While no longer a new technology, PCR has repeatedly proven its worth as advancements in its utility and purpose continue to evolve. Innovations such as multiplexing help further scientific goals focused on obtaining more information from smaller or more complex resources, providing multi-dimensional insights into cancer, infectious disease, genetic mutations, and immune response at a molecular level. Using this powerful technique offers advantages over other less quantitative methods, and developments in this area continue to broaden its range.
The multiplex advantage
Multiplex PCR enables the simultaneous amplification and detection of multiple target sequences in a single reaction. Combining several assays within one mix can result in cost-savings, but more importantly, it allows researchers to obtain comprehensive data from limited amounts of starting material. The use of multiplex PCR to enrich small and poor quantities of nucleic acid is a strong advantage in areas such as forensics, or before next-generation sequencing (NGS) to amplify miniscule amounts of DNA, offers Steve Chiu, DNA amplification product marketing manager at New England Biolabs.
The quantity and quality of starting material can influence the success of a PCR reaction. Digital PCR (dPCR), in particular, tends to be less susceptible to the effects of inhibitory substances present in more complex samples, notes Madhuri Ganta, MBA, Ph.D., senior product manager, digital biology center at Bio-Rad Laboratories, making multiplex analysis more effective when compared to alternate techniques. Microarrays provide a broader scope of analytes but are only semi-quantitative and designed for low throughput. NGS can offer higher multiplexing capability but is less cost-effective and more time-consuming for analyzing low target numbers.
Bio-Rad’s QX200 ddPCR System combines water-oil emulsion droplet technology with microfluidics.
While cost, time, and material savings are all significant advantages of the technique, multiplex PCR also offers the unique ability to optimize and directly compare controls for all samples in a single reaction. “The use of artificial single-target standards, for example, may not be reflective of multiplexing in a real-world sample. Careful consideration must be given to type of template, primer and template interactions, and ratios of templates as all of these factors affect PCR efficiencies,” explains Thomas Parks, scientist at Advanced Biotechnologies. Optimized multiplex PCR ideally allows detection of multiple targets in the same reaction with the same sample, thus eliminating the need to normalize for sample input between reactions.
Realizing the impact of technological advancement
As the technique expands its reach, companies are working to improve multiplexing components and develop targeted assays for easier optimization. NEB has developed several products and tools to support multiplex PCR, including standalone enzymes, master mixes, and kits already optimized for robust and highly specific reactions. “Products feature either Taq-based polymerases used for routine PCR applications where polymerase fidelity is not of concern, or Family B polymerases such as Q5 High Fidelity DNA Polymerase for instances where high-fidelity amplification is required, like uniform amplification of NGS libraries. Q5 features the highest fidelity available on the market today at 280x that of standard Taq,” remarks Chiu.
Progress in altering the parameters that affect PCR efficiency and end-point fluorescence expands the number of simultaneously detected targets included per assay. “Typically, researchers are limited by the number of available fluorophore colors. We can now distinguish between two targets using the same color by diluting one of the two assays, altering the fluorescence intensity of the positive droplets in dPCR. Despite the dilution, assays retain the ability to quantify the target when used in multiplexed reactions,” explains Ganta. Researchers can also use alternate fluorophores to increase the number of targets to be measured. Bio-Rad’s QX200 Droplet Digital PCR system, for example, is calibrated for FAM and HEX/VIC but can also detect extra fluorophores that do not align in a perfectly orthogonal matrix, allowing further separation of targets.
Enhanced and more representative controls have also helped multiplex PCR become more specific. Rather than synthetic templates that consist only of defined gene regions, Advanced Biotechnologies has developed highly purified whole-genome quantitative pathogen controls, which are more representative of actual clinical isolates in character and performance. These controls can function as quantitative standards for pathogen detection in different regions of the entire genome.
To more closely mimic a patient sample, Parks recommends Advanced Biotechnologies’ infected cell DNAs to multiplex pathogen and cell markers together in a single control. New offerings also include custom quantification of targets within these controls by digital PCR. “Given their highly purified nature and relevant concentrations, our controls can be mixed together to make quantitative standards for multiplex panel assays. These mixed standards can even be used in the differentiation of closely related viruses, HSV-1 and HSV-2 for example, provided the proper gene regions are chosen for PCR,” Parks explains.
