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  • [Comparison of nucleic acid extraction efficiency using different commercial kits and qPCR. Effect of inhibitors].

[Comparison of nucleic acid extraction efficiency using different commercial kits and qPCR. Effect of inhibitors].

Revista Argentina de microbiologia (2012-10-30)
Hugo R Poma, Carolina Davies, Dolores Gutiérrez Cacciabue, María C Mora, Miguel Á Basombrío, Verónica B Rajal
ABSTRACT

The detection of specific nucleic acid (NA) sequences by PCR has revolutionized the biological and medical sciences. Real-time PCR (qPCR) opened up the possibility of obtaining quantitative results. NA extraction is a decisive step prior to qPCR since it may produce either the removal or co-extraction of inhibitory substances of the enzymatic reaction, which in turn affects the amplification efficiency. In the present work we compared the commercial NA extraction kits from Qiagen, Invitrogen and Macherey-Nagel, which were used to extract DNA from mice blood artificially infected with Trypanosoma cruzi and PP7 RNA, Pseudomonas aeruginosa bacteriophage, in spiked aqueous matrices. NA recovery efficiency in samples without inhibitors was similar for the three extraction kits. However, the Invitrogen kit was the only one that remained unaffected in the presence of inhibitors in the samples.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Guanidine thiocyanate, ≥97% (titration)
Sigma-Aldrich
Guanidine thiocyanate, Molecular Biology, ≥99%
Sigma-Aldrich
Guanidine thiocyanate solution, BioUltra, Molecular Biology, ~6 M in H2O
Sigma-Aldrich
Guanidine thiocyanate, BioReagent, Molecular Biology, ≥99%
Sigma-Aldrich
Guanidine thiocyanate, BioUltra, Molecular Biology, ≥99.0% (AT)
Sigma-Aldrich
Guanidine thiocyanate, Vetec, reagent grade, ≥97%