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Multiplex Molecular Testing
Molecular multiplex assays have the ability to significantly lower the cost of testing multiple targets at one time. Compared to singleplex reactions, multiplex requires less reagents, less time, and less labor to achieve the same result. However, the performance of a molecular assay depends on its ability to accurately detect low levels of a pathogen, and in multiplex analysis, this challenge grows substantially. The 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, its length, GC content, flanking regions and secondary structures also may add to the imbalance of the reaction and impact on Ct values. 1
To overcome these challenges, it is important to use a master mix optimized for multiplexing to ensure that Ct value remains the same and the assay retains its high sensitivity for each of its targets. Meridian’s Fast 1-Step RT-qPCR Mix (Catalog MDX032) is designed for multiplex RT-qPCR diagnostic tests and for high-throughput, automated platforms. The amount of polymerase, dNTP, and Mg 2+ has been optimized and the mix’s buffering capacity (to stop the pH changing) has been enhanced to provide optimal performance. Another major concern for multiplexing assays, especially ones on high-complexity platforms that involve the manipulation of amplified PCR products, is the potential for false-negative results from PCR inhibition or false-positive results from PCR amplicon carryover. Most assay protocols require DNA or RNA extraction prior to testing in order to remove PCR inhibitors found in clinical samples such as nasopharyngeal or sputum specimens. However, these purification methods are problematic, can cause sample loss, and are not completely effective at removing all inhibitors. In addition, during the height of the first wave of the COVID-19 pandemic, the huge demand for molecular testing created an RNA extraction reagent shortage. Faced with the challenge of overcoming this supply shortage, assay manufacturers looked for alternative methods that rely on direct detection, entirely avoiding the need to purify the DNA or RNA. To address these issues, Meridian focused on creating inhibitor-tolerant molecular master mixes that allow clinical crude specimens to be run directly on a PCR machine, without performing purification or extraction first. Meridian’s first direct detection mix, Inhibitor- Tolerant RT-qPCR Mix (Catalog MDX016) was designed for qualitative multiplex assays using crude lysates or inhibitor-rich samples from different sources. The mixes
can be used for direct amplification to substantially reduce steps in the assay workflow and improve assay turn-around times.
Figure 2. Schematic of direct detection from clinical sample
Compared to singleplex reactions, multiplex requires less reagents, less time, and less labor to
achieve the same result.
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