key: cord-0906567-p8rsagx2 authors: Asghari, E.; Höving, A.; van Heijningen, P.; Kiel, A.; Kralemann-Köhler, A.; Lütkemeyer, M.; Storm, J.; Vollmer, T.; Knabbe, C.; Kaltschmidt, B.; de Vos, G.; Kaltschmidt, C. title: Ultra-fast one-step RT-PCR protocol for the detection of SARS-CoV-2 date: 2020-06-26 journal: nan DOI: 10.1101/2020.06.25.20137398 sha: cf3c5953c885bfe383a7a9b5257b3300f5c24e90 doc_id: 906567 cord_uid: p8rsagx2 The COVID-19 pandemic resulted in lockdowns all over the world thus affecting nearly all aspects of social life and also had a huge impact on global economies. Since vaccines and therapies are still not available for the population, prevention becomes desperately needed. One important aspect for prevention is the identification and subsequent isolation of contagious specimens. The currently used methods for diagnostics are time consuming and also hindered by the limited availability of reagents and reaction costs, thus presenting a bottle neck for prevention of COVID-19 spread. Here, we present a new ultra-fast test method which is ten times faster than conventional diagnostic tests using real time quantitative PCR (RT-qPCR). In addition, this ultra-fast method is easy to handle as well as cost effective. We translated published SARS-CoV-2 testing protocols from the Centers of Disease Control and Prevention (Atlanta, Georgia, USA) and the Charite Berlin (Germany) to the NEXTGENPCR (NGPCR) machine and combined it with a fluorescence-based endpoint measurement. Fluorescence was measured with a commercial blue light scanner. We confirmed the NEXTGENPCR results with commercially available positive controls. In addition, we isolated RNA from SARS-CoV-2 infected patients and achieved similar results to clinical RT-qPCR assays. Here, we could show correlation between the results obtained by NEXTGENPCR and conventional RT-qPCR. human residual material to evaluate suitability of an in vitro diagnostic medical device ( §24). The need for informed consent and ethical approval was waived since all materials used were waste from routine laboratory diagnostics. The COVID-19 pandemic resulted in lockdowns all over the world thus affecting nearly all aspects of social life and also had a huge impact on global economies. Since vaccines and therapies are still not available for the population, prevention becomes desperately needed. One important aspect for prevention is the identification and subsequent isolation of contagious specimens. The currently used methods for diagnostics are time consuming and also hindered by the limited availability of reagents and reaction costs, thus presenting a bottle neck for prevention of COVID-19 spread. Here, we present a new ultra-fast test method which is ten times faster than conventional diagnostic tests using real time quantitative PCR (RT-qPCR). In addition, this ultra-fast method is easy to handle as well as cost effective. We translated published SARS-CoV-2 testing protocols from the Centers of Disease Control and Prevention (Atlanta, Georgia, USA) and the Charité Berlin (Germany) to the NEXTGENPCR (NGPCR) machine and combined it with a fluorescence-based endpoint measurement. Fluorescence was measured with a commercial blue light scanner. We confirmed the NEXTGENPCR results with commercially available positive controls. In addition, we isolated RNA from SARS-CoV-2 infected patients and achieved similar results to clinical RT-qPCR assays. Here, we could show correlation between the results obtained by NEXTGENPCR and conventional RT-qPCR. The COVID-19 pandemic of late 2019 is a major threat to people's health worldwide, with no approved therapeutic drugs or vaccines available 1 shows that testing capacity can be a key in controlling outbreaks 5 . The most prevalent testing method for the SARS-CoV-2 virus currently is the use of one-step reverse transcription quantitative polymerase chain reaction (RT-qPCR) 6,7 . Despite RT-qPCR being a generally well-established diagnostic technique 8 , testing for SARS-CoV-2 is currently limited in many regions 9 . Two current issues limiting the testing capacities are the availability of the reagents 10 and the technical capability of the qPCR machines limiting the total amount of possible tests per day. The NEXTGENPCR (NGPCR) machine is a new platform capable of performing polymerase chain reactions in short time frames 11 . A mechanical system moves a reaction plate between three zones of heating at different temperatures. Each zone consists of two blocks at equal temperature. Samples are contained in thin plastic foils, which are pressed between the two perpendicular blocks in a zone, ensuring sample mixing. This allows instant temperature changes of the sample, thus removing the limitations of classical Peltier element-based PCR machines. The reaction is no longer slowed down by the ramping rates at different temperatures. The NEXTGENPCR machine combines ultrafast cycling with the possibility to perform a RT-step within the same program, before the PCR reaction. In this study, we adapted two commonly used SARS-CoV-2 diagnosis protocols for usage with the NEXTGENPCR machine. Both assays use hydrolysis probes (ex. TaqMan), making them suitable for fluorescence-based endpoint measurements to determine amplification. The primer/probe sets for the detection of the viral nucleocapsid 1 and 2 (N1 and N2) genes from "Centers for Disease Control and Table 3 ). 