High multiplex quantitative PCR: what is needed right now

Real-time polymerase chain reaction (PCR) has become the gold standard for molecular diagnostics.The method offers excellent sensitivity and specificity and is highly adaptable to a simple automated instrument platform. These advantages have driven broad adoption of real-time PCR for numerous diagnostic applications in infectious disease, oncology, and genetic disease testing.¹ While real-time PCR has become the preferred analytical method for molecular diagnostic procedures, discovery of new targets using high density techniques, such as microarrays and next generation sequencing, has highlighted the need for a high multiplex method. 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.² Multiplexing is challenging, but provides a more standardized result, with equivalent cycling conditions, reagents, samples and handling. However, the performance requirements of these assays have uncovered the limited multiplexing capabilities of current real-time PCR platforms.³

These limitations are attributable to two main issues. The first is inherent in the current detection technology employed 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.³ Second, sequence-based interactions, commonly referred to as “multiplex interference,” are particularly problematic.⁴

With the coming tide of companion diagnostics, enabling diagnostics and fusion gene variant panels, singleplex, low multiplex, and semi-quantitative methods will not be sufficient.

Several methods have been developed to address these deficiencies. However, they are based on parallel individual (i.e., singleplex) amplifications in miniaturized reaction wells.⁵ As these are no longer truly multiplex assays, a large amount of nucleic acid is required in the reaction mixture, and small reaction volumes lead to a narrowing of the dynamic range and lower sensitivity. As a consequence, these limitations preclude broad application of these methods for diagnostic purposes, and they have been relegated to the status of “Research Use Only” applications. Additional methods, such as bead capture, probe-based direct measurement, or hybridization arrays, do not utilize PCR, and are commonly used in both research as well as diagnostic applications. Like the parallel methods, these methods may suffer from sensitivity issues and a narrow dynamic range and are not considered fully quantitative. With the coming tide of companion diagnostics, enabling diagnostics and fusion gene variant panels, singleplex, low multiplex, and semi-quantitative methods will not be sufficient. A method of doing high multiplex qPCR is needed now.

Recently, a surge in diagnostic tools startup companies has led to the launching of a handful of interesting platforms. Two such systems are presented here. A system produced by Idaho Technologies has a strong focus on usability. This is a closed system, with infectious disease detection panels provided as pouches.⁶ Each pouch contains not only the primers specific to the targets, but also the reagents needed for sample prep, reverse transcription, and PCR. The pouches are freeze dried, and the user simply rehydrates them and adds sample. This is a true sample-to-answer solution. Idaho Technologies currently sells a respiratory panel that provides results for 21 different respiratory pathogens. Although the system has the capability to provide a quantitative result, that is not needed in the infectious disease applications that are currently being run on the system. There may be two main drawbacks to this technology, however: only one sample can be run at a time; and it is a closed system. It represents one end of the multiplexing spectrum, and it is simple to use, albeit limited to the content that Idaho Technologies pushes forward through its development pipeline.

The second platform that can be used for high multiplex qPCR based testing, from PrimeraDx, is based on Scalable Target Amplification Routine (STAR) technology, which represents the marriage of PCR and capillary electrophoresis (CE).⁴ The engineers and scientists at PrimeraDx have patented a real-time sampling method of the PCR so that molecules of amplified product are injected into a capillary and the CE is run concurrently with the PCR. The injection and subsequent CE happens every other cycle of the PCR, and provides amplification data on all amplicons in the reaction.

In brief, the PrimeraDx system automatically separates all amplified products across two dimensions: size and time. The user simply sets up an end-labeled PCR, enters in the sample information and run definitions, and selects run. The system is an open platform, allowing users to design their own assays using a suite of tools provided by PrimeraDx and running as many as 144 samples through the system every day. Also, PrimeraDx has begun clinical trials and is currently seeking 510(k) clearance of a C. difficile assay to be run on the system; as of now the system is for investigational use only. Because it can be used in an open mode and is entering clinical trials in a closed mode, the software has been designed to run in either open or IVD mode, with no interference between the two modes.

Theoretically, the system can be used for assays that are as high as 100-plex, and because it is based on very simple chemistry, it is easy to perform multi-target type (multimodal) assays. PrimeraDx has shown in proof-of-concept studies that it is easy to design assays to detect fusion genes and gain-of-function point mutations in the same well from the same slice of FFPE tissue. Also, assays have been tested that test for gene expression biomarkers and microRNAs in the same well. This quantitative multimodal high multiplex capability is exactly what is needed for oncology-based companion diagnostics.

Andrew T. Bond, PhD, is Director of Marketing and Customer Support for Massachusetts-based PrimeraDx.

References

1. Logan J, et al. Real-time PCR, current technology and applications; Caister Academic Press; 2009.
2. Sotiriou C, Pusztai L. Gene-expression signatures in breast cancer. N Engl J Med. 2009;360:790-800.
3. Gunson RN, et al. Using multiplex real time PCR in order to streamline a routine diagnostic service. J Clin Virol. 2008;43:372-375.
4. Garcia E, et al. Scalable transcriptional analysis routine—multiplexed quantitative real-time polymerase chain reaction platform for gene expression analysis and molecular diagnostics. J Mol Diagnostics. 2005;7:444-454.
5. Spurgeon SL, et al. High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS ONE. 2008;3:e1662.
6. Poristz MA, et al. FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection. PloS ONE. 2011; 6:e26047.