Current Trends in Medical Diagnostic Methods Novel CE-Marked SARS-CoV-2 IVD Assay Solves the Problem for Genomic DNA Contamination

The COVID-19 pandemic created a worldwide need for rapid and accurate testing of individuals for the SARS-CoV-2 virus. One of the earliest Reverse Transcription Polymerase Chain Reaction (RT-PCR) test designs for detection of SARS-CoV-2 in clinical Nasopharyngeal/Oropharyngeal (NP/OP) samples was published by the United States Centre for Disease Control (US-CDC). The internal control RPP30 primers and probe target exon 1 of the human RPP30 gene and can therefore result in amplification of both complementary DNA (cDNA) and genomic DNA (gDNA). In the event of lack of RNA in the clinical samples, the risk of false negative results is increased. In order to support the global efforts against COVID-19, Yourgene Health developed the Clarigene TM SARS-CoV-2 test, a CE-marked real-time RT-PCR in vitro Diagnostic (IVD) assay for detection of SARS-CoV-2 RNA sequences in NP/OP samples. The Clarigene TM SARS-CoV-2 test incorporates re-designed RPP30 primer sequences which only detect human RPP30 RNA sequences, as opposed to RNA and gDNA, thereby serving as a reliable internal control for sample collection and RNA extraction, as well as verifying the performance of the RT-PCR. Here, we demonstrate the high accuracy (100% sensitivity, 100% specificity) and limit of detection (5 copies/reaction) of the Clarigene TM SARS-CoV-2 test. We also provide evidence for the optimised design of the RPP30 primers and show that the Clarigene TM SARS-CoV-2 test can identify individuals infected with the novel UK SARS-CoV-2 variant B.1.1.7. Since the emergence of the SARS-CoV-2 B.1.1.7 variant, associated with increased infectivity, we have extended our testing regime to include in silico and in vitro analysis of SARS-CoV-2 strains circulating in the current population to ensure the ability of Clarigene TM SARS-CoV-2 test to detect them.


Introduction
At the end of 2019 a novel zoonotic human virus emerged in the Chinese city of Wuhan, Hubei province [1]. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) strain, causing Coronavirus disease 2019 (COVID-19), rapidly spread across China and the rest of the world, leading to declaration of worldwide pandemic by the World Health Organisation (WHO) on March 11, 2020. Since then, the SARS-CoV-2 strain has been heavily studied and great insights have been gained into its genetics, phylogeny and epidemiology [2][3][4][5]. A critical need for rapid and accurate testing emerged to support the public healthcare efforts to identify symptomatic and asymptomatic individuals, enable them to isolate and thus, reduce the spread of SARS-CoV-2.
Yourgene Health, an international molecular diagnostics company based in Manchester, UK, mobilised its expertise in early March 2020, in order to support the global efforts against COVID-19. We present here a novel high-throughput diagnostic test, Clarigene TM SARS-CoV-2 test, able to detect the presence of SARS-CoV-2 in NP/OP patient samples.
The assay incorporates RT-PCR internal control primers and probe targeting a region of exon 1 of the human RPP30 gene. However, the primers cannot distinguish between gDNA and reverse transcribed RNA, as they do not span the exon boundary [6]. In the event that gDNA is amplified in the absence of RNA (caused for example by a poor swabbing technique, failed RNA extraction, failed reverse transcription step or a combination of all three) the conclusion could be drawn that a reaction has worked as intended and therefore would have detected the virus, if present. This has the potential to lead to false negative calls and in turn infected individuals would not be advised to self-isolate resulting in an increased transmission rate.
Clarigene TM SARS-CoV-2 test is a real-time RT-PCR assay that amplifies Nucleocapsid (N) gene and Envelope (E) gene RNA sequences from the SARS-CoV-2 genome. The Clarigene TM SARS-CoV-2 internal RPP30 control primers are re-designed to span the exon 1-exon 2 boundary, thus ensuring only RPP30 RNA is amplified.
Additionally, a novel variant of SARS-COV-2 was identified in the UK towards the end of 2020. Studies by Public Health England (PHE) revealed that the B.1.1.7 lineage is associated with higher transmission rate and is present in regions with higher incidence of COVID-19 in Southern England, but there is no evidence for increased fatality of patients [7]. The B.1.1.7 lineage contains 23 mutations (13 non-synonymous, 4 deletions and 6 synonymous) and was deemed a variant of concern by PHE on December 21. A significant impact on diagnostic tests targeting the Spike gene (S gene) of SARS-COV-2 was observed, because the accumulation of seven mutations in the region prevented detection of the S gene target in individuals positive for SARS-COV-2 infection [8]. Although, the Clarigene TM SARS-CoV-2 test does not target the S gene and therefore is not affected by these mutations, one of the mutations in B.1.1.7 is located in the N gene, which is a target in the Clarigene TM SARS-CoV-2 test. The impact on this variant was assessed and it was found that the Clarigene TM SARS-CoV-2 test successfully detects COVID-19 in the presence or absence of the C28977T (p.S235F) mutation in the N gene ensuring maintained detection levels with the emergent strain.
Clarigene TM SARS-CoV-2 test offers a CE-marked solution for SARS-CoV-2 detection with high specificity, sensitivity and fast turnaround time compatible with automation for highthroughput laboratory testing.

