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

Nengwu Si, Jun Li, Hongfei Gao, Yunjing Li, Shanshan Zhai, Fang Xiao, Li Zhang (), Gang Wu and Yuhua Wu ()
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Nengwu Si: Key Laboratory of Agricultural Genetically Modified Organisms Traceability of the Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China
Jun Li: Key Laboratory of Agricultural Genetically Modified Organisms Traceability of the Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China
Hongfei Gao: Key Laboratory of Agricultural Genetically Modified Organisms Traceability of the Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China
Yunjing Li: Key Laboratory of Agricultural Genetically Modified Organisms Traceability of the Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China
Shanshan Zhai: Key Laboratory of Agricultural Genetically Modified Organisms Traceability of the Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China
Fang Xiao: Key Laboratory of Agricultural Genetically Modified Organisms Traceability of the Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China
Li Zhang: College of Life Science, South-Central Minzu University, Wuhan 430074, China
Gang Wu: Key Laboratory of Agricultural Genetically Modified Organisms Traceability of the Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China
Yuhua Wu: Key Laboratory of Agricultural Genetically Modified Organisms Traceability of the Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China

Agriculture, 2023, vol. 13, issue 1, 1-11

Abstract: 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.

Keywords: digital PCR; real-time quantitative PCR; genomic DNA; plasmid DNA; quantification (search for similar items in EconPapers)
JEL-codes: Q1 Q10 Q11 Q12 Q13 Q14 Q15 Q16 Q17 Q18 (search for similar items in EconPapers)
Date: 2023
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