by Professor Stephen Bustin

Remodelling qPCR as a tool for molecular diagnostics

PCR is an incredibly powerful tool in the field of molecular diagnostics, and its current role is embedded in the clinical laboratory where it is indispensable for non-time-critical routine testing. However, point-of-care testing via lateral flow devices, as seen in the recent COVID-19 pandemic, allows fast, localized specimen analysis. This article discusses how recent developments in PCR technology allowing much faster turn-around times mean that it could soon become the go-to choice for ‘while you wait’ molecular diagnostics.

PCR: the basics

PCR [1] is arguably the most consequential molecular technology invented and has had a momentous impact on biological, medical, veterinary, and agricultural research. Its chemistry is extraordinarily simple, requiring only a buffer, two oligonucleotides complementary to opposite strands of a target DNA molecule and a heat-stable polymerase. The DNA is heated, thus separating the two strands, then cooled, allowing the oligonucleotides to bind to specific sequences on the DNA. The polymerase primes DNA synthesis from the oligonucleotides, thus amplifying the sequences in-between up to a billion-fold using multiple cycles of heating and cooling (Fig. 1). Whereas the chemistry is simple, PCR requires a dedicated thermocycler capable of reaching and accurately holding these tempera-tures. In the past, these instruments were fairly large, complex and required a supporting infrastructure, making them ideal laboratory-based tools with limited appeal for point-of-care (POC) applications.

Real-time PCR

In its real-time guise, known as quantitative PCR (qPCR), amplific-ation products are detected after each amplification cycle using DNA-specific dyes or fluorophore-labelled probes that are specific for the amplified sequences. Its high specificity, coupled to the ability to amplify minute amounts of DNA has made probe-based qPCR an indispensable technology for diagnostic applications, for example making it possible to test accurately for inherited diseases, detect cancer-associated mutations or reliably detect pathogens. When preceded by a reverse transcription (RT) step, the method is designated RT-qPCR and is widely used to detect RNA viruses as well as to identify gene expression patterns of specific cancer types or profiles associated with specific outcomes, such as response to therapy or prognosis [2].

Role of molecular diagnostics in disease diagnosis and treatment

The significant global health challenges that mankind faces regarding climate change, antimicrobial resistance and zoonotic diseases are urgent drivers of the need to establish a comprehensive molecular diagnostic testing infrastructure. The three are linked, with climate change exacerbating the emergence and spread of zoonotic diseases and widespread overuse of antimicrobial drugs in agriculture resulting in antimicrobial resistance, making it more difficult to treat zoonotic diseases [3]. This testing infrastructure includes a wide choice of requirements with regards to technologies,

Figure 1. Steps in the PCR process
PCR targets a small section of a target DNA, highlighted in colour. The amplification process is a three-step process and requires an instrument able to cycle between temperatures. Step 1 involves a denaturation temperature, generally 95°C, primers bind to their target during step 2 (usually between 55°C and 60°C) and Taq polymerase synthesizes new DNA during step 3 (around 70°C). Steps 2 and 3 are often combined in probe-based molecular diagnostic assays, with the annealing/polymerization carried out at 60°C. This cycling is repeated around 30–40 times, resulting in million to billion-fold amplification of the original target.

structures, technical expertise and timelines. Consequently, molecular diagnostics is not a ‘one size fits all’ but a combination of complementary technologies that all have their specific uses. Solutions range from time-consuming, high-throughput, lab-based testing to near-instant POC assessment, with the latter’s flexibility, localized use and speed of testing making it an essential component of that infrastructure (Fig. 2).

Figure 2. An effective molecular diagnostic infrastructure requires a complementary approach
This involves a wide range of methods ranging from expensive laboratorybased central testing facilities staffed by trained laboratory staff to inexpensive point-of-care (POC) methods for use in doctors’ surgeries or at home. At the moment, PCR-based methods dominate the former but are absent from the latter.

