Effect of the Matrix and Target on the Accurate Quantification of Genomic and Plasmid DNA by Digital Polymerase Chain Reaction

In polymerase chain reaction (PCR)-based nucleic acid quantification, the DNA template type, primer/probe sequence, and instrument platform such as real-time quantitative PCR (qPCR) and digital PCR (dPCR) affect the accuracy and reliability of quantitative results. In this study, a plasmid DNA (pDNA...

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Bibliographic Details
Published in:Agriculture (Basel) 2023-01, Vol.13 (1), p.127
Main Authors: Si, Nengwu, Li, Jun, Gao, Hongfei, Li, Yunjing, Zhai, Shanshan, Xiao, Fang, Zhang, Li, Wu, Gang, Wu, Yuhua
Format: Article
Language:English
Subjects:
Assaying
Calibration
Deoxyribonucleic acid
digital PCR
DNA
Genes
Genetic modification
Genetically modified organisms
genomic DNA
Genomics
Nucleic acids
Nucleotide sequence
plasmid DNA
Plasmids
Polymerase chain reaction
Quality control
quantification
Rapeseed
real-time quantitative PCR
Screening
Target detection
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Summary:In polymerase chain reaction (PCR)-based nucleic acid quantification, the DNA template type, primer/probe sequence, and instrument platform such as real-time quantitative PCR (qPCR) and digital PCR (dPCR) affect the accuracy and reliability of quantitative results. In this study, a plasmid DNA (pDNA) pBI121-screening, genetically modified (GM) rice SDrice genomic DNA (gDNA), and GM rapeseed SDrape gDNA, all carrying the same 11 screening elements, were used to prepare samples of different levels of gDNA and pDNA in a non-GM gDNA background. The comparison of the dPCR assays targeting the 11 screening elements revealed that the primer/probe set is a key factor that affects the accuracy of dPCR quantification. The optimal PCR method for the 11 screening elements was screened out from among the validated qPCR methods. The accuracy of the qPCR quantification of the low-level pDNA and gDNA test samples was low when pDNA was used as a calibrator, whereas that of the dPCR quantification was high and not affected by variations in template type and detection target. The validated dPCR assays targeting one or two elements can be randomly selected to characterize multiple-target pDNA reference materials (RMs). Low-level pDNA RMs with certified values can be used as quality controls for dPCR assays to avoid significant bias in gDNA quantification.
ISSN:2077-0472
2077-0472
DOI:10.3390/agriculture13010127