The AMG is built upon technologies described in the previous web pages, including NASBA and the sensor module of the Hand-Held analyzer. The AMG is designed to sample ambient seawater with a syringe pump, filter and extract RNA, partially purify the RNA, and perform NASBA using the RNA eluate. All modules of the sensor unit have been tested independently and have been show to be functional, producing results comparable to laboratory-based protocols. The operation of the sensor can be divided into two basic modules as outlined below.
Sample collection, purification and elution module: A comparison of the extraction wheel module against a commercially available column kit was performed using K. brevis cells. Automated nucleic acid extraction was performed by capturing and lysing K. brevis on the AMG custom filtration/purification columns. NASBA was performed in an EasyQ reader using a 5Ál aliquot of the eluted RNA as the template (click here to view Figure 1). Slightly longer CT times are observed in the AMG samples, this is primarily due to retention of some liquid in the column matrix (approximately 60Ál are added to the column to achieve a 30Ál eluate). In the final AMG configuration the entire eluate will be used in the NASBA reaction, thereby overcoming this dilution effect.
Amplification/detection module: Figure 2 (click here to view Figure 2) shows NASBA amplification plots of 1x106 copies of K. brevis in vitro transcribed rbcL RNA and negative (no template controls). Amplification was performed in a 20Ál reaction in the AMG amplification/detection chamber. For comparison, control reactions were run in an EasyQ detector.
Manufacturing of the waterproof pressure vessel for the sensor module and battery pack is being performed in house. Integration of the wireless communication link into the sensor electronics is almost complete. Valve, fluidic injector and carousel logic have been described and programmed in process subroutines termed: sample collection, lysis, purification, elution, cleaning/rinsing and negative control. Calling and linking each individual subroutine automates the system. Mechanical alignment of the system has been achieved allowing for reproducible fluidic connections during each stage of the purification process. To further ensure correct mechanical alignment, two levels of optical couplers are being added. This will ensure that each carousel is aligned with a home position at the start of each sample, and that each injector is correctly aligned before initiating a fluidic connection.
Field deployments of the AMG