By Ayisha Malik, Cole-Parmer, EMEA
World renowned scientist chooses Techne and PCRMax for research
With the second wave of COVID-19 cases sweeping through Europe, there is an increasing requirement for accurate, fast and reliable virus testing; not only for individual diagnosis but for the development of strategies aimed at alleviating the crippling social restrictions that we have had to endure over the past few months. World renowned scientist, Dr. Stephen Bustin from Anglia Ruskin University, and his colleagues developed a 20-minute COVID-19 detection test – Cov2-ID; aimed at fast tracking our return to a busy and crowded norm.
According to Bustin’s recent publication – CoV2-ID, a MIQE- compliant sub-20-minute 5-plex RTPCR assay targeting SARS-CoV-2 for the Diagnosis of COVID-19 , which is currently under peer-review, successful COVID-19 diagnostic assays need to be capable of addressing three main functions – identifying the presence or absence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with COVID-19 symptoms, determining viral load in marginal results, and developing screening programmes that can successfully monitor the spread of the virus. Successful virus testing and screening is often based on specific, reliable, sensitive and rapid assays. CoV2-ID can be adapted to fit any of the three situations described above. To fulfil the requirements of each of these cases, the assay design needs to be adjusted; for the first scenario, a binary positive or negative readout would suffice, but when treatment decisions are guided by the viral load , assay design needs to be able to address quantitative assessment. Finally, for widespread screening, cost, throughput and simplicity play an essential role.
The CoV2-ID test
The global pandemic has had a detrimental effect on all aspects of our lives. SARS-CoV-2 was quickly identified as the agent for the COVID-19 disease, but the current diagnostic regimen highlighted many inadequacies in the system.
Using the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, Bustin’s team developed a 5-plex real time quantitative polymerase chain reaction (RT-qPCR) test. It simultaneously detects three viral targets (Nsp10, Nsp12 and N), a human internal control, and artificial RNA sequences for quality assessment, employing multiple cycle florescence detection for rapid analysis. The robust and consistent test produced 100% specific and 100% sensitive results for all samples tested in the lab. Additionally, the assay design was able to detect the D614G gene, a mutation in the SARS-CoV2 virus that was isolated in every case in the Essex (UK) area between April and June 2020.
The CoV2-ID test has garnered much interest because it has the potential to revolutionise how we test and screen COVID-19 cases. It offers faster testing, with increased specificity and sensitivity, amenities for reducing cost and increasing throughput.
PCR reaction times (RT) can be reduced by shortening denaturation and polymerisation times and altering their temperatures. With the right instruments, the high-speed qPCR protocol used in the CoV2-ID test can be completed in 15 minutes. To further reduce processing time, reagent usage, and increase throughput, the assay was optimised to run in multiplex.
To improve throughput, Bustin’s team gradually reduced RT to 1 minute, and both denaturation and polymerisation times to 1 second each. Although the annealing/ polymerisation usually takes around 6 seconds in similar protocols, it is to be noted that the fluorescence scanning involved in this step is what requires most if that time, five out of the 6 seconds to be exact. Without the need to scan the reaction after each cycle, the overall assay time was slashed to 16 minutes. Furthermore, the cooling step, which tends to be the slowest part of the assay was shortened by reducing the gap between denaturation and annealing/polymerisation temperatures; the team was able to bring down total assay time to 14-minutes 11-seconds.
The CoV2-ID test can also reduce run time during quantitative analysis using a multiple cycle fluorescence detection (MCFD) protocol. When compared to standard PCR methods that take 43 minutes to complete, the MCFD run took just over 22 minutes.
According to Bustin’s research, the reductions in run times is especially achievable when using fast cycling machines such as thePCRMax® Eco 48 system or Techne® PrimePro 48 system but it can also be achieved on instruments that are not designed to run as fast.
He wrote, “On a fast instrument – PCRMax® and Techne®, run times can be reduced to as little as eight minutes, which is significantly faster than any other methodology.”
