Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

Abstract

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model

“…Lin et al [8] reported a similar finding, with a 3-fold increase in the NK603/ zSSIIb gene(s) using the eukaryotic system, maize. Interestingly, little attention has been paid to the effect of circular plasmid standards in bacterial and archaeal systems which commonly have genomes and plasmids that are circular, although linear forms are found in some cases [9].…”

Utilization of a real-time PCR assay for diagnosis of Babesia microti infection in clinical practice

“…By contrast, the analytical sensitivity using samples with intact B. microti parasites in infected RBCs was 2.7 parasites/l, which was ∼7-fold less sensitive than that established using samples spiked with plasmid DNA. Both reductions in DNA extraction efficiency and reduced PCR amplification have been well documented using intact organisms containing large genomic DNAs versus extracellular, small plasmid DNA fragments (Lin et al, 2011;Yun et al, 2006). Imprecision in the measurement of parasitemia in a patient sample and in the estimation of the plasmid copy numbers in DNA used for the spiking may also contribute to the observed difference (College of American Pathologist, 2013;Applied Biosystems, 2003).…”

“…In these reports, the analytical sensitivity of real-time PCR assays was most often assessed by spiking plasmid DNA to negative blood samples, which may result in inaccurate results due to the potential for preferred amplification by PCR of small plasmid DNA fragments, compared with the same target sequence contained within the complete genome of a microorganism (Lin et al, 2011;Yun et al, 2006). Moreover, there are only very limited data available on the performance of real-time PCR for diagnosis and monitoring of treatment in patients with babesiosis in routine clinical practice.…”

Comparison of a quantitative PCR assay with peripheral blood smear examination for detection and quantitation of Babesia microti infection in humans

“…Slight imprecision in measurements of the concentration of the plasmid DNA used in the preparation of the external standard could adversely affect the accuracy of estimates of the target DNA in patient samples, since just 1 ng of the plasmid DNA (3080 bp) is equivalent to 3.01 × 10 8 copies of target DNA. In addition, it is possible that the complexity of B. microti genomic DNA (~6.5 Mbp) in patient samples, compared with that of the cloned plasma DNA (3080 bp) used as the external standard, led to an underestimation of the quantity of the target DNA in the patient samples (Lin et al, 2011). Other factors that might have contributed to a reduction in the estimated quantity of the Babesia DNA target in blood include incomplete lysis of erythrocytes and/or of the parasite itself or inefficient PCR amplification; however, none of these would have been anticipated since the standard DNA extraction protocol used in this study lyses leukocytes well (Qiagen, 2012), and the PCR was found to be highly efficient with a calculated efficiency of~95% (Wang et al, 2015).…”