Chandler D P, Schuck B L, Brockman F J, Bruckner-Lea C J
Environmental Microbiology Group, Battelle Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, Mail Stop P7-50, Richland, WA 99352, USA.
Talanta. 1999 Aug 9;49(5):969-83. doi: 10.1016/s0039-9140(99)00074-0.
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 microbial/nucleic acid detector.
我们已将亲和纯化概念与新型可再生表面微柱相结合，应用于顺序注射系统，以直接从粗土壤提取物中自动快速分离和纯化核酸。将纳摩尔浓度的查佩利地杆菌DNA加入到含有皮摩尔浓度竞争性DNA、腐殖酸和其他可溶性土壤成分的清洁溶液或粗土壤提取物中。16S rDNA靶标（包括天然的和添加的）在不到20分钟的时间内被纯化并洗脱，其形式适合直接进行聚合酶链反应（PCR）扩增和检测。该自动化系统的提取效率等同于使用相同试剂进行4小时的批量反应。在此处采用的条件下，估计的分离和纯化效率最高为30%，与通过标准技术处理土壤/沉积物所获得的效率相当，在含有10⁹个基因组竞争背景的土壤提取物中，地杆菌靶标的检测限为1.7阿托摩尔（10⁶个拷贝）。本论文首次报道了使用顺序注射流体系统和可再生微柱技术从环境样品中自动纯化核酸，并提供了一个优化和加速集成微生物/核酸检测器开发的优秀平台。
