A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples.

Pengyu Zhu,Chenguang Wang,Wentao Xu,Yunbo Luo,Kunlun Huang

doi:10.3390/ijms17030402

Pengyu Zhu, Chenguang Wang + Show 3 more

Open Access

https://doi.org/10.3390/ijms17030402

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Abstract

Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems.

Highlights

Polymerase chain reaction (PCR) is considered one of the most popular detection tools used for molecular diagnosis and commercial detection
The single-plex chamber-based dPCR (cdPCR) detection method has good linearity when the genetically modified organism (GMO) content is between 0.57% and 100% [25], indicating that this method can cover approximately three orders of magnitude
As the digital PCR was regarded as an absolute quantitative detection tool, the potential elements that might influence the absolute quantitative detection have been evaluated in our study

Summary

Introduction

Polymerase chain reaction (PCR) is considered one of the most popular detection tools used for molecular diagnosis and commercial detection. Different PCR-based methodologies have been developed and are widely used, including qualitative PCR [1,2,3,4,5], quantitative PCR [6,7,8,9,10], competitive real-time PCR [11,12], etc. Qualitative PCR is the most widely used since it is cost-effective and easy to perform. While qualitative PCR can be regarded as a screening method, it is only capable of achieving a yes-or-no judgment. Quantitative PCR, known as qPCR or real-time PCR, is considered to be the most popular method for absolute quantitation; it is the “gold standard” for absolute quantitation. The potential PCR inhibitors can have a significant influence on qPCR [13]

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Discussion

Conclusion