Clinical samples
Extracted NP/OP clinical samples were purchased from Tissue solutions (Glasgow, UK) and used to demonstrate accuracy of the Clarigene TM SARS-CoV-2 test as part of clinical validation for CE marked IVD test. The samples have been previously tested with 2019-nCoV real-time RT-PCR RdRp gene assay [9]. All samples were supplied with evidence for patient consent.

Re-design of RPP30 Reverse Primer
Primers and probes targeting N gene and E gene from the SARS-CoV-2 genome were adapted from Vogels, et al. [10], who investigated the sensitivity and specificity of publicly available SARS-CoV-2 primers and probe sets. The forward primer and probe sequences for the RPP30 internal control are the same as the US-CDC published sequences (https://www.cdc.gov/ coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). The reverse primer was re-designed to span the exon 1-exon 2 junction of the human RPP30 gene in order to distinguish between cDNA and gDNA sequences ( Figure 1A). The Clarigene TM SARS-CoV-2 test is a multiplex real-time RT-PCR assay detecting the three targets in a single reaction well with each probe conjugated to a different reporter dye: FAM (N gene probe), SUN (E gene probe) and Cy5 (RPP30 probe) ( Figure 1B). To confirm the selectivity of the re-designed RPP30 primers and probe for cDNA, and to provide evidence that the US-CDC RPP30 primers and probe set cannot distinguish between gDNA and cDNA, human RPP30 gDNA (72.5 copies/ µL) and cDNA (72.5 copies/µL) sample material was tested with both the re-designed and the US-CDC RPP30 primer and probe sets. The reaction setup followed the Clarigene TM SARS-CoV-2 test instructions for use (see 'Real-Time RT-PCR reaction setup' subsection for an overview). The experiment was carried out on a LightCycler 480 II (Roche Life Science, Penzberg, Germany) in a 96-well format (LightCycler ® 480 Multiwell Plate 96, Roche Life Science, Penzberg, Germany).

Positive control
The Clarigene TM SARS-CoV-2 test is supplied with an exogenous positive control manufactured by Yourgene Health, containing equimolar concentration of N gene and E gene plasmids, diluted to 200 copies/µL (1000 copies/reaction).

Real-Time RT-PCR Reaction Setup
Reaction set-up followed the Clarigene TM SASR-CoV-2 instructions for use. Briefly, each reaction contained 11 µL RT-PCR master mix, 5.06 µL Primers and Probes mix, 0.44 µL of ROX reference dye and 5 µL of sample material. Samples were run in a 96-well format (MicroAmp ™ Optical 96-Well Reaction Plate, Thermo Fisher Scientific, Waltham, USA) on QuantStudio 7 Flex (Thermo Fisher Scientific, Waltham, USA) with the programme settings displayed in Table 1

SARS-CoV-2 lineage B.1.1.7
The ability of the Clarigene TM SASR-CoV-2 test to detect the mutant N gene (N mut ) in the recently-characterised B.1.1.7 lineage of SARS-CoV-2 [8] was assessed both in silico and in vitro. The Clarigene TM SASR-CoV-2 N gene primers and probe were mapped to the N mut gene sequence using the Geneious software (8.1.9). In vitro analysis involved obtaining two gBlock Gene Fragments (Integrated DNA Technologies, Coralville, USA), one with the C28977T (p.S235F) variant and a wild-type N gene (N wt ). Both were tested in parallel at 5 copies/reaction, 50 copies/reaction and 500 copies/reaction. All samples were run in 4 replicates.

