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Automated high multiplex qPCR platform for simultaneous detection and quantification of multiple nucleic acid targets
 
Louis Hlousek1, Sergey Voronov1, Vesselin Diankov1, Amy B. Leblang1, Patrick J. Wells1, Donna M. Ford*1, Jork Nolling1, Kyle W. Hart1, Patricio A. Espinoza1, Michael R. Bristol1, Gregory J. Tsongalis2, Belinda Yen-Lieberman3, Vladimir I. Slepnev**1, Lilly I. Kong1, and Ming-Chou Lee***1
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Supplementary Material




Figure 3. ICEPlex operating range. (Click to enlarge)


ICEPlex target size operating range

Since the ICEPlex system uses size differences to distinguish and track specific amplicons, it is essential to evaluate the amplicons size operating range. Figure 4 demonstrates that the ICEPlex system was capable of resolving up to at least 52 amplicons over the size range of 100 to 500 bp when injected directly from PCR buffer. At the smaller sizes, the fragments were separated by 5 bp, whereas the largest two amplicons were spaced 10 bp apart.




Figure 4. ICEPlex target size range. (Click to enlarge)


The MLPA system was expected to yield approximately equal peak areas for most of these amplicons when using control DNA (13). According to the vendor however, heights of amplicon peaks can decrease as much as 3-fold with increasing fragment size. This reduction was estimated by comparing the peak heights at the smaller sizes compared with the largest sizes, and indeed this effect was seen with the ICEPlex system. However, the peaks also became somewhat broader, and when peak areas were compared with their corresponding fragment sizes, there was a minimal negative correlation with an r2 value of only 0.145 (data not shown).

ICEPlex two-color capability

To increase the number of potential multiplex reactions on the ICEPlex system, two different dyes were used concurrently. Figure 5 shows the simultaneous measurement of 16 amplicons from the AlloMap panel using both FAM and TYE channels. The data indicate that the two-color capabilities of the system permit close approximation of amplicon peaks, permitting increased “density” of different amplicons within a constrained range of sizes.




Figure 5. ICEPlex two-color capability. (Click to enlarge)


Comparison of ICEPlex with real-time PCR TaqMan data

Detection outcomes using the ICEPlex system were compared with several TaqMan assays from Argene, an alternative real-time technology. Viral particles were spiked into target-free plasma, and the extracted DNAs were used for testing. For EBV, the prototype viral assay using ICEPlex was more sensitive than TaqMan, yielding a log-linear slope of 1.46 (r2 = 0.988). The ICEPlex CMV results were slightly lower than those using the TaqMan approach (slope = 0.90, r2 = 0.984), potentially due to slight variations in the detected amplification efficiencies of the two systems that use different primer and probe sets. Overall, the ICEPlex system yielded results comparable with the TaqMan platform.

ICEPlex cross-contamination tests

Using the ViraQuant assay, the potential for well-to-well cross-contamination was examined by alternating negative and positive samples in a checkerboard arrangement in a full 48-well PCR plate. This was repeated multiple times on two different instruments, and none of the 168 negative wells (24 per plate from seven runs) generated any positive signal. This confirmed that the traditional oil-covered system used in the ICEPlex system was efficient in preventing well-to-well contamination. In order to examine the possibility of run-to-run carryover from the high levels of end-point amplicons, another full 48-well PCR plate alternating negative and positive samples in the opposite checkerboard arrangement was run immediately after the previous experiment. The arrangement of each subsequent plate alternated, such that a negative sample always followed the location of a positive in the previous run. Under the cycling and sampling conditions used, none of the 24 negative wells per plate showed a positive signal above the limit of detection, which ranged from 10 to 40 copies/reaction of the five viral targets.

Currently available molecular diagnostic platforms either are quantitative with limited multiplexing capability or are capable of multiplexing thousands of genes but significantly lacking in sensitivity and quantitative ability (2-4). Based on STAR technology (8), ICEPlex is designed to be an automated, high-throughput molecular testing platform integrating PCR thermocycling and CE separation, to allow quantitative multiplexing of dozens of targets in a single reaction well. Similar to real-time PCR, STAR uses the concept of kinetic PCR (14), wherein the quantification is based on the number of cycles required for a target amplicon to reach a preselected threshold (Ct). Here however, detection and quantification are achieved by sampling through electrokinetic injection of the DNA samples directly from PCRs into the CE device. In contrast to TaqMan real-time PCR, STAR does not require fluorogenic probes and secondary labeling. Rather, direct fluorescently labeled PCR products are separated and detected by CE. The use of two-color detection further expands the number of targets that can be measured from a single sample.

The optical detection range of the ICEPlex system is over three orders of magnitude (Figure 2), and its DNA target detection range covers at least seven orders of magnitude (Figure 3). Thus, the system has the capability of detecting target molecules over a wide range of input concentrations. Furthermore, the ability to separate more than 50 DNA fragments from a single mixture (Figure 4), and use of multiple color channels for detection (Figure 5) demonstrate that ICEPlex is an extremely versatile platform. Additionally, this high level of multiplex capability permits the inclusion of a variety of internal molecular controls, in addition to the desired target analytes, to ensure accurate and quantitative results.

STAR unites two well-characterized technologies, PCR and CE, and ICEPlex effectively automates the process via a TC module, a CE module, electrokinetic injection, and a straightforward and innovative software interface. By using a direct labeling approach, some of the complexities of designing multiplex assays that have been encountered with more commonplace probe-based chemistries, such as TaqMan, have been overcome. It should be possible to use the ICEPlex system for a variety of applications, including detection and quantification of microorganisms and fusion genes, SNP genotyping, as well as gene expression and microRNA profiling. Thus, the ICEPlex technology is applicable to many fields of investigation, from basic research and clinical testing, to environmental monitoring, biohazard detection, and bioterrorism surveillance. We are currently exploring a number of different technological approaches and applications to fully realize the potential of this novel molecular platform.

Acknowledgments

We would like to thank Benjamin Stone for his participation in directing and conducting experiments; Martin Verhoef and Matthew McManus for the direction, support, and critical review of this manuscript. We also like to thank the following for helpful comments and review on the manuscript or technical input: Andrew Bond, Dan Bowman, James Fett, Laurence McCarthy, George Serbedzija, Gregory Storch, Bert Top, Vlado Dancik, and Sarah Girald.

Competing interests

PrimeraDx owns patents on and is the commercial manufacturer of the ICEPlex instrument system.

Correspondence
Address correspondence to Lilly Kong, PrimeraDx, Inc., 171 Forbes Blvd, Suite 1000, Mansfield, MA, USA. Email: [email protected]


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