How much does the nucleic acid extraction method affect the Real-time PCR efficiency?

HUGO R. POMA; CAROLINA DAVIES; DOLORES GUTIÉRREZ CACCIABUE; MARÍA C. MORA; MIGUEL A. BASOMBRÍO; VERÓNICA B. RAJAL

Río de Janeiro

Simposio; Latin American Symposium of Environmental Virology; 2010

Nucleic acids (from a variety of microorganisms) are currently analyzed for different purposes such as the detection of contamination in water and food, the diagnosis of disease from clinical samples, gene expression, phylogenetic and forensic analysis, among others. The nucleic acid extraction is oftentimes performed before PCR determinations, and its efficiency and behaviour in the presence of inhibitors will affect the results from PCR. There are many extraction kits available at the market, however, very little had been done in terms of assessing the extension of the impact on quantitative results from real-time PCR. The aim of this work was to assess whether the extraction with different commercial kits had a direct effect on real-time PCR efficiency. We extracted nucleic acids from different matrixes with kits from Qiagen, Invitrogen, and Macherey-Nagel. Samples were chosen to illustrate different situations: a) extraction of RNA from the Pseudomonas areuginosa bacteriophage PP7 in water with and without PCR inhibitors (tannins), amplified with Taqman probes, and b) DNA from Trypanosoma cruzi from blood samples, amplified with SybrGreen. Our results showed that when the samples had no inhibitors the three kits had similar qPCR efficiency. Conversely, inhibitors present in the matrixes studied had no effect on real-time PCR efficiency only when the extraction procedure was carried out with the kit from Invitrogen.