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A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment.

Fu W, Zhu P, Wang C, Huang K, Du Z, Tian W, Wang Q, Wang H, Xu W, Zhu S - Sci Rep (2015)

Bottom Line: However, to use this improved method, the samples must be pretreated, which could introduce inaccuracy into the results.The results showed that the limit of detection of our method was 0.1%, which was lower than the labeling threshold level of the EU.In conclusion, the method in our study would allow more sensitive, specific and stable screening detection of the GMO content of international trading products.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100029, China.

ABSTRACT
Digital PCR has developed rapidly since it was first reported in the 1990 s. It was recently reported that an improved method facilitated the detection of genetically modified organisms (GMOs). However, to use this improved method, the samples must be pretreated, which could introduce inaccuracy into the results. In our study, we explored a pretreatment-free digital PCR detection method for the screening for GMOs. We chose the CaMV35s promoter and the NOS terminator as the templates in our assay. To determine the specificity of our method, 9 events of GMOs were collected, including MON810, MON863, TC1507, MIR604, MIR162, GA21, T25, NK603 and Bt176. Moreover, the sensitivity, intra-laboratory and inter-laboratory reproducibility of our detection method were assessed. The results showed that the limit of detection of our method was 0.1%, which was lower than the labeling threshold level of the EU. The specificity and stability among the 9 events were consistent, respectively. The intra-laboratory and inter-laboratory reproducibility were both good. Finally, the perfect fitness for the detection of eight double-blind samples indicated the good practicability of our method. In conclusion, the method in our study would allow more sensitive, specific and stable screening detection of the GMO content of international trading products.

No MeSH data available.


The amplification hot map of the verification of ddPCR with different GMO content.(A–F) represented the different GMO content of 1%, 0.5%, 0.4%, 0.3%, 0.2%, and 0.1%. The positive droplets were highlighted by the red cycles.
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f4: The amplification hot map of the verification of ddPCR with different GMO content.(A–F) represented the different GMO content of 1%, 0.5%, 0.4%, 0.3%, 0.2%, and 0.1%. The positive droplets were highlighted by the red cycles.

Mentions: Previous studies showed that pretreating genomic DNA samples was essential to achieving reliable dPCR results. A recent study8 showed that pretreatments could be omitted from the ddPCR protocol; however, some researchers17 were concerned that without pretreatments, the stability of the method would be uncertain. For practical detection applications, including pretreatment steps makes the assays inconvenient and may introduce uncertainty into the final results. To comply with China’s labeling laws, routine qualitative detection methods must have good stability and specificity. The dPCR is the best choice for the detection of the low-abundance targets due to the partitioning of the initial PCR sample. Therefore, in this study, a stable pretreatment-free dPCR method for GMO screening was explored. Due to the production of random breaks in the genomic DNA during sample extraction, quantitative detection cannot be achieved by the screening elements. Thus, the target samples were serially diluted to evaluate the stability and reliability of our method. The linear curve of the theoretical copy number of transgenes compared with the practical number of copies of the screening elements per microliter is shown in Fig. 4. Based on the correlation coefficient of this curve, the linearity value of our method was 0.9989, showed that the good isolation of undigested genomic DNA by chambers even the GMO content is extremly low. This result suggested that the pretreatment-free dPCR detection method developed in this study had good sensitivity and linearity even when the GMO content was very low.


A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment.

Fu W, Zhu P, Wang C, Huang K, Du Z, Tian W, Wang Q, Wang H, Xu W, Zhu S - Sci Rep (2015)

The amplification hot map of the verification of ddPCR with different GMO content.(A–F) represented the different GMO content of 1%, 0.5%, 0.4%, 0.3%, 0.2%, and 0.1%. The positive droplets were highlighted by the red cycles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4530665&req=5

f4: The amplification hot map of the verification of ddPCR with different GMO content.(A–F) represented the different GMO content of 1%, 0.5%, 0.4%, 0.3%, 0.2%, and 0.1%. The positive droplets were highlighted by the red cycles.
Mentions: Previous studies showed that pretreating genomic DNA samples was essential to achieving reliable dPCR results. A recent study8 showed that pretreatments could be omitted from the ddPCR protocol; however, some researchers17 were concerned that without pretreatments, the stability of the method would be uncertain. For practical detection applications, including pretreatment steps makes the assays inconvenient and may introduce uncertainty into the final results. To comply with China’s labeling laws, routine qualitative detection methods must have good stability and specificity. The dPCR is the best choice for the detection of the low-abundance targets due to the partitioning of the initial PCR sample. Therefore, in this study, a stable pretreatment-free dPCR method for GMO screening was explored. Due to the production of random breaks in the genomic DNA during sample extraction, quantitative detection cannot be achieved by the screening elements. Thus, the target samples were serially diluted to evaluate the stability and reliability of our method. The linear curve of the theoretical copy number of transgenes compared with the practical number of copies of the screening elements per microliter is shown in Fig. 4. Based on the correlation coefficient of this curve, the linearity value of our method was 0.9989, showed that the good isolation of undigested genomic DNA by chambers even the GMO content is extremly low. This result suggested that the pretreatment-free dPCR detection method developed in this study had good sensitivity and linearity even when the GMO content was very low.

Bottom Line: However, to use this improved method, the samples must be pretreated, which could introduce inaccuracy into the results.The results showed that the limit of detection of our method was 0.1%, which was lower than the labeling threshold level of the EU.In conclusion, the method in our study would allow more sensitive, specific and stable screening detection of the GMO content of international trading products.

View Article: PubMed Central - PubMed

Affiliation: The Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100029, China.

ABSTRACT
Digital PCR has developed rapidly since it was first reported in the 1990 s. It was recently reported that an improved method facilitated the detection of genetically modified organisms (GMOs). However, to use this improved method, the samples must be pretreated, which could introduce inaccuracy into the results. In our study, we explored a pretreatment-free digital PCR detection method for the screening for GMOs. We chose the CaMV35s promoter and the NOS terminator as the templates in our assay. To determine the specificity of our method, 9 events of GMOs were collected, including MON810, MON863, TC1507, MIR604, MIR162, GA21, T25, NK603 and Bt176. Moreover, the sensitivity, intra-laboratory and inter-laboratory reproducibility of our detection method were assessed. The results showed that the limit of detection of our method was 0.1%, which was lower than the labeling threshold level of the EU. The specificity and stability among the 9 events were consistent, respectively. The intra-laboratory and inter-laboratory reproducibility were both good. Finally, the perfect fitness for the detection of eight double-blind samples indicated the good practicability of our method. In conclusion, the method in our study would allow more sensitive, specific and stable screening detection of the GMO content of international trading products.

No MeSH data available.