Rheonix CARD® Technology: An Innovative and Fully Automated Molecular Diagnostic Device

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A versatile microfluidic platform for the evolving molecular diagnostics industry is described. It incorporates low-cost Rheonix CARD (Chemistry And Reagent Device) technology to analyze a variety of clinical specimens. A patented lamination process incorporates all pumps, valves, microchannels, and reaction compartments into an inexpensive disposable plastic device. Once an untreated clinical specimen is introduced, all assay steps, including cell lysis, nucleic acid purification, multiplex polymerase chain reaction (PCR), and end point analysis, are automatically performed. Three distinct CARD assays are described, which use either a low-density microarray for multiplex detection of amplicons or an integrated primer extension assay to detect single-nucleotide polymorphisms (SNPs) of interest. The sexually transmitted infections CARD is able to simultaneously detect 4 sexually transmitted infectious agents (Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, and Trichomonas vaginalis). Human C33A cervical epithelial cells were spiked with different levels of genomic DNA from the 4 species of interest, singly or in combination, and applied to the CARD device. Using multiplex PCR amplification of the targets followed by microarray detection, the CARD device was able to correctly detect a minimum of 10 copies of each of the 4 pathogens. The human papillomavirus (HPV) CARD was able to detect and distinguish 20 different clinically relevant HPV types using cloned HPV DNA. In addition, the HPV CARD could identify HPV types in vaginal specimens previously demonstrated to contain high-risk or low-risk HPV using a currently commercially available testing method. Finally, the detection of specific SNP associated with warfarin dosing sensitivity was achieved on the Warfarin Genotyping CARD by analyzing human buccal swabs. Once multiplex PCR was completed, the SNPs were detected using a primer extension assay.

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