Solutions for Common Challenges in Developing PCR-Based Diagnostic Assays

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Introduction

In vitro diagnostics (IVDs) are tests done on human samples, such as blood, saliva, or tissues, to monitor a patient’s overall health and detect diseases. The IVDs market is currently worth $89 billion and is expected to reach $118 billion by 2027. Within this market, molecular diagnostics (MDx) tests are the fastest-growing segment; they are being increasingly used in various fields, including oncology, infectious diseases, clinical chemistry, and chemical genetics. There are different types of MDx techniques in the market and no technique is a silver bullet. Instead, users must consider two important factors to determine which technique best suits their needs: time and multiplexing capabilities (Figure 1). Techniques with low multiplexing capabilities include PCR-based methods, isothermal amplification, and in situ hybridization. The first two techniques offer shorter workflows leading to faster results, while in situ hybridization has a long workflow time. Techniques with high multiplexing capabilities include microarrays and sequencing. These techniques allow for the detection of thousands of genes simultaneously; however, they involve many steps and take hours or days to complete.

PCR/qPCR vs isothermal amplification

PCR-based methods allow researchers to rapidly amplify copies of DNA to generate millions (or sometimes billions) of copies of the same DNA fragment. The technique involves short synthetic DNA fragments, i.e., primers, which determine the DNA sequence to be amplified, followed by multiple rounds of temperature cycling to amplify the target DNA sequences. Real-time PCR (or quantitative PCR, qPCR) is a variation of PCR that incorporates a fluorescent reporter, either as a dye-based or probe-based experiment. In this way, the amount of DNA amplified correlates with the fluorescence signal. These techniques are particularly well suited for applications including, gene expression analysis, snip genotyping, copy number variation, genetically modified organism detection, pathogen detection, and drug efficacy monitoring.

Isothermal amplification, on the other hand, involves the continuous exponential amplification of nucleic acid sequences at a constant temperature. Common isothermal amplification methods include strand displacement amplification, rolling cycle amplification, and loop-mediated isothermal amplification. The products of these reactions can be used in subsequent testing methods such as lateral flow. Some of the common differences between these two techniques are summarized in Figure 2.

Choosing an amplification method depends on the individual’s application and available equipment. While PCR is widely used for amplification, the method requires access to a thermocycler. This can be a disadvantage or a deal breaker for point-of-care and diagnostic applications. But if a thermocycler is available, PCR amplification is a relatively straightforward process and can be a better choice for quantifying the detection of rare transcripts.

PCR-based assays: challenges and solutions

The use of PCR experiments has evolved over the years, but developing PCR-based assays still comes with certain challenges. The section below highlights six of the most common challenges as well as some of the solutions available in the marketplace to overcome them.

1) Non-specific amplification

A common source of nonspecific amplification is the formation of primer dimers. One possible solution is to use reagents that prevent the formation of primer dimers such as Hot start polymerase. In this technique, the Taq Polymerase is inhibited during the reaction setup and later activated in a heat activation step. Using a Hot start polymerase makes it possible to setup the reaction at room temperature, avoiding nonspecific amplification and primer dimer formation. FlashTaq Hot start polymerase has one of the fastest activation times on the market (only two minutes at 95°C), resulting in faster cycling, fewer program changes, and effective inhibition of primer dimer formation (Figure 3). FlashTaq Hot start polymerase is a great solution for anyone looking to prevent nonspecific amplification.

Multiplex PCR experiments allow for the simultaneous amplification of multiple targets in a single reaction using different primers for each target. In multiplex PCR experiments, off-target amplification and primer dimer formation can be common occurrences. The primers must be highly selective for the target sequences, otherwise, they can form primer dimers, or amplify off-target sequences. The solution to this problem is therefore twofold. First, the primers must be designed using algorithms that account for seed sequence specificity. Second, the DNA polymerase must be modified to ensure that all primers are completely melted before the polymerase becomes active. The FASTaq HotStart polymerase has extremely low activity at ambient temperatures, stringent activation parameters, and the ability to do fast thermocycling (Figure 3). These features make it an ideal enzyme for anyone interested in multiplexing PCR experiments.

2) Slow reaction kinetics

PCR experiments sometimes have slow reaction kinetics and therefore take a long time to perform. The slow reaction kinetics can be overcome using reagents and enzymes specifically formulated for fast reaction kinetics. For example, the SPRINTaq Master Mix is an advanced polymerase and buffer formulation able to withstand rapid PCR cycling conditions (45 cycles in 15/20 minutes). The kit is available either as a standard or Hot start modified version and allows the end user to obtain high-quality reproducible amplification data. Figure 5 shows the performance of the SPRINTaq Master Mix in a qPCR experiment. In this experiment, the total time to complete 45 cycles was 18 minutes and 50 seconds, outperforming standard and competitor master mixes. This technology has been optimized for use in several types of PCR. For example, endpoint PCR and qPCR formulations successfully amplified targeted DNA in 16 minutes using 45 cycles. This technology meets the demands for point-of-care molecular testing in a variety of settings including human diagnostics, environmental science, agriculture, and veterinary medicine.