Limit of Detection
The lowest concentration of SARS-CoV-2 viral targets at which the Positive Percent Agreement (PPA) is ≥95% was determined by serial dilution of synthetic RNA from SARSrelated Coronavirus 2 (BEI, Manassas, USA). The tested target concentrations include 100 copies/reaction, 50 copies/reaction, 25 copies/reaction, 10 copies/reaction, 5 copies/reaction and 2.5 copies/reaction. Preliminary limit-of-detection experiment was also carried out by serial dilution of BEI synthetic RNA to target concentrations of 5 copies/reaction, 25 copies/reaction, 50 copies/ reaction, 100 copies/reaction and 2000 copies/reaction.

Accuracy (Sensitivity and Specificity)
A cohort of 92 clinical samples, 46 positives and 46 negatives, was tested in a blinded study to determine the ability of the Clarigene TM SARS-CoV-2 test to accurately detect the presence of SARS-CoV-2 viral sequences in clinically derived sample material.

Repeatability
The robustness and repeatability of the Clarigene TM SARS-CoV-2 test was assessed by testing synthetic sample material at 3 copies/µL and human RNA, negative for SARS-CoV-2, at 0.25 ng/ µL in thirty replicates per sample. The experiment was repeated three times in total over a 24-hour period.

Reproducibility
In total, 432 positive contrived samples (216 at 1 copies/ µL and 216 at 3 copies/ µL) and 216 negative synthetic samples were tested in order to demonstrate the inter-and intra-assay reproducibility of Clarigene TM SARS-CoV-2 test. Three operators used three reagents lots over a three day-period testing synthetic sample material at 1 copies/ µL and 3 copies/ µL, and human RNA, negative for SARS-CoV-2, at 0.25 ng/µL in eight replicates per sample.

Cross Reactivity
In silico analysis of the primer sequences contained within the Clarigene TM SARS-CoV-2 test was carried out using the Basic Local Alignment Search Tool (BLAST). Assay primers were tested for sequence similarity to a range of common respiratory pathogens ( Table 2).

Respiratory Pathogen
In  In order to determine if the primers can specifically amplify only SARS-CoV-2 viral sequences in vitro, three respiratory specimen panels were purchased from ZeptoMetrix (Buffalo, USA), NATtrol Respiratory Verification Panel 2, NATtrol Blood Culture Identification 2 Panel and NATtrol Respiratory Pathogen Panel 1 and tested with Clarigene TM SARS-CoV-2 test.

Re-design of RPP30 Reverse Primer
The Clarigene TM SARS-CoV-2 test RPP30 primers and probe exclusively amplified cDNA sequences of the human RPP30 gene (Figure 2). In contrast, the US-CDC primers and probe set amplified both cDNA and gDNA sequences of the human RPP30 gene.

SARS-CoV-2 lineage B.1.1.7
The in silico analysis identified that the C28977T (p.S235F) mutation falls within the 5'-end of the N gene reverse primer ( Figure  3). In vitro testing showed that the Clarigene TM SARS-CoV-2 test successfully detected N mut gene of the B.1.1.7 lineage of SARS-CoV-2. The mean Ct values for N mut at 5 copies/reaction, 50 copies/reaction and 500 copies/reaction were 36.19, 33.07 and 29.91, respectively. The mean Ct values for N wt for 5 copies/reaction, 50 copies/reaction and 500 copies/reaction were 36.15, 32.99 and 29.95, respectively. A paired-sample t-test was conducted to compare the mean Ct between N wt and N mut . There was no significant difference in the scores for N wt (mean: 33.03, standard deviation: 3.10) and N mut (mean: 33.06, standard deviation: 3.14); t = 0.7559, p = 0.5286.

Limit of Detection
The analytical sensitivity of the ClarigeneTM SARS-CoV-2 test was determined by testing synthetic SARS-CoV-2 RNA transcripts at varying concentrations. The results showed that the assay can detect SARS-CoV-2 targets at an input concentration of 5 copies/ reaction 95% of the time (Table 5). Additionally, the amplification plot of the preliminary limit-of-detection is shown in Figure 4.

