Quantitative PCR (qPCR) using real-time detection of amplification is limited to a small number of targets within a single reaction. The ICEPlex system, using our scalable target analysis routine (STAR) technology, was developed to provide an automated, high multiplexing PCR solution. ICEPlex combines PCR thermal cycling with dynamic, sequential amplicon separation by capillary electrophoresis and two-color quantitative detection in a single integrated system. In contrast to probe-based qPCR, ICEPlex directly measures amplicon accumulation through incorporation of labeled primers. Three orders of magnitude of optical detection range and at least 7 logs of detectable target concentration range are demonstrated. The system can separate more than 50 amplicons per color channel, ranging from 100 to 500 bases, providing broad multiplexing capabilities for a wide spectrum of nucleic acid amplification applications. ICEPlex can be used for analysis of viral DNA or RNA targets, detection of genetic variants, and for reverse-transcriptase PCR gene expression panels.

Real-time PCR has become the gold standard for molecular diagnostics. The method offers excellent sensitivity and specificity and is adaptable to a simple automated instrument platform (1-3). These advantages have driven broad adoption of real-time PCR for numerous diagnostic applications in infectious disease, oncology, and genetic diseases (4). Recently, a new generation of tests based on quantification of expression levels of a panel of genes has emerged as a promising diagnostic and prognostic tool (5). However, the performance requirements of these assays have uncovered the limited multiplexing capabilities of current real-time PCR platforms (6, 7). These are attributable to two main issues. The first is inherent to the current detection technology used in real-time PCR, in which different fluorescent dyes are used to distinguish distinct nucleic acid targets. A significant overlap in fluorescent dye excitation and emission spectra leads to a complex computational problem, particularly when the targeted nucleic acids differ in their abundance. This limits multiplexing to three or four quantifiable color channels (6, 7). Second, sequence-based interactions, commonly referred to as “multiplex interference” (4, 8), can be problematic. Several methods have been developed to address these deficiencies. However, they are based on parallel individual (i.e., singleplex) amplifications in miniaturized reaction wells (9). As these are no longer truly multiplex assays, larger amounts of nucleic acid are required in the reaction mixture, and small reaction volumes lead to lower sensitivity. In addition, preloaded assay formats lack flexibility in customization of assay design and per-sample cost, and time requirements are expected to be significantly higher compared with multiplex reactions. As a consequence, these limitations restrict broad application of these methods for diagnostic purposes. We have previously described scalable target analysis routine (STAR) technology as an alternative real-time multiplexing approach (8). The technology is based on the continuous sampling of PCR reactions containing fluorescently labeled primers during sequential cycles of amplification. This is followed by size-based detection of amplified products by capillary electrophoresis (CE) and reconstruction of the amplification kinetics using real-time PCR algorithms to quantify the amount of material in the initial sample. Using STAR technology, as well as an innovative approach to primer design, we have significantly improved multiplexing capabilities over limits previously associated with chemistries using fluorogenic probes, such as TaqMan. With the addition of our automated bench top system, ICEPlex, we have created an innovative platform enabling simultaneous quantification of multiple targets from a single reaction. Herein we describe performancecharacteristics of this platform, which integrates direct electrokinetic injection from a PCR amplification reaction mixture with real-time CE separation and detection.

Materials and methods

System description

ICEPlex comprises several key elements (Supplementary Figure 1): an operating infrastructure with computer control, a PCR thermal cycler (TC), robotics and fluidics, and a CE system. The software elements include the user interface, consumables management, instrument control, data analysis, and reporting.

Figure 1. Performance of the ViraQuant assay on the ICEPlex platform. (Click to enlarge)

The TC heating block accommodates standard 96-well PCR plates and is temperature controlled in four zones to ensure uniformity. It incorporates a weight-calibrated and spring-loaded hold-down lid to provide uniform clamping and thermal contact between the plate and the block. The lid is perforated to allow insertion of the cannulae from the CE cartridge, for sampling from each well during cycling.

The system computer controls a three-axis robotic motor system, the fluidic pumps, and valves necessary to fill capillaries with gel and to pump CE buffer and system decontamination solution. All of these functions are preprogrammed and occur without operator intervention. The system monitors fluid levels and alerts the operator to replenish consumables and empty the waste reservoir when necessary.

The CE system itself consists of a prefilled disposable 384-well CE plate, a 96-well idle tray, a capillary cartridge, a gel and buffer filling system, and two high voltage power supplies.