Role of qPCR within molecular diagnostics

Traditional diagnostic qPCR assays are typically performed in centralized laboratories using high-throughput nucleic acid extraction robots and qPCR instruments. Testing relies on a complex infrastructure and workflow that involves sampling, storage, shipment of samples to the laboratory, handling of numerous samples by laboratory staff, nucleic acid extraction, target amplification, data interpretation and finally notification of the results to the patient or GP. This arrangement allows for efficient and accurate mass screening using trained personnel and has the advantage of being relatively easy to standardize. This makes it appropriate for routine testing that may not be time-critical or for population screening such as was used during the COVID-19 pandemic where high testing capacity, consistent accuracy, efficient use of resources, and efficient tracking and reporting were critical parameters. Conversely, however, this structure also provides numerous opportunities for disruptions and errors that may delay diagnosis and treatment in other, less turbulent settings. Its most important drawback, though, is that even under the most favourable conditions, it takes, at the very least, hours and, at worst, many days to return a result.

qPCR and point of care testing

The COVID-19 pandemic has brought POC testing to everyone’s attention, as user-administered, relatively simple lateral-flow-device tests based on antigen recognition have become familiar tools in everyday life. These tests require no elaborate instrumentation, report results within minutes and are inexpensive. Their major drawbacks are their variable sensitivity and specificity as well as current lack of multiplexing capacity [4]. One possible alternative is based on isothermal amplification methods [5], which amplify their targets at a constant temperature and do not require instruments capable of thermal cycling. However, the assay designs are complex as they require multiple primers and/or enzymes, they can be prone to non-specific amplification and, critically, they still require around 20 minutes to complete a test run. Although this is faster than established PCR testing, it is still not an ideal solution for many scenarios such as, for example, testing a queue of visitors to a care home or a GP’s surgery or intraoperative testing.

The importance of qPCR as a diagnostic tool was highlighted by the speed with which it was possible to develop a SARS-CoV-2 diagnostic test. The virus genome sequence was posted online on 11 January 2020 and the first commercial qPCR kit authorized for emergency use was released on 12 March. The main issue that has prevented qPCR from becoming a viable POC technology is the time that it takes from sampling to obtaining a result when using a conventional PCR-based test. First, there is the requirement for target purification, as qPCR assays are sensitive to inhibitors commonly present in biological fluids and plant or soil samples. Second, most conventional thermocyclers are not sufficiently portable, too slow or too expensive to be creditable POC devices. Third, current PCR protocols have not evolved much in the last 30 years and still use disproportionately long denaturation and annealing/polymerization times [6]. However, hel-
ped by innovations, experience and, most importantly, funding released during the COVID-19 pandemic, these issues are being addressed.

1. Sampling and extraction

There are numerous methods that result in the sensitive detection of respiratory viruses such as SARS-CoV-2 without an extraction step. These can be combined with devices such as a microfluidics cartridge that allows lysis and amplification to be carried out without user intervention, resulting in detection performance that is comparable to a laboratory-based test [7]. Figure 3 shows the results of an RT-qPCR assay carried out without extraction on SARS-CoV-2 RNA from saliva. The RNA was enriched using a capture oligonucleotide attached to a magnetic bead and the complex was immobilized to the side of a microfuge tube placed next to a magnet. The supernatant was removed, and RT-qPCR master mix was added and a rapid RT-qPCR reaction was carried out in 17 minutes. A comparison with a control RNA target demonstrates comparable sensitivity and reproducibility. Future refinement will allow this approach to be extended to other, more demanding sample types such as blood or stool and pathogens such as fungi [8].

2. Thermocyclers and reagents

Despite their limitations as POC devices, PCR instruments have improved dramatically over the last 30 years, with a range of different sizes, designs and speeds available. Nevertheless, the cycling times of conventional PCR instruments are limited by the time it takes to ramp up and down between cycling temperatures [9]. A wide range of novel thermocycler concepts is being tested that includes evolutionary solutions such as contact thermocyclers, some of which have been around for a long time [10], as well as more radical non-contact instruments such as plasmonic thermocyclers [11] and flexible systems able to use real-time and end-point detection by RT-qPCR as well as isothermal amplification [12]. These have the

Figure 3. Results of an RT-qPCR assay carried out without extraction on SARS-CoV-2 RNA from saliva

