ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

Real-time reverse transcription polymerase chain reaction (RT-PCR), a standard method recommended for the diagnosis of coronavirus disease 2019 (COVID-19) requires 2–4 h to get the result. Although antigen test kit (ATK) is used for COVID-19 screening within 15–30 min, the drawback is its limited sensitivity. Hence, a rapid one-step quadruplex real-time RT-PCR assay: termed ƩS COVID-19 targeting ORF1ab, ORF3a, and N genes of SARS-CoV-2; and Avocado sunblotch viroid (ASBVd) as an internal control was developed. Based on strategies including designing high melting temperature primers with short amplicons, applying a fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing/extension temperatures; the assay could be accomplished within 25 min. The limit of detection of ORF1ab, ORF3a, and N genes were 1.835, 1.310, and 1 copy/reaction, respectively. Validation was performed in 205 combined nasopharyngeal and oropharyngeal swabs. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.8%, 100%, 100%, and 97.1%, respectively with 96.7% accuracy. Cohen’s Kappa was 0.93. The newly developed rapid real-time RT-PCR assay was highly sensitive, specific, and fast, making it suitable for use as an alternative method to support laboratory diagnosis of COVID-19 in outpatient and emergency departments.


Development of rapid real-time RT-PCR (ƩS COVID-19)
A newly developed one-step quadruplex real-time RT-PCR assay was designed to target ORF1ab, ORF3a, and N genes of SARS-CoV-2, and ASBVd as an internal control (Fig. 2).The initial setting used the standard protocol as described in the previous section.The total run time was 67 min 56 s.
To develop a rapid real-time RT-PCR assay, the protocol was first adjusted to a 5 min RT step followed by 1 min initial denaturation and 40 cycles of 2 s denaturation at 92 °C and 4 s annealing/extension at 60 °C.In addition, the fast mode of QuantStudio 5 which automatically adjusted the ramp rate from 1.6 °C/s in standard mode to 4.13 °C/s denaturation and 3.16 °C/s annealing was also applied.The performance of this adjusted setting was comparable with the initial setting as shown in Fig. 3.
Then, the assay was investigated step by step whether it could preserve its performance under extreme conditions.Hold time in each step including RT, initial denaturation, cycling denaturation, and annealing/extension was minimized.
Starting with RT, because the assay used WarmStart Luna Reverse Transcriptase (New England Biolabs, USA) which required incubation at 55 °C to ensure full activation, the idea of omitting the RT step by just letting the RT reaction occur while preparing the master mix at room temperature was not possible.Therefore, 5 min of RT time was compared with 4 min, 2 min, and 30 s using reference materials.Although the statistically significant difference in mean ± SEM Ct values was found, the mean difference was less than one cycle in ORF1ab (0.840) and N (0.756).Therefore, the reduction of RT time from 5 min down to 30 s very slightly affected the performance of the assay for all 3 SARS-CoV-2 gene targets.The most effect was found in ASBVd, the internal control, but the mean difference was still less than two cycles (Fig. 4A, Supplementary Table S2).Since the test was developed for use with the clinical specimen, five clinical specimens (two positive and three negative samples) were run instead of using the reference materials at RT time of 4 min and 30 s.The results revealed that 4 min was significantly better than 30 s for all 3 SARS-CoV-2 gene targets (Fig. 4B).As a result, the 4 min RT time was selected.
