Oligonucleotide batch quality has a limited impact on quantitative real-time PCR

Abstract

Quantitative real-time PCR (qPCR) is currently the method of choice for sequence-specific quantification of DNA. PCR methods require the design and application of appropriate oligonucleotide primers and probes. Such oligonucleotides are available from different manufacturers but also in different purity grades. Three event-specific qPCR methods for the quantification of genetically modified organisms (GMOs) were chosen, namely for maize NK603, maize MIR604 and soya 356043, to assess a possible impact of the choice of the source and the purity grade of oligonucleotides on qPCR results. Coefficients of determination (R2), slopes and y-intercepts obtained from calibration curves using oligonucleotides of different suppliers and purity grades were evaluated statistically. The same oligonucleotides were used to quantify the DNA content of GMO in Certified Reference Materials (CRMs) ERM-BF415f (maize NK603), ERM-BF423d (maize MIR604) and ERM-BF425d (soya 356043) by qPCR. The calibration curves for the measurements of all GM events with oligonucleotides from all manufacturers and purity grades were highly linear. The lowest R2 observed in this study was 0.997. No considerable differences were observed for mean R2 from calibration curves generated with oligonucleotides from different manufacturers and purity grades. Mean slopes and mean y-intercepts of calibration curves obtained using oligonucleotides from different manufacturers and purity grades produced clustering patterns varying by PCR target. However, no tendency in qPCR method performance which could be due to the source or purity grade of the tested oligonucleotides could be identified across the six analysed PCR targets. A statistical comparison of slopes was performed testing the equivalence of all analysed calibration curves. Though for the measurements of some of the investigated PCR targets slopes were significantly different, no effect of oligonucleotide manufacturer or purity grade could be shown. Furthermore, for every one of the tested CRMs (ERM-BF415f, ERM-BF423d and ERM-BF425d), consistent DNA copy number ratios were obtained using different sources and purity grades of oligonucleotides, in each case, with a relative standard deviation of the respective mean DNA copy number ratio below 4 %. This study demonstrated that the choice of oligonucleotide manufacturer and purity grade has little impact on qPCR measurement results. Consequently, no additional uncertainty component has to be accounted for in the uncertainty budget of qPCR measurement results. However, the oligonucleotide quality can have an effect on qPCR calibration, especially in qPCR methods that tend to be less robust.