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

Quantitative real-time PCR (qPCR) is currently the method of choice for sequence specific quantification of DNA. The design of oligonucleotide primers and probes is essential. Oligonucleotides are available not only 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 manufacturer and 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 and compared. 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. All calibration curves for all events and all suppliers 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 suppliers and purity grades. For mean slopes and mean y-intercepts of calibration curves group distribution patterns per PCR target were observed. However, for all PCR targets no tendency in performance between suppliers and purity grades could be identified. A statistical comparison of slopes was performed testing the equivalence of all analysed calibration curves. Though slopes were significantly different for some of the investigated PCR targets, no effect of oligonucleotide supplier or purity grade could be shown. Also, the estimated GMO content for the three CRMs ERM-BF415f, ERM-BF423d and ERM-BF425d using different sources and purity grades of oligonucleotides were in agreement with each other with relative standard deviations below 4 %. With this study we demonstrate that the choice of oligonucleotide manufacturer and purity grade has little impact on qPCR measurement results. Consequently, we suggest that no additional uncertainty component has to be accounted for in the uncertainty budget of qPCR measurement results. However, oligonucleotide quality can have an effect on qPCR calibration especially in qPCR methods that tend to be less robust.

JEYNOV Boyan;  MEYER Wibke;  MERVEILLIE An;  TRAPMANN Stefanie;  CORBISIER Philippe;  EMONS Hendrik; 

2013-01-09

SPRINGER

JRC70965

1438-2377,   

http://rd.springer.com/article/10.1007/s00217-012-1863-z#,    https://publications.jrc.ec.europa.eu/repository/handle/JRC70965,   

10.1007/s00217-012-1863-z,