The most common identification method for SARS-CoV-2 is PCR. However, the reported sensitivity for many of these assays is around 500 viral copies per reaction, which is significantly lower than what is achievable via this technology. The need for increased sensitivity lead to Bustin’s 5-plex CoV2-ID assay design; a robust method that is capable of consistently detecting two copies of viral RNA, with a limit of detection of a single copy.
Bustin combined, viral targets – Nsp10, N-gene, JUN, and EICAS2 panels, to form his initial assay. Quantification cycle (Cq) values from the assay, which represent the cycle number where the PCR amplification meets a predefined mathematical criterion, were comparable to ones obtained in multiplex reactions.
More investigation highlighted that Droplet Digital PCR (ddPCR), a method where two viral genes were targeted using the same flurophore, could increase assay sensitivity by another 80%. This was further enhanced by targeting all three viral genes with the same probe.
Although targeting different genes with the same probe eliminates the opportunity for extracting quantification information, it represents a valid improvement for COVID-19 diagnostic tests. To identify COVID-19 positive cases, the test does not need to be able to distinguish between different viral targets but rather reliably detect the presence of viral genes in the sample.
To prevent false positive results, viral detection assays need to consider a number of steps that ensures specificity. CoV2-ID does so, firstly, by simultaneously targeting multiple SARS-CoV-2 genes (Nsp10, Nsp12 and N). This is in line with the World Health Organization (WHO) guidelines that mandate the use of at least two genetic targets in such diagnostic assays. Having multiple targets that confirm the presence of the same virus means that there is a reduced chance of errors; even if the mechanism fails to correctly function with one of the genetic targets, the others are in place to provide required results.
Probes and primes used in any assay need to be carefully chosen to ensure they are only compatible with the targets in question. Neither the primers nor the probes used in CoV2-ID can amplify or detect other sequences that are present in the reaction mixture, thus, preventing inaccurate readings.
The assay is also designed to include a human control target; this helps confirm the presence of human cells in patient samples and for monitoring contamination.
Finally, inhibition control artificial sequences (EICAS) are also added to the reaction to ensure proper performance of the CoV2-ID assay and to detect any sample induced reaction inhibition.
To further validate these findings, Bustin’s team investigated 10 patient samples using a commercial diagnostic kit (Sansure Biotech), with comparable results, confirming the reliability of his technique.
False negatives seem to be a common problem in COVID-19 testing. This can stem from sampling difficulties, poor RNA recovery, the presence of PCR inhibitors, and human error. Therefore, it is extremely important to ensure the RT-qPCR assays do not induce further errors.
The potential for viruses to accumulate mutations in their primer binding sites to produce false negative results can be very high. The CoV2- ID test investigates three different viral targets simultaneously, ensuring virus detection rates can be maintained, even when new mutations reduce primer binding efficiencies for one or two of the targets. Additionally, by testing for a human gene and EICAS panel of genes, CoV2-ID minimises the potential for contamination and other errors.
While it may be desirable to always measure SARS-CoV-2 viral load, there is considerable confusion around how this data can be leveraged. Quantitative PCR data, which is routinely reported as Cq values, are affected by numerous external parameters. Therefore, in the absence of certified standard controls, the inclusion of an RNA control template, quantified by ddPCR, allows not just for the monitoring of the RT-qPCR reaction but also the quantification of viral load in each sample. However, we must note that this data will always be laboratory specific and cannot be freely shared and utilised across different labs and machines without further optimisation.
Future of the Cov2-ID test
Like other viruses, SARS-CoV-2 is also constantly acquiring new mutations. While there has been no evidence of the evolution of distinct phenotypic changes as of yet, the Spike protein amino acid, D614, which was present in the original variant of the virus has been replaced by D614G to become the most prevalent form in the global pandemic. This mutation increases infectivity and may even increase the severity of infection symptoms. Several location-specific mutations that have now been identified, interferes with the efficiencies of established diagnostic protocols; producing mismatches with published primer and probe sequences. Hence, it is important to routinely verify mutations in primer- and probe-binding regions of the viral genome and modify the actual probes and primers being used in diagnostic assays to ensure testing reliability into the future.