Accuracy (Sensitivity and Specificity)
The accuracy of the Clarigene TM SARS-CoV-2 test was assessed by testing 92 pre-extracted NP/OP clinical samples, which have been previously tested with a commercially available EUA approved test (see methods). The results showed 100% Positive Percent Agreement (PPA) for all 46 positive samples and showed 100% Positive Percent Agreement (PPA) for all 46 negative samples. Thus, the overall rate of agreement was determined to be 100% (Table 3).  Table 3: Accuracy of the Clarigene TM SARS-CoV-2 test.

Repeatability
The repeatability of the Clarigene TM SARS-CoV-2 test was demonstrated by testing contrived SARS-CoV-2 positive (at 15 copies/ reaction) and negative samples in thirty replicates three times over a 24-hour period. All samples (90 positive and 90 negative) were correctly identified, showing 100% overall agreement between all replicates.

Reproducibility
The reproducibility of the Clarigene TM SARS-CoV-2 test was determined by testing 24 replicates of SARS-CoV-2 samples at 1 copy/µL and 3 copies/µL, and 24 replicates of SARS-CoV-2 negative samples across three manufactured batches. As appropriate, variability was also assessed over three days and across three operators (n=216). The study output showed 100% PPA among all three operators, reagent lots and sample replicates. Despite the presence of one inconclusive result, which will be subjected to re-test criteria in a clinical setting, NPA of 100% was achieved for the negative synthetic samples (Table 4).

Cross Reactivity
In silico analysis of the Clarigene TM SARS-CoV-2 test primers showed several instances of ≥80% sequence similarity with other respiratory pathogens. The cases where both forward and reverse primers were highly similar to a different pathogen were investigated in detail. The analysis showed that the distance between the forward and reverse primers in all instances is >70 kb, thus making potential amplification highly unlikely under standard assay conditions. Potential cross-reactivity with E gene sequences from SARS was detected.
Wet lab testing of several commercially available panels of pathogens from the same genetic family and other organisms likely present in a respiratory specimen were assessed with the Clarigene TM SARS-CoV-2 test. Amplification of the E gene target (<40 Ct) in the presence of SARS coronavirus was demonstrated, however, the N-gene target was not amplified, therefore this cross reactivity would lead to an inconclusive result. No additional cross reactivity was detected for any of the other pathogens tested (

Discussion
One of the early developed tests in the COVID-19 pandemic was the US-CDC the 2019-nCoV Real-Time RT-PCR Diagnostic Panel, which targets two N gene regions of the SARS-CoV-2 genome and the human RPP30 gene in a singleplex format. The RPP30 targets serve as an endogenous control for verification of the assay performance. However, concerns have emerged regarding the primer design. Reports from the literature highlight that both gDNA and cDNA sequences will be amplified because the forward and reverse primers align to a region of exon 1 of the RPP30 gene [6,11]. This can prove problematic in instances where a negative test result has been reported in the absence of RNA in the patient sample. The high abundance of gDNA in clinical samples would allow the RPP30 control to produce a strong signal in such cases, thereby resulting in false negative results. This issue could be alleviated by the addition of a DNase I treatment step after RNA extraction. However, it would also extend the SARS-CoV-2 sample processing pipeline by adding an additional step in the workflow.
The Clarigene TM SARS-CoV-2 test is an easy to use multiplex assay which allows detection of all targets in a single reaction well, thus saving reagent cost and time. The re-designed internal control primers and probe set, ensure clear distinction between true negative and false negative results, arising from failed RT step, poor swabbing technique and failed extraction. Moreover, the studies presented here show the high analytical sensitivity, high accuracy, reproducibility and repeatability, as well as the high specificity of the assay targets (ensured by requirement of N gene and E gene target detection), summarised as Performance Characteristics (Table 4). The Clarigene TM SARS-CoV-2 test was also shown to successfully detect the novel SARS-CoV-2 variant which was identified in the UK in late 2020. We continue to monitor the emergence of SARS-CoV-2 strains within the population and perform adequate in silico and in vitro investigations to ensure that Clarigene TM SARS-CoV-2 test successfully detects COVID-19.