In addition to denaturing the target RNA template, an initial denaturation step was required for two other purposes.First, it allowed full activation of a Hot Start Taq DNA polymerase if used.Second, it inactivated a reverse transcriptase to avoid interference with subsequent PCR steps.Since the assay used an aptamer-based Hot Start Taq DNA polymerase (New England Biolabs, USA) swiftly becoming active once the incubation temperature was above 45 °C, we speculated that the hold time of this step might also be shortened.Thus, the initial denaturation time was varied from 1 min to 30 and 2 s.The results showed that the performance of the assay using a 2 s initial denaturation was not inferior to the default 1 min setting for all three SARS-CoV-2 gene targets.(Fig. 5A, Supplementary Table S3).These findings suggested that the full function of the Hot Start Taq DNA polymerase was gained without any interference between RT and PCR when such a short hold time was used.Thus, the 2 s initial denaturation time was chosen.
From previous knowledge native Taq DNA polymerase has the extension rate of 24 nucleotides per second at 55 °C and up to 45-60 nucleotides per second at 70-75°C 30,31 .To amplify the targets sized 76-105 bp, the annealing/extension time was supposed to be at least 2-3 s.However, considering the fact that most conventional real-time PCR instruments will spend a few seconds longer for plate read in each cycle, we hypothesized that amplification of such short products might be efficient with a 1 s annealing/extension time setting.Therefore, the cycling denaturation and annealing/extension times of 2 s and 4 s were compared with 1 s each.The results clearly showed that the performance of 1 s each was not significantly different from that of 2 s and 4 s (Fig. 5B, Supplementary Table S4).Therefore, 1 s denaturation and 1 s annealing/extension were selected.
Aside from incubation at various steps, real-time RT-PCR assay wastes substantial time ramping between denaturation and annealing/extension temperatures.Hence, the range between denaturation and annealing/ extension temperatures should be reduced to shorten the run time.This could be achieved via two approaches, i.e., increment of the annealing/extension temperature and decrement of the denaturation temperature.
At first, the annealing/extension temperature was varied from 60 to 65 °C.The findings showed that the temperature could be raised to as high as 65 °C without detrimental effects on SARS-CoV-2 ORF3a and N gene targets.Meanwhile, only 60 and 61 °C worked best for the SARS-CoV-2 ORF1ab gene target and the internal control, without the delayed Ct (Fig. 5C, Supplementary Table S5).Lastly, we predicted the melting temperature (Tm) of each amplicon using uMelt Quartz and found that the Tm of SARS-CoV-2 ORF1ab, ORF3a, and N amplicons were estimated as 84.5, 84, and 84 °C, respectively.At the same time, the predicted Tm of the ASBVd amplicon was 81.5 °C (Supplementary Fig. S1).We hypothesized that lowering the denaturation temperature down to 85 °C might be sufficient for an effective amplification.Thereby, the denaturation temperature was varied from 92 to 90, 88, 85, and 82 °C.The findings revealed that the denaturation temperature as low as 85 °C still gave an efficient amplification of all targets not different from the higher temperatures (Fig. 5D, Supplementary Fig. S2, Supplementary Table S6).Thus, denaturation at 85 °C was opted.
Altogether, the extreme setting of our newly developed assay named "Super Speed inSpector of COVID-19" or "ƩS COVID-19" was a 4 min RT step at 55 °C followed by 2 s initial denaturation at 95 °C and 40 cycles of 1 s denaturation at 85 °C and 1 s annealing/extension at 60 °C.Total run time was reduced to 24 min 46 s (more than 60% reduction compared with the standard protocol) (Fig. 6).

