Automated nucleic acid isolation and purification from soil extracts using renewable affinity microcolumns in a sequential injection system

摘要

We have combined affinity purification concepts with novel renewable-surface microcolumns in a sequential injection system for the automated and rapid isolation and purification of nucleic acids directly from crude soil extracts. Geobacter chapellii DNA was spiked at femtomolar concentrations into clean solutions or crude soil extracts containing picomolar concentrations of competitive DNA, humic acids and other soluble soil constituents. The 16S rDNA targets (indigenous and spiked) were purified and eluted in less than 20 min in a form suitable for direct polymerase chain reaction (PCR) amplification and detection. The extraction efficiency of the automated system was equivalent to a 4-h batch reaction using identical reagents. The estimated efficiency of isolation and purification was maximally 30% under the conditions employed here, with levels comparable to those obtained with soils/sediments processed by standard techniques, and a detection limit of 1.7 attamoles (10~6 copies) Geobacter target in a soil extract containing a competitive background of 10~9 genomes. This manuscript represents the first report of automated nucleic acid purification from an environmental sample using sequential injection fluidic systems and renewable microcolumn technology, and provides an excellent platform from which to optimize and accelerate the development of an integrated microbialucleic acid detector.