Determination of Genotypes Using a Fully Automated Molecular Detection System

Abstract

The EncompassMDx workstation (Rheonix, Inc, Ithaca, New York) is a molecular detection system that can automatically determine the genotypes of individuals in an unattended manner. Considerably less technical expertise was required to achieve results identical to those obtained using more complex, time-consuming, and expensive bidirectional DNA sequencing. This optimized system may dramatically simplify and reduce the costs of pharmacogenomics testing, thus leading to more-widespread use.

A modifiable microarray-based universal sensor: providing sample-to-results automation

“…To demonstrate the versatility of the system, DNA was isolated from a variety of samples exactly as previously described [25, 26]. Detection of the infectious agents, Chlamydia trachomatis , Neisseria gonorrhoeae and Trichomonas vaginalis was demonstrated by spiking the organisms into phosphate buffered saline either individually or combined prior processing on the CARD cartridge.…”

“…The buccal swab samples were obtained using FLOQSwabs™ purchased from Copan Diagnostics, Inc. (Murrieta, CA) and suspended in phosphate buffered saline. DNA was then automatically isolated on the CARD cartridge from the samples and PCR amplified exactly as previously described [25, 26]. Finally, in order to demonstrate a higher multiplex detection of multiple targets, 5′ biotinylated DNA sequences, corresponding to multiple human papilloma virus (HPV) subtypes and other sexually transmitted infection (STI) agents were purchased from Integrated DNA Technologies and served as surrogate amplicons.…”

“…For both PCR reaction vessels, the thermal cycling conditions included an initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 48 °C for 30 s, and extension at 72 °C for 30 s. The final extension was carried out for an additional 5 min at 72 °C. For SNP PCR reactions, the primer pairs were biotinylated at both termini [26] in order to facilitate detection via streptavidinylated horse radish peroxidase.…”

“…In both cases, the ultimate detection of the various amplicons was achieved by incubation with streptavidinylated horse radish peroxidase (HRP), followed by 3, 3′,5, 5′-tetramethylbenzydine (TMB) for color development. The signal generated by incubation of HRP with TMB was detected after six minutes by allowing the workstation's onboard Cognex (Natick, MA) CMOS camera to capture the images which were then processed by the instruments imaging software [26].…”

“…Although there has been considerable opposition to the new draft guidance documents by CLIA laboratories and multiple laboratory associations, it is apparent that changes to oversight of LDTs, either by the FDA, the Centers of Medicare and Medicaid Services (CMS) or both is likely to occur in the future. In order to overcome the limitations of current commercial systems and to provide universal “plug and play” functionality, we set out to expand the capabilities of our current fully automated system [25, 26, 27] to allow end users to automatically functionalize DNA arrays contained within generic microfluidic cartridges interfaced with a programmable workstation. Another goal of this effort was to allow the system to achieve the required functionalization of the universal array while the rest of the assay was underway, thus eliminating any delay in having the DNA arrays functionalized and available for hybridization.…”