Specificity of ƩS COVID-19
To determine the specificity, 22 respiratory samples positive for other 16 common respiratory viruses detected using either BioFire Respiratory 2.1 Panel or QIAstat-Dx Respiratory SARS-CoV-2 Panel were tested with ƩS COVID-19.The aforementioned respiratory viruses included coronavirus OC43, coronavirus 229E, coronavirus Fig. 3.The performance of the real-time RT-PCR assay in the adjusted setting compared with the initial setting (the standard protocol).The mean ± SEM cycle threshold (Ct) values from an experiment with triplicate amplification were compared between two settings in each SARS-CoV-2 gene target (ORF1ab, ORF3a, and N genes) and the internal control (ASBVd).An unpaired t-test was used for comparison analysis.The significant difference was when p < 0.05.NL63, coronavirus HKU1, influenza A(H1N1)pdm09 virus, influenza A(H3N2) virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, rhinovirus/enterovirus, adenovirus, and human bocavirus.The results confirmed that the newly developed assay had no cross-reactivity with any of these common respiratory pathogens (Fig. 8).

Discussion
Accurate diagnosis of COVID-19 is crucial for patient management as well as infection control measures.Realtime RT-PCR is a standard method recommended for the definite diagnosis of COVID-19.Nevertheless, at least 2-4 h are required before obtaining the result which is not suitable for medical service in outpatient and emergency departments.In recent years, several rapid real-time RT-PCR assays for SARS-CoV-2 detection were developed in response to COVID-19 pandemic.These assays could be completed within 20-30 min while maintaining the sensitivity as high as conventional real-time RT-PCR.Most of these assays required an operation on a specially designed microfluidic device which allowed the reaction mixture to immediately move between the denaturation and annealing/extension chambers.For instance, Cobas Liat System (Roche, Switzerland) and  PicoGene PCR1100 (Nippon Sheet Glass, Japan) could generate the results within 20 min by this approach 24,32,33 while Q-POC (QuantuMDx Group, UK) could do the same in approximately 30 min 34,35 .However, most of these platforms could process just one clinical specimen per run which might be a bottleneck for medical services during an epidemic season.Therefore, this study developed a one-step quadruplex rapid real-time RT-PCR assay for SARS-CoV-2 detection named "ƩS COVID-19", operating on an opened conventional real-time RT-PCR instrument.In addition to its rapid run time of less than 25 min, the newly developed assay could expand the throughput to as high as 94 samples per run.
The basis underlying the development of the rapid real-time RT-PCR assay comprised 4 main steps.First, primers with the highest Tm (60-65 °C or over) and short amplicons (approximately 70-100 bp) were designed.Usage of primers with high Tm would allow the adoption of two-step PCR instead of three-step PCR which would greatly reduce total run time.At the same time, short amplicons would aid in minimizing hold time in the next step.Second, a fast ramp rate was applied.In the standard mode of QuantStudio 5 used in this study, the ramp rate was constant at 1.6 °C/s.When the fast mode was opted, the ramp rates were automatically adjusted to 4.13 °C/s in the denaturation step and 3.16 °C/s in the annealing step.For some real-time PCR instruments that do not have fast mode, e.g., CFX96 real-time PCR detection system (Bio-Rad Laboratories, USA), fast ramp rate can also be applied by manually changing the ramp rate setting for each step to the maximum (5 °C/s).Third, hold time in each step (RT, initial denaturation, cycling denaturation, and annealing/extension) was minimized.In this study, although RT time could be decreased to as short as 30 s for the reference materials, a 4 min RT time was required when testing with the clinical specimens.This discrepancy was also observed by a previous study showing that a 30 s RT time was sufficient for pure SARS-CoV-2 RNA but at least 5 min was needed for extraction-free clinical specimens.This might be affected by the presence of inhibitors in the patient's respiratory samples 36 .In this study, the amplification of short amplicons (ranging from 76 to 105 bp) using the ultra-short hold time (1 s denaturation and 1 s annealing/extension time) and a Hot Start Taq DNA Polymerase was successfully implemented.Several previous studies also reported the success of using this ultra-short setting in the development of rapid real-time RT-PCR assays for animal viruses 37,38 and SARS-CoV-2 39,40 .Lastly, the range between denaturation and annealing/extension temperatures was narrowed.This could be done by lowering the denaturation temperature and/or raising the annealing/extension temperature.To our knowledge, this was the first study that introduced the approach of predicting the Tm of the amplicons and determining the possible lowest denaturation temperature to be used in the assay.By this approach, lowering the denaturation temperature down to 85 °C which was much lower than typical PCR (94-96 °C) was enabled.