The development of multiplex panels and screening kits offers new capabilities for detecting multiple mutations or mRNA fusions in a single test. For example, Bio-Rad’s ddPCR KRAS G12/G13 Multiplex Screening Kit detects seven KRAS mutations, known to be drivers of colorectal cancers. Bio-Rad also offers multiplex mRNA fusions assays that detect 4-8 mRNA fusions from a single well. These assays expand liquid biopsy and FFPE sample analyses that typically pose challenges with their limited sample size or the presence of PCR inhibitors. “Bio-Rad contributes to ongoing research that supports the use of multiplex PCR, helping share best practices and new applications that can benefit research and medicine,” Ganta comments.
Getting a helping hand from software
In order to reduce complexity and oversight in multiplexing experiments, NEB provides the frequently used NEB Tm calculator, an analytical tool used to assist in primer design. The use of several primers in each reaction makes primer design complicated and a critical factor to successful multiplex PCR, notes Chiu. The calculator provides recommended melting temperatures for primers and annealing temperatures based on the primer sequence, concentration, and DNA polymerase selected.
Some companies like PREMIER Biosoft focus solely on assay development and analysis aspects that can be solved using software. “Next-generation sequencing (NGS) has fueled advances in molecular medicine by enabling study of genomic variants that code for specific traits and disease. Whole-exome and whole-genome sequencing also remain prohibitively expensive. A number of resequencing strategies exist for validation that relies on multiplex PCR to capture the variants in a single PCR reaction in a cost-effective method. We decided to author PrimerPlex and Beacon Designer software products to address this specific need of research,” explains Shonali Paul, COO at PREMIER Biosoft.
PrimerPlex designs optimal and compatible multiplex primer sets under uniform reaction conditions for up to 130 sequences, minimizing potential Tm mismatches to ensure specific amplification and high signal strength. Since primer specificity is important when multiplexing, primers can be BLAST searched directly from PrimerPlex against any genomic database at NCBI and automatically interpreted, notes Paul. Identified homologies can then be avoided during primer design. Software products such as PrimerPlex and Beacon Designer, which designs specific oligos for all major qPCR assays (supporting TaqMan and Beacon-based multiplex reactions), offer an automated approach for fast multiplex PCR assay development.
The expanding world of multiplexing
Multiplex PCR is commonly used in pathogen identification, genomic feature detection and analysis, forensic studies, and enrichment techniques as well as in assessing and verifying genomic DNA changes and off-target effects from CRISPR gene editing. “Detecting multiple pathogens in a single test has far-reaching implications in food safety and Agbio applications such as cannabis safety testing. Pathogens found in cannabis plants include E. coli, Salmonella and Aspergillus, and a sensitive, specific, reliable, and validated test would be useful,” adds Chiu.
“Multiplex PCR in infectious disease screening is also making great headway in the clinical laboratory, including utility for point-of-care testing in doctors’ offices and in the field,” comments Parks. Diagnostic companies are designing multiplex PCR assays for respiratory illness, gastro-intestinal screening, encephalitis, and mosquito-borne vectors that screen for common disease-causing pathogens. In fact, pathogen panels for multiplex field identification of Zika, Dengue, and Chikungunya viruses are already deployed in outbreak and endemic areas such as Brazil for effective disease monitoring.
Incorporating multiplex PCR assays into liquid biopsy diagnostics is assisting in improved diagnosis of particular types of cancers at earlier stages, detecting (sometimes multiple) mutations and other genetic variations. “Further integration of multiplexed PCR in liquid biopsy analysis, particularly for those malignancies that have poor early diagnosis or rapid treatment failure, will have huge impacts on the standard of healthcare in the cancer field. This will depend on the discovery of new targets associated with early-stage diagnosis and prognostic indicators of treatment efficacy,” explains Parks.
Rebecca Margraf, Ph.D., principal investigator, research and development in genetics at the ARUP Institute for Clinical & Experimental Pathology at ARUP Laboratories, found an innovative way to use multiplex dPCR to count copies of a genetic biomarker for isolated non-syndromic hearing loss, where 11% of cases are associated with a large deletion in the STRC gene. This deletion is difficult to detect since it is nearly 99% identical to the pseudogene pSTRC. For a more targeted approach, Margraf and her team developed a new STRC assay using Bio-Rad’s QX200 Droplet Digital PCR system, composed of four target assays to three specific divergent sites within STRC and one location in the nearby CATSPER2 gene. Margraf multiplexed the assays, running two target assays and the reference assay in one reaction. This type of multiplex assay development will impact not only STRC deletion identification but could be applied to other areas where it is imperative to distinguish between similar genes.
The need for multiplexing will only grow as assays become more complex with new understanding of disease processes and the need for fast yet accurate detection and identification. New technologies employing multiplexing will continue to advance the development of new therapeutic targets, the understanding of disease pathogenesis, and much more.