fig. 3).

The experimental results show that the amplification curves of the internal standard gene RBE4 and the exogenous gene TT51-1 of the transgenic rice TT51-1 are very good, the R2 of the standard curve of the internal standard gene RBE4 is 0.9976, the slope is -3.33, and the amplification efficiency is 99%;

The effect of the exogenous gene amplification curve is also very good, the R2 of the standard curve is 0.9982, the slope is -3.24, and the amplification efficiency is 104%.

The results showed that the amplification effect and stability of internal standard gene and exogenous gene were good, which was suitable for quantitative detection of transgenic rice TT51-1.

Example 2 Quantitative detection of transgenic TT51-1 components in rice powder

1. Materials and methods

1. The sample to be tested is the standard substance of transgenic rice TT51-1 rice seed powder with a mass fraction of 2% (developed by China Institute of Metrology)

2. Accurately weigh the sample to be tested and the quality control product (transgenic rice TT51-1 rice seed powder standard substance with a mass fraction of 1% and 5% content) with a balance

Genomic DNA Purification Kit(美国普罗麦格公司)提取待测样品和质控品的基因组DNA，并用紫外分光光度计和Picogreen试剂盒分别测定所提DNA的纯度和浓度。3. Using the Plant Genome Extraction Kit

Genomic DNA Purification Kit (Promega, USA) was used to extract the genomic DNA of the sample to be tested and the quality control product, and the purity and concentration of the extracted DNA were determined with a UV spectrophotometer and a Picogreen kit, respectively.

4. Use the standard substance (plasmid molecular standard substance containing about 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² copy/ml of TT51-1 and RBE4 gene fragments), test sample DNA and quality control DNA as The templates were prepared according to Table 1 for the PCR system, and then the fluorescent quantitative PCR amplification of the internal standard gene RBE4 and the exogenous gene TT51-1 were performed respectively.

2. Experimental results

1. See Table 8 and Table 9 for the Ct values of the internal standard gene and exogenous gene of the standard substance and the sample to be tested.

Table 8 standard product as the Ct value of the internal standard gene and exogenous gene of standard curve

Table 9 The Ct value of the genomic DNA of the test sample after PCR amplification

2. Quantitative detection results of the samples to be tested

The samples to be tested were quantified by the standard curve made by the standard. The results show that the concentration of the sample to be tested is within the linear range of the standard curve, which can better determine the content ratio of the exogenous gene in the target sample. The results are shown in Figure 4 and Figure 5, where the abscissa in the figure is the initial concentration of the template The logarithm of , the ordinate is the Ct value. It can be seen from Table 7 and Figure 4 that according to formula 1: y=-3.4663x+28.287, the internal standard gene concentration of the sample to be tested can be calculated as 19130 copy/ul. It can be known from Table 7 and Figure 5 that according to formula 2: y=-3.3934x+27.611, the exogenous gene concentration of the sample to be tested can be calculated as 393 copy/ul. From the formula three: exogenous gene content ratio of the sample to be tested=(exogenous gene concentration/internal standard gene concentration)*100%, it can be seen that the exogenous gene content ratio of the sample to be tested is 2.05%.

Claims (9)

1. a transgenic rice TT51-1 nucleic acid quantitative detection kit, including fluorescent quantitative PCR reagent, PCR enzyme premixed solution in the kit, is characterized in that also containing following composition: The upstream primers, downstream primers, and probes of the internal standard gene RBE4 are SEQ ID NO: 1 ~ SEQ ID NO: 3 The sequences of the upstream primer, downstream primer and probe of the exogenous gene TT51-1 are SEQ ID NO: 4～SEQ ID NO: 6; The quality control product is the standard substance of transgenic rice TT51-1 seed powder with a content of 1% and 5%; The standard product is a plasmid molecule standard substance containing TT51-1 and RBE4 gene fragments at different concentrations, and the quantity is 3-8. 2. The kit according to claim 1, wherein the number of said standard products is 4-6. 3. The kit according to claim 1, wherein the number of said standard products is 5, and the concentrations of TT51-1 and RBE4 gene fragments are respectively 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , and 10 ² copy/ml. 4. A method for quantitative detection of transgenic rice TT51-1 nucleic acid, characterized in that: 1) Accurately weigh the quality control product and the sample to be tested, and the quality control product is as described in claim 1; 2) Extract the genomic DNA of the quality control product and the sample to be tested separately, the purity of the obtained DNA should be A ₂₆₀ 1.8-2.0, and the concentration should be greater than 20ng/μL; 3) The upstream primer, downstream primer, probe of the internal standard gene RBE4, and the upstream primer, downstream primer, and probe of the exogenous gene TT51-1 were dissolved in deionized water to prepare a working solution with a concentration of 10 μM; 4) Each standard product, quality control product DNA and sample DNA to be tested are subjected to fluorescent quantitative PCR amplification respectively, and described standard product is as described in claim 1; 5) Perform fluorescence quantitative PCR detection, draw a standard curve of internal standard gene and exogenous gene according to the different indicated concentrations and measured Ct values of the standard, and quantify the sample to be tested through the standard curve. 5. The detection method according to claim 4, wherein the number of said standards is 5, and the concentrations of TT51-1 and RBE4 gene fragments are 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² copy/ml. 6. the described detection method of claim 4, the PCR reaction system used in described fluorescent quantitative PCR amplification is as follows: RBE4 reaction system: PCR enzyme master mix 12.5ul, upstream primer (200nM) 0.5ul, downstream primer (200nM) 0.5ul, probe (100nM) 0.25ul, TT51-1 genomic DNA 5ul, deionized water to make up 25ul system; TT51-1 reaction system: PCR enzyme master mix 12.5ul, upstream primer (400nM) 1ul, downstream primer (400nM) 1ul, probe (200nM) 0.5ul, TT51-1 genomic DNA 5ul, deionized water to make up 25ul system. 7. The detection method according to claim 4, wherein the reaction procedure in the fluorescence quantitative PCR amplification is as follows: the first step is 94° C. for 5 minutes; the second step is 45 cycles of 94° C. for 15 seconds and 60° C. for 60 seconds. 8. the described detection method of claim 4, said testing sample is quantified by this standard curve, is to calculate respectively the copy number of exogenous gene and internal standard gene according to the equation of the standard curve of exogenous gene and internal standard gene, Finally, calculate the copy number of exogenous gene/copy number of internal standard gene to obtain quantitative detection results. 9. The application of RBE4 as an internal standard gene in the quantitative detection of transgenic rice TT51-1.

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