Error estimation in environmental DNA targets quantification due to PCR efficiencies differences between real samples and standards

PÉREZ, L.M.; FITTIPALDI, M.; ADRADOS, B.; MORATÓ, J.; CODONY, F.

FOLIA MICROBIOLOGICA

SPRINGER

Año: 2013 vol. 58 p. 657 - 662

0015-5632

In real-time PCR the amplification and subsequent fluorescence detection of a genetic target occurs simultaneously during each cycle. PCR efficiency is driven by the amplification process; theory indicates that DNA duplicates in each cycle, but different factors which are related to reagents performance, methodological procedure and sample DNA quality, frequently have a considerable impact on DNA amplification. Absolute quantification is achieved using a standard curve constructed by amplifying known amounts of a target DNA; being the current approach utilized in environmental microbiology. Nevertheless, PCR efficiency obtained using good quality standard DNAs from pure cultures, plasmids, or cDNA, may be different than that obtained using DNA from environmental samples. This fact contributes to an analytical bias and consequently introduces a quantification error. In the present work, we have addressed this problem by analyzing a large number of environmental samples for the detection of Bacteroides spp., Legionella pneumophila or Adenovirus spp. We demonstrated the importance of calculating the PCR efficiency for both, standard and sample DNAs, in order to correctly quantify the amount of microbial target in environmental samples. We noted that environmental samples with low concentration of target DNA significantly differ in the PCR performance from that calculated using standards, leading to substantial bias in the quantified DNA concentration. Therefore, the results herein showed, point out the paramount importance of knowing the PCR efficiency for each procedure and every particular kind of environmental sample in order to optimize the complete workflow or, at least, to correct the final results with an appropriate factor.