Upon applying all the aforementioned approaches, the assay's run time could be shortened to less than 25 min which was more than 60% reduction compared with the standard real-time RT-PCR protocol.The speed of ƩS COVID-19 was close to other rapid real-time RT-PCR assays for SARS-CoV-2 detection.In a previous study, Lownik JC et al. reported the development of a rapid monoplex real-time RT-PCR assay for SARS-CoV-2 detection using CDC N1 primers and probe.Their assay relied on 5 min RT time and 10 s annealing/extension time.Nonetheless, operating on a capillary-based LightCycler 1.5 instrument (Roche, Switzerland) which had a much faster ramp rate (20 °C/s) allowed the assay to be completed in 20 min 36 .In another study, Milosevic J et al. reported the development of three parallel rapid real-time RT-PCR assays for SARS-CoV-2 detection using CDC N1, N2, and human RNase P (RP) primers and probes.Although their assays also depended on 40 cycles of 1 s denaturation and 1 s annealing/extension time, the denaturation temperature was set at 95 °C.These assays were operated on CFX96 real-time PCR detection system and could be completed in 30 min, a bit longer than ours 40 .In another study, Bustin S et al. reported the development of a multiplex rapid RT-PCR called CoV2-ID targeting 3 SARS-CoV-2 genes (NSP10, NSP12, and N).The assay also carried on 40 cycles of 1 s denaturation and 1 s annealing/extension steps, at 95 °C and 60 °C, respectively.Nevertheless, they used multiple cycle fluorescence detection (MCFD) which detected signal at only cycles 8 (for baseline), 15, 20, 25, 30, and 35 instead of real-time detection.In this way, the CoV2-ID could be completed in less than 20 min 39 .Since SARS-CoV-2 mutated continuously, three conserved SARS-CoV-2 gene targets were included in the newly developed assay to ensure that the performance of the assay was not affected if one of these gene targets mutated.In the previous study, CoV2-ID also included 3 SARS-CoV-2 gene targets (NSP10, NSP12, and N).Nonetheless, the NSP12 target was intentionally added in order to improve the sensitivity of the assay and was detected in the same channel as the NSP10 target 39 .
The newly developed assay could detect SARS-CoV-2 ORF1ab, ORF3a, and N gene targets as low as 1.835, 1.310, and 1 copy/reaction, respectively.This assay had high diagnostic sensitivity (92.8%), specificity (100%), and accuracy (96.7%) when compared with FDA EUA approved Cobas SARS-CoV-2 System.The performance of ƩS COVID-19 was comparable to rapid real-time RT-PCR assays developed by other researcher groups.The extraction-free LightCycler SARS-CoV-2 assay using CDC N1 primers and probe was reported to reach the LOD of 3 copies/reaction.It had a positive percent agreement (PPA) of 97.6% and a negative percent agreement   36 .Three parallel CFX96 SARS-CoV-2 assays using CDC N1, N2, and RP primers and probes were reported to yield the LOD of 25 copies/reaction for both N1 and N2 targets.They had a PPA of 100%, NPA of 100%, and overall percent agreement (OPA) of 100% when compared with Xpert Xpress SARS-CoV-2 (Cepheid, USA) 40 .The CoV2-ID was reported to have the LOD of 2 and 5 copies/reaction for NSP10 and NSP12, respectively.The assay had a sensitivity of 100%, specificity of 100%, and accuracy of 100% when compared with the VIASURE SARS-CoV-2 real-time PCR detection kit (Certest Biotec, Spain) 39 .In addition, this was the first study that described the use of a viroid RNA as an internal control for human virus testing.Single-stranded viroid RNA is extensively self-complementary forming a robust secondary structure 41,42 .Its secondary structure seemed to provide more durability and resistance to RNases than hostderived RNA 43 .In addition to its native difficult template, this study designed the internal control amplicon size larger than the target amplicons and successfully utilized it as the internal control for the non-competitive rapid real-time RT-PCR.In a previous study, Botermans M et al. also reported the usage of a viroid as an internal control in multiplex real-time RT-PCR for the detection of other viroids causing diseases in plants 43 .In another study, Dinkle KE et al. reported the use of hepatitis D virus (HDV) RNA, which also had the rod-like secondary structure, as an internal control for nested multiplex RT-PCR of other respiratory viruses 44 .
Apart from switching to using a real-time PCR instrument with a faster ramp rate, there is still room to improve the performance and versatility of our rapid real-time RT-PCR assay.The former; amplification of longer amplicons might be enabled if an alternative high-speed DNA polymerase, such as KAPA2G Fast, Klen-taq1, and KOD polymerases, was used [45][46][47] .The latter, hold time might be further shortened by usage of a higher concentration of primers (5-20 μM) in a smaller reaction volume (5-10 μl) 36,45,48 .However, these approaches could come at the expense of a higher cost.
In conclusion, based on strategies including designing primers with high Tm and short amplicons, applying fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing/extension temperatures; we could develop a one-step quadruplex rapid real-time RT-PCR assay for SARS-CoV-2 detection that could be accomplished within 25 min.The newly developed rapid real-time RT-PCR assay was highly sensitive, specific, and fast suitable for use as an alternative method to support laboratory diagnosis of COVID-19 in outpatient and emergency departments.

