Multiplex molecular diagnostics: shifting the paradigm

Feb. 17, 2013

Up to now, the widespread adoption of molecular diagnostics (MDx) has been hampered by high test prices and limited detection capabilities. Today, the industry is on the cusp of a paradigm shift to new MDx tests that promise to be significantly less costly while simultaneously increasing the number of targets per test through high multiplexing. This powerful value combination has the potential to drive growth in the market, particularly in areas such as infectious disease testing, genetic analysis, and personalized medicine.

The clinical value of new, multiplex MDx tests has been demonstrated in instances such as the 2009 H1N1 influenza pandemic, BCR-Abl genotyping in adult leukemia, and real-time detection of the causative pathogens of sepsis. Advanced multiplex MDx technology will also enable novel personalized medicine approaches such as “multi-omic” tests that can scrutinize a patient’s biological map of biomarkers to guide individualized therapeutic regimens.

To successfully introduce highly multiplexed MDx into the everyday clinical algorithm, new multiplex polymerase chain reaction (PCR) technologies must be developed that can affordably and consistently identify multiple pathogens, resistance mutations carried by pathogens, cancer-associated mutant genes, or host-related gene expressions associated with chronic diseases.

In molecular diagnostics, value depends on accurately detecting low levels of pathogens and genetic mutations in clinical samples that are limited in amount or difficult to obtain. Consequently, detecting less than 100 targets is often extremely difficult. When applied to multiplexed reactions (Table 1), that challenge grows substantially, especially in relation to the design, development, and validation of the diagnostics. Oligonucleotide chemistry and design still present issues for both reaction kinetics and target specificity. This is particularly the case in high false-positive rates due to cross-reacting targets.

Table 1. Benefits of complex multiplexing reactions

While there have been advances in PCR technology, including improvements in enzyme capabilities and bioinformatics applications, there have been no significant developments in the chemistry of PCR and molecular diagnostics since the initial development of real-time PCR more than 20 years ago.

Real-time PCR is still widely considered the gold standard for clinical diagnostics because of its versatility, speed, and ease of use; however, those benefits come at a price. Oligonucleotide chemistry and design still present issues for both reaction kinetics and target specificity. In particular, target specificity is a major concern for target detection because of the false positives associated with cross-reacting targets. Furthermore, competition between real target and cross-reacting species, as well as potential primer-primer interactions, exhaust reaction components and reduce the assay’s robustness, specificity, and sensitivity.

In a single reaction, temperature settings, target denaturation, and reaction components can all be optimized for the single target, but accounting for these many parameters in a multiplex reaction is complex. Therefore, multiple primer pairs must be designed with high specificity for individual targets, while functioning under generic assay conditions. Table 2 outlines several requirements that must be addressed to successfully validate multiplex reactions.

Table 2. Limitations of current approaches to multiplexing”

Despite these challenges, instrumentation is currently viewed as the major limiting factor in real-time PCR multiplexing. Existing instrumentation is typically limited to four to six fluorescent channels with common understanding of one channel—one analyte.

With that limitation, a maximum number of four to six primer pairs must be carefully designed to match annealing temperature and oligonucleotide size while minimizing secondary structures, complementary 3′ ends, and cross-reactivity to non-specific targets. Likewise, primer and fluorescently-labeled probes must be designed to minimize primer-probe interactions, while maximizing probe-target interactions, quencher function, and/or fluorescent probe interactions that result in the real-time readout.

Additionally, overall variability in the levels of the targets can result in preferential amplification of one target over another, as well as PCR drift—stochastic variation caused by low template concentration. Variability in the physicochemical characteristics of the amplified sequences, GC content, flanking regions and secondary structures also may add to the imbalance of the reaction and the subsequent outcomes.

Although predictive molecular modeling, bioinformatics, and instrument performance are important features in the overall success of multiplex real-time PCR, the significance of the optimized reaction conditions cannot be overstated. Innovations in the chemistry and reaction conditions to circumvent the limiting issues are key to leapfrogging incremental advances to take multiplexing PCR capabilities to the next level.

For example, technologies such as dual priming oligonucleotides (DPO) and tagging oligonucleotide cleavage and extension (TOCE) enable multiplex assays on existing real time platforms that are currently found in most clinical laboratories. Using the standard four-color real time PCR instrument, TOCE chemistry allows for simultaneous detection of multiple analytes in a single color channel, with up to 20 analytes from a single sample in a single reaction. This new and emerging technology is redefining multiplexing by fully exploiting the number of analytes that can be detected in a single reaction, using current, real-time instrumentation.

The incorporation of technologies such as DPO and TOCE into real-time PCR assay design and development has the potential to help the industry overcome the design constraints inherent in current multiplex assays. DPO- and TOCE-based multiplex PCR assays combine the cost-effectiveness of identifying multiple analytes in a single reaction with the ease of development, increased specificity, enhanced sensitivity, and high level of reproducibility necessary to validate assays for in vitro diagnostics.

Jong-Yoon Chun, PhD, is founder and CEO of Seegene, Inc., provider of Dual Priming Oligonucleotides (DPO™) and Tagging Oligonucleotide Cleavage and Extension (TOCE™) technologies.