3) Inhibition by sample contaminants

Samples usually contain contaminants that can inhibit PCR and ultimately reduce analytical and diagnostic sensitivity. These compounds can, for example, interfere with the enzymatic activity of the DNA polymerases, bind directly to the DNA (preventing amplification) or interfere with essential cofactors. This challenge can be overcome using reagents that are tolerant to inhibition such as – InhibiTaq Master Mix – which contains reagents specifically developed to permit robust qPCR performance in the presence of contaminants found in a wide range of samples (Figure 6). InhibiTaq Master Mix can be a solution for anyone who is facing challenges with working with difficult sample types.

4) Carryover contamination

PCR can detect a single copy gene from a single cell. This extreme sensitivity is also the basis of one of its potential problems: even a single molecule from a previous amplification can lead to a false positive result. Carryover contamination can be minimized by using uracil DNA glycosylases (UDG/UNG) and dUTPs during the PCR reaction. Using this workflow, any residual products from previous PCR amplifications that are found in a new reaction will be digested by the UDG enzyme. Figure 7 shows the workflow of this method. The first step involves adding dUTPs to the reaction mixture of the first PCR experiment so that uracil will be incorporated randomly into the amplicons. In the second PCR experiment, the reaction mixture is pre- treated with UDG. During this incubation step, UDG digests any uracil-containing amplicons left from the previous experiment. After this, the temperature is raised to activate the hot start DNA polymerase and simultaneously inactivate the UDG so that only the target nucleic acids of the second experiment will be amplified. UDG can be added to any PCR master mix, thus saving time by eliminating initial pipetting steps and further carryover contamination.

5) Rapid RNA MDx

In many cases, as happened during the SARS-COV2 pandemic, the need for rapid RNA diagnostics is key. Quick diagnosis of RNA viral-based infectious diseases requires tools to rapidly amplify RNA targets. For PCR workflows, there are two methods to reverse transcribe RNA into DNA. On one hand, there’s a two-step process where RNA is first reverse transcribed into cDNA products and then that cDNA is transferred to a second tube for subsequent PCR. On the other hand, a one-step RT-PCR allows for both major reactions (reverse transcription and PCR) to be carried out in a single tube. Many labs, especially clinical labs, performing RNA-based MDx assays want a simple, one-step master mix design where all enzymes (RT, Taq, UDG) and reagents (buffer, salts, dNTPs, etc.) are included in a single mix rather than as separate reagents/steps. One-step systems, like QuantiTASE, are good for quick step-ups and limited hands-on time. The reaction takes place in a single closed tube reducing contamination. One-step workflows enable faster qualitative diagnosis in point-of-care settings.

6) Long-term ambient storage

Reagents for molecular diagnostics need to be kept cold. This is sometimes more difficult than it sounds. Many locations may not have adequate freezer space, or the end-user of a point-point-of-care device may be in a doctor’s office, urgent care center, or even in their own home. Lyophilization offers a solution to these challenges by providing an alternative for long-term shelf-life at ambient temperatures. Lyophilization removes water from the product without the need of heating the product excessively. The method involves freezing and placement of product under vacuum and can be done in situ (single reaction vessels) or in bulk bead format depending on the specific needs. The benefits of lyophilized products include reduced refrigerated requirements, lower shipping costs, simplified product use, and longer shelf life. Lyophilized products are easier to use and maintain functionality. Lyophilized beads offer significant advantages for molecular diagnostics as they are easy to handle, reduce pipetting errors, eliminate contamination and waste, and are designed for single use.

Customizing solutions to meet your needs

In today’s market, one size does not fit all, and the needs for many molecular diagnostic tests go beyond standard catalog products. Fortis’ domain expertise in custom PCR-based applications and enzyme development addresses unmet needs in the molecular diagnostic market. Fortis is able to develop and deliver highly customized reagents and enzymes for a customer’s specific application, including:

• Custom scale services, e.g., reagents in a different volume than the standard
• Custom QC services, e.g., the modification of a reagent that involves changes in QC processes
• Custom formulation, e.g., a reagent in a different concentration
• Custom enzyme projects that require validation, function activity testing and customized QC processes

All products are manufactured under ISO 13485 certification.

A case study

Around three years ago, a customer approached Fortis to develop a rapid thermal cycling enzyme for a multiplex application. Fortis developed the enzyme FASTaq polymerase and a customized master mix. Later, during the SARS-COV2 pandemic, the customer requirements pivoted to support COVID testing. As a result, Fortis pivoted with them and develop a Reverse Transcriptase Master Mix for their COVID testing workflow. More recently, the same customer needed a UDG enzyme that inactivated 55°C. Fortis was able to develop a robust UDG enzyme for the customer RT application and help save them money as well. This customer’s journey spanned custom formulations and enzymes, custom QC capabilities and custom manufacturing (Figure 9).

Conclusion

Finding the right manufacturing partner can be challenging. It is important to identify a manufacturer that can meet both current and future needs, and consistently deliver a quality product on time. Fortis provides customized solutions for various MDx applications to help customers overcome common challenges faced during the development of (PCR)-based assays. As a result, they not only deliver high-quality specialized enzyme-based reagents, but they also build long-term relationships with their customers.

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