Use of Magnetic Bead Resin and Automated Liquid Handler Extraction Methods to Robotically Isolate Nucleic Acids of Biological Agent Simulates

摘要

The events that occurred following the mailing of Bacillus anthracis- laced envelopes through the postal system highlight the need to perform biological screening on large numbers of environmental samples. High-throughput screening that relies on integrated robotic systems to speed analysis has been undertaken to handle the surge in samples requiring testing in events involving weapons of mass destruction. These automated screening systems require DNA extraction methods capable of handling environmental samples that contain inhibitors and have target organisms at low concentrations. This study describes the development of a method for testing paramagnetic bead-based resin for use with an automated liquid handler and environmental samples. The DNA purification kits for food and blood were capable of purifying DNA from bacteria used to simulate biological weapons. An optimized hybrid protocol combining components from these two kits gave the best purification results, as determined by real time-polymerase chain reaction. Using this protocol, the gram-negative bacteria Erwinia herbicola could be detected as low as 1 CFU/ml starting concentration, and Bacillus subtilis var. niger spores were detectable at 10,000 CFU/ml. When combined with bead beating, these spores were consistently detectable at 100 CFU/ml. Furthermore, the automated approach significantly shortens analysis time versus manual operations, and has the capacity to purify nucleic acid from a variety of environmental samples.