Quantification of DNA of genetically modified organisms using real-time PCR technique in Vietnam

Quantification of DNA of genetically modified organisms using real-time PCR technique in Vietnam​ (Illustrative image)

According to the Annex on the technical procedure for qualitative and quantitative analysis of Deoxyribonucleic Acid issued with Circular 13/2013/TT-BTNMT, it is stipulated that Real-time PCR can be understood as kinetic PCR reactions where analytical data are obtained through PCR reactions. Consequently, the replication of specific genes and the detection of the products can be combined in the same step through the use of fluorescent substances in the reaction. The fluorescence intensity represents the concentration of the created PCR product. The reaction is determined after time points (or PCR cycles) at which the target sequence replication is first detected. This value is referred to as the cycle threshold (Ct), which is the point at which the fluorescence intensity surpasses the baseline fluorescence value. The more the target DNA is amplified, the faster the fluorescence signal appears, thereby resulting in a lower Ct value.

In real-time PCR, double-stranded DNA binding dyes (such as SYBR Green) are often used to emit fluorescence, along with specific sequence probes or primers labeled with fluorescence (such as TaqMan PCR). Special cyclic thermal devices (PCR machines) equipped with a fluorescence signal detection module are used to monitor the reaction progress as amplification occurs. The measured fluorescence signal mirrors the quantity of product amplified in each cycle.

In quantifying DNA of genetically modified organisms and products derived from them, it is essential to identify a standard gene - a reference gene (an endogenous gene of the transgenic organism) to standardize the method. This standard gene must meet the conditions of: i) having a highly specific sequence; ii) existing as a single copy in the diploid genome; iii) being stably expressed across different lines of the same species. Therefore, in quantitative real-time PCR, two types of reactions are required for the target gene and the reference gene. The replication efficiency of the standard samples and the research samples is considered identical for this method.

To establish the standard curve for the target gene, total DNA must be extracted from the standard sample (DNA extracted from certified standard material such as DNA from genetically modified organisms or plasmid DNA carrying the target gene sequence) and the negative control sample (certified material known not to contain the target gene sequence).

For example, in quantifying DNA from products derived from Bt11 transgenic corn, to establish the standard curve for the target gene, total DNA from Bt11 transgenic corn (100% Bt, certified valid sample) and non-transgenic corn is extracted and quantified. Dilute the DNA of Bt11 into samples (M1-M6) with different haploid genome copy numbers as follows: M1:20000; M2:5000; M3: 1250; M4: 312; M5: 78; and M6: 19 (noting that the haploid genome of corn has a mass of 2.725 pg). Mix the diluted DNA of Bt11 with non-transgenic corn DNA to ensure equal DNA amounts in each sample. To establish the standard curve for the reference gene - adh1, extract, quantify and dilute total DNA from non-transgenic corn into samples (S1-S6) with different haploid genome copy numbers as follows: S1: 183486; S2: 61162; S3: 20387; S4: 6796; S5: 2265; and S6: 755 (noting that the haploid genome of corn has a mass of 2.725 pg).

More details can be found in Circular 13/2013/TT-BTNMT, which comes into effect in Vietnam from August 5, 2013.

Le Vy

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