Primer and probe design
One hundred and fourteen SARS-CoV-2 nucleotide sequences were retrieved from the GenBank and Global Initiative on Sharing Avian Influenza Data (GISAID) databases (their accession numbers were listed in Supplementary Table S1).Three pairs of primers and three hydrolysis probes targeting the conserved regions of ORF1ab, ORF3a, and N genes of SARS-CoV-2 were designed using the Primer-Blast program (Fig. 2) 49 .A pair of primers and a hydrolysis probe specific to Avocado sunblotch viroid (ASBVd), which was used as an exogenous internal control, were designed using the PrimerQuest program (Integrated DNA Technologies, USA) available from https:// www.idtdna.com/ SciTo ols.All primers and probes were synthesized by Macrogen (South Korea).The sequences of the primers and probes were shown in Table 2.The melting temperature of each amplicon was predicted using the uMELT Quartz program 50 .

Reference materials
Three RNA transcripts of SARS-CoV-2 ORF1ab, ORF3a, and N genes were transcribed in vitro by HiScribe T7 quick high yield RNA synthesis kit (New England Biolabs, USA) using synthetic DNA fragments (based on SARS-CoV-2 reference genome, isolate Wuhan-Hu-1, GenBank accession number NC_045512.2) appended with T7 promoter sequence (Macrogen, South Korea) as their templates.One microgram of the template DNA was mixed with NTP Buffer Mix (6.7 mM each) and 2 μl of T7 RNA Polymerase Mix.Nuclease-free water was

Fig. 2 .
Fig. 2. The SARS-CoV-2 genome was targeted by three sets of primers and probes specific for ORF1ab, ORF3a, and N genes.Red half arrows represented forward and reverse primers.Green half arrow represented probes.The figure was created with BioRender.com.

Fig. 4 .
Fig. 4. The performance of the adjusted real-time RT-PCR assay (A) The RT time was varied from 5 to 4 min, 2 min, and 30 s.The comparison of mean ± SEM Ct values of SARS-CoV-2 gene targets (ORF1ab, ORF3a, and N genes) and the internal control (ASBVd) was analyzed among the different RT time settings using the reference materials; (B) Comparing the mean ± SEM Ct values of SARS-CoV-2 gene targets in clinical samples at RT time between 4 min and 30 s.The significant level was calculated using an unpaired t-test and one-way ANOVA with Turkey's multiple comparison test.The asterisk indicated the statistically significant difference.*p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 5 .
Fig. 5.The performance of the real-time RT-PCR assay.(A) when varying initial denaturation time from 1 min to 30 and 2 s; (B) when cycling denaturation and annealing/extension time was compared between 2-4 s and 1-1 s; (C) when varying annealing/extension temperatures from 60 to 65 °C; (D) when varying denaturation temperatures from 82 to 85, 88, 90, and 92 °C.The comparison of mean ± SEM Ct values of SARS-CoV-2 gene targets (ORF1ab, ORF3a, and N genes) and the internal control (ASBVd) was analyzed among the different conditions.The significant difference was calculated using an unpaired t-test and one-way ANOVA with Turkey's multiple comparison test.The asterisk indicated the statistically significant difference.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig. 7 .
Fig. 7.The sensitivity of the rapid real-time RT-PCR assay (ƩS COVID-19).The sensitivity of ƩS COVID-19 was determined using the reference SARS-CoV-2 RNA ranging from 10 6 to 1 copies/reaction.Forty cycles of amplification were done in 20 replicates for each concentration of the reference RNA.The amplification plots and the standard curve of the (A) ORF1ab, (B) ORF3a, and (C) N genes of the SARS-CoV-2.The error bar represented the mean ± SEM.

Table 2 .
The primers and probes used in the rapid real-time RT-PCR assay (ƩS COVID-19).F forward, R reverse, P probe.