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 . One possible alternative is based on isothermal amplification methods , 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 . 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 . 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 .
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 . 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 , as well as more radical non-contact instruments such as plasmonic thermocyclers  and flexible systems able to use real-time and end-point detection by RT-qPCR as well as isothermal amplification . These have the