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IDENTIFYING POTENTIAL SOURCES OF BACKGROUND CONTAMINATION IN RT-PCR
Citation:
REVETTA, R. P., M. M. KEINANEN, AND J. SANTO-DOMINGO. IDENTIFYING POTENTIAL SOURCES OF BACKGROUND CONTAMINATION IN RT-PCR. Presented at American Society for Microbiology Annual Meeting, Atlanta, GA, June 04 - 09, 2005.
Impact/Purpose:
To inform the public.
Description:
Extraction of nucleic acids from low biomass samples, such as drinking water, is particularly sensitive to potential background contamination because the contaminating material is minimally diluted by the sample. The presence of bacterial DNA in Taq DNA polymerase is well established and while several methods have been used to reduce this contamination, none are 100% effective. This fact, coupled with the sensitivity of PCR make contamination a potentially serious issue. When analyzing drinking water samples for 16S rRNA using RT-PCR with a commercially available kit, we observed false positive results which led us to examine the source of the contamination. We report here an analysis of reagents supplied with a commercially available RT-PCR kit for potential DNA contamination. Buffer, dNTP mix, enzyme mix, and RNase-free water were tested in negative controls, using conventional PCR with 16S rDNBA eubacterial primers and resolved with agarose gel electrophoresis. No PCR products were detected when primers 8f and 338r were used at 25 cycles. However, PCR products of the expected size were obtained from the enzyme mix at 35 and 40 cycles. PCR products were also observed from the enzyme mix at 35 cycles using primers 8f and 519r. When primers 8f and 685r were used, bands were observed in the buffer, enzyme mix and water at 35 cycles. Negative controls containing only PCR master mix without template (buffer, Taq enzyme, dNTP mix, primers, water) did not produce PCR products. While contaminants may originate from various sources including operator technique, source of reagents, water, etc. researchers analyzing drinking water, and other low biomass samples must be aware of this issue and analyze potential background contamination sources in order to minimize false-positive results.