Curtis Knox & Terri McDonnell on the development of novel direct amplification reagents and protocols
Covid-19 has challenged – and stressed – every area of our healthcare systems. As the pandemic emerged, scientists took up the challenge, putting many existing projects on hold and turning their expertise to understanding the virus structure and pathogenesis, investigating binding targets for drug development, mapping the virus RNA in the search for vaccines and developing new diagnostic tests.
The effort has seen unprecedented levels of global cooperation, and it has produced remarkable – and rapid – progress in every area. Here, we highlight one area of this work – the search for alternative diagnostic testing methods – and discuss the development of a novel direct amplification reagent and protocols that enable rapid viral reverse-transcription polymerase chain reaction (RT-PCR) testing, without the need for viral RNA extraction.
Spotlight on testing
Testing is our primary tool to understand the pandemic and how it is being contained – or is spreading. Without it we would have no way to identify infected individuals or inform the treatment that they receive. It supports the isolation of those affected and the tracing and quarantining of their contacts. Moreover, it helps with the allocation of medical resources, and enables assessment of interventions such as social distancing as they are implemented.
In practice, mass testing for SARS-CoV-2 allows governments and officials to isolate those who test positive, limit the spread of disease and help to determine when it is safe to relax restrictions. Figure 1 shows the total number of Covid-19 tests per 1,000 head of population for most countries in the world (updated 1 March 2021).
But many countries continue to struggle with testing capacity. One reason is that the ‘gold standard’ test to detect SARS-CoV-2 is based on a core-lab technique, RT-PCR, that requires trained personnel, specific chemical supplies and expensive instruments. It takes between one and four hours to provide results and is often available only in labs that are part of a centralised service.
Importantly, in many regions, including the USA test providers have reported a severe shortage of test kits and required materials – from nasopharyngeal swabs to chemicals and reagents – because of supply-chain problems.
Against this background, some in the research community have focused on replacing RT-PCR with other technologies in putative ‘rapid’ tests. Others have concentrated instead on improving the RT-PCR assay and protocol: to accelerate testing capacity, simplify protocols, and reduce time to result.
Overcoming extraction challenges – Promega scientists step up
A significant element of standard RT-PCR protocols is the work needed to extract the nucleic acid – RNA in the case of SARS-CoV-2 – from the sample ahead of amplification and analysis. There are several liquid handling steps, plus the need for incubation and centrifugation steps. Even without the supply chain challenges specific to ramping up COVID-19 testing, these provide a barrier and a bottleneck.
A research team at Promega – with considerable expertise in sample preparation and nucleic acid amplification, and long experience with challenging samples such as forensic crime scene evidence – set out to overcome these bottlenecks. They sought to circumvent the extraction steps so that RT-PCR could be conducted directly on lysed samples – a so-called direct amplification method.
Issues around PCR inhibition – from sample matrix effects, or collection and storage materials, for example, and a loss of assay sensitivity – because any concentration effect form the extraction step is removed – needed to be overcome. Achieving this, would allow labs to realise the benefits of a direct method, namely:
• Purification wash steps, multiple reagents, buffers and plastic consumables are eliminated from the workflow
• Labs avoid potential throughput limitations from supply constraints
• Time in the lab is reduced (this varies depending on previous extraction method, and whether it was automated or manual)
• Direct amplification workflows are high-throughput / automation friendly across multiple platforms
The project was conceived in the company’s US R&D headquarters, where the direct lysis reagents were developed and initial protocols were established using inactivated viral targets (RSV A and Influenza B) to optimise lysis conditions and confirm reproducibility. Early samples showed viral RNA detection at levels as low as 10 copies/ul using the GoTaq 1-step Probe RT-qPCR system.
Next stage experiments used synthetic SARS-CoV-2 RNA targets in the original CDC Emergency Use Authorization assay, again with a successful outcome.
To complete this proof of principle series of studies, it was important to show that testing reagents safely inactivate viral particles, thereby simplifying the laboratory process. These experiments were supported by Dr Thomas Klimkait’s team from the Molecular Virology Group, Department of Biomedicine, University of Basel, Switzerland. In viral inactivation studies there, Dr Klimkait’s lab found that the new XpressAmp Lysis Buffer successfully inactivated the SARS-CoV-2 virus (Figure 2).
A new workflow for saliva samples
In a further reaction to supply shortages of nasopharyngeal swabs, and with a view to creating a simpler sampling process, scientists investigated if a saliva sample could provide appropriate, equivalent results to the standard test.
Workers at Yale School of Public Health have studied the use of saliva as a source of genetic material for many years and led the way in considering its potential in testing for Covid-19. Their Yale SalivaDirect protocol emerged as the result of this research, and it was issued an Emergency Use Authorization (EUA) from the US Food and Drug Administration (FDA) in August 2020. As with swab samples and XpressAmp, the SalivaDirect protocol relies on RT-PCR to amplify and analyse the lysed samples so it is only for use in authorised laboratories. In addition, the Yale EUA protocol relies on a proteinase K digestion of the sample prior to amplification, adding time and steps to the protocol, and opening the test up to shortages of proteinase K.
To test if XpressAmp could process saliva samples with similar performance as the Yale protocol – but without the need for the Proteinase K reagent – Promega scientists ran a series of comparisons.
Figure 4 summarises the experimental protocol and shows the results of using the XpressAmp Lysis Buffer with saliva samples in a comparison with the Yale protocol that included the Proteinase K digestion step. In an RT-PCR assay a positive reaction is detected by accumulation of a fluorescent signal. The Cq (quantification cycle) is defined as the number of cycles required for the fluorescent signal to cross the threshold and exceed background level. Cq levels are inversely proportional to the amount of target nucleic acid in the sample – the lower the Cq level the greater the amount of target nucleic acid in the sample.
Using an inactivated SARS-CoV-2 virus model, similar and more reproducible Cq values were reported with the XpressAmp direct amplification method. The conclusion of this, and other tests, provide solid evidence that the new XpressAmp lysis buffer is suitable for use as a direct amplification method with saliva samples.
The SARS-CoV-2 pandemic has stimulated innovation throughout the scientific community. A crucial focus for this research has been to improve and streamline the ‘gold-standard’ test for detecting the virus – RT-PCR. The Promega project described here has resulted in the commercial release of a new sample preparation reagents (XpressAmp) that looks set to allow rapid viral testing with direct amplification become a predictable, 10-minute, routine workflow – saving time and easing bottlenecks.
Curtis Knox & Terri McDonnell are with Promega