Four saliva samples containing SARS-CoV-2 RNA were mixed with target-specific capture oligonucleotides attached to magnetic beads. The solutions were heated to 95°C for 1 minute, then placed in a rack with a magnet. After 3 minutes the supernatants were replaced with a 1-step RT-qPCR master mix and subjected to a rapid cycling protocol (1 second at 95°C/1 second at 65°C). The same concentration of four pure RNA samples were amplified at the same time and acted as positive controls. the RNA extracted from saliva recorded the same quantification cycles (blue) as the untreated control RNA (red).

potential to achieve the accuracy and sensitivity required to compete with laboratory-based diagnostic solutions, but at a fraction of the time. However, the costs of these devices and associated consumables to the end user are unknown and may not be within reach of ordinary households. One interesting and affordable solution that could be a game changer is a home-testing, disposable cartridge-based PCR system that is about to be launched commercially at a cost of around USD300 for the instrument and around USD10 per lysis/amplification cassette (https://codiagnostics.com/products/co-dx-pcr-home-testing-platform/). At this cost such a system could make home-testing for, for example, flu, norovirus or respiratory syncytial virus a routine part of the ‘going off to school’ ritual for parents during a local outbreak.

There is a wide choice of reverse transcriptases and their choice, quality and speed has improved significantly over the last 20 years [13]. Similarly, Taq polymerases have become faster and more processive [14], oligonucleotide quality has improved [15] and buffers have been optimized to increase Taq polymerase extension rates [16]. Yet, surprisingly, PCR protocols have remained virtually unchanged for 30 years. The paper describing the original hydrolysis probe experiments used a cycling protocol of 20 seconds at 95°C/60 seconds at 60°C [17] and cycling times and temperatures have not changed much since then, taking 27 minutes even when used on the fastest thermocyclers [18].

This means that there is considerable scope to modify qPCR protocols with the aim of exploiting the POC potential of modern instruments and reagents. It is worth pointing out that diagnostic tests have different priorities to those relevant for research applications. The latter include the generation of a representative RNA pool, ensuring unbiased amplification, fidelity and yield, whereas the former require sensitivity, specificity and speed. First, the RT reaction completes in seconds [19] and there is no need to include a long, dedicated RT step when targeting RNA [20]. Second, one obvious way of reducing the time it takes to complete a qPCR assay is to reduce denaturation and polymerization times, as has been demonstrated for SARS-CoV-2 [21] as well as a range of cellular genes [6]. Depending on the instrument, this reduces qPCR run times from a 45–55-minute range to 15–25 minutes. But even this can be bettered, with the fastest PCR methods able to complete 30 cycles in less than 20 seconds [22], making so-called extreme PCR (ePCR) [23] the obvious choice for molecular diagnostics ‘while you wait’ [24]. This approach has been used to complete detection of a Zika virus target in 2 minutes [19].

Conclusion

The COVID-19 pandemic has ensconced POC testing as an important mainstay of the public health response to this and future risks from infectious diseases. The threats posed by climate change, antimicrobial resistance and zoonosis are focusing attention on the need to develop a fit-for-purpose testing infrastructure capable of a rapid, accurate and cost-effective reaction. Recent hardware innovations such as microfluidics and lab-on-a-chip technology as well as chemistry improvements hold out the promise that the sensitivity and specificity of laboratory-based PCR-based testing can be enhanced by ultrafast assay times, making the technology a viable POC alternative. The simplicity of assay design, potential for low cost and capacity to multiplex for the simultaneous detection of multiple pathogens couples an efficient use of resources with a greater chance of achieving a comprehensive, specific diagnosis. Finally, it is important that an effective future-proof regulatory framework is in place. This will safeguard not only that POC tests are safe, reliable and effective but help to ensure compliance with regulatory requirements and standards, thus enhancing the market acceptance of POC diagnostic tests.

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The author

Stephen Bustin PhD
Medical Technology Research Centre, Anglia Ruskin University, Chelmsford CM1 1SQ, Essex UK

E-mail: Stephen.bustin@aru.ac.uk