5 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. Each clinical RNA sample assayed in-house was run in duplicate using 1 µl of the eluate as a template. Direct reverse transcription with the qScript™ XLT ToughMix® from Quanta Biosciences (Beverly, Mass., USA) was performed. As incubation time for the RT-step, 5 min at 55°C was used in the NEXTGENPCR machines and 10 min in the RT-qPCR machine. The reagent volumes in the CDC Assay and the Drosten Assay were used as instructed by the manufacturers. The optimized protocol used for the NEXTGENPCR is shown in Fig. 2 F. For the RT-qPCR we used a Corbett Rotor-Gene 6000 (Qiagen, Hilden, Germany) with the settings shown in Table 4 . 6 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. For each assay, additional plates with no template controls (NTCs) were prepared and the mean of the NTCs plus three times the standard deviation was set as the threshold for quantification. Absolute fluorescence values measure from 0 to 256. If only one of the measured wells showed a grey level above the threshold level, the value of the well qualifying as negative was excluded from further analysis and the sample was called positive. If both wells showed grey levels below the threshold value, the sample was called negative and no quantification value was shown in the quantification graphs. 7 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. . To visualize the workflow used in this study we made a graphical abstract (Fig 1) . In For measuring the hydrolysed fluorescent probes, a fluorescence scanner (MicroTek Bio1000F) obtained from MBS was used (Fig. 1 C) . PCR plates were scanned (Fig. 1 D) and quantified with QuickDetect™ ( Fig. 1 E) , which automatically recognizes the liquid within each well and quantifies fluorescence in 256 grey levels. The software scores each well, positive (red) or negative (green), based on user-defined threshold values. A typical result of the quantification is shown in Fig. 1 F. 8 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We performed an optimization in the NEXTGENPCR machine of the CDC panel protocol using the according plasmid standard (Fig. 2 A, B, C) . We determined that the protocols using 35 and 40 cycles are sufficient to detect at least 2000 plasmid copies, see figure 2 b and c. 40 and 45 cycles were efficient in amplification of as low as 40 copies. However, at the end of 45 cycles, some NTC controls appeared positive. We therefore conclude that limitation of cycle number to 40 in endpoint PCR might be a way to avoid false positive results. In these experiments with 40 cycles PCR amplification time was 10 min. 9 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. . https://doi.org/10.1101/2020.06.25.20137398 doi: medRxiv preprint The optimization of the Drosten panel was performed using synthetic RNA (Twist Synthetic SARS-CoV-2 RNA Control 1, Twist Bioscience San Francisco), thus in contrast to the CDC panel optimization included an RT step. Comparison between annealing at 58°C (Fig.2 D) and 61°C (Fig.2 E) yielded no obvious difference in amplification efficiency. Like 45 cycles (Fig.2 D, E) , 40 cycles (Fig.2 F) 10 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. . https://doi.org/10.1101/2020.06.25.20137398 doi: medRxiv preprint Additionally, we tested the reproducibility of the protocol used in Fig. 2 C on five different NEXTGENPCR machines (Fig. 3 A-E) . We were able to reproduce identical results on all five machines. Furthermore, we excluded the occurrence of edge effects by testing different positions on the plate (Fig. 3 F) using the protocol shown in Fig.2 F. Taken together, the reactions seem to be robust and transferable. 11 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. directly observable by eye in the endpoint NEXTGENPCR assay (Fig.4 B) . Qualitative scoring by visual inspection of the NEXTGENPCR tests becomes difficult with samples that have a low amount of viral RNA (copies with corresponding Cq values above 28; see Fig. 4 samples 2,4,5,6, and 7) . QuickDetect™ will also score lower fluorescent values and discriminate between positives and negatives. Thus, we conclude the endpoint-based method is a great simplification, allowing for rapid diagnosis of high load samples. The official recommendation for RT-qPCR assay positivity from the CDC are Cq values below 40 cycles 6 , rendering the CDC non template controls positive in our hands (Fig. 5 A) . All NTC wells in this plate were called positive using the NEXTGENPCR method (in the range of 1 synthetic RNA-copy). This could be caused 13 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. . by contamination, maybe resulting from the absence of a clean room environment in our assays. The threshold for the NEXTGENPCR could be adjusted accordingly. For the RT-qPCR of the Drosten panel, the threshold for positivity can be set at the recommended 40 cycles. As the CDC N1 assay turned positive in the NTC with a Cq value of 36.5, we suggest a threshold of 36. For the CDC N2 assay we suggest a threshold of 38 cycles based on similar reasoning. The sensitivity of the Drosten-and the CDC RT-qPCR as well as tests performed on the NEXTGENPCR machine ranged between 1 to 10 synthetic RNA copies (Fig. 5 ). 14 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. . https://doi.org/10.1101/2020.06.25.20137398 doi: medRxiv preprint Samples were scored positive if either replicate exceeded the thresholds set according to the values determined in Figure 5 . The RT-qPCR assay using the Drosten panel called all of the samples positive (Fig. 6 A) , resulting in 100% correlation with the results from an accredited clinical diagnostic laboratory (Fig. 4 A) . The NEXTGENPCR method using the Drosten panel resulted in 9 of 10 positive samples being correctly called (sample 4 was called negative). (Fig. 6 B, E) . Using the CDC N1 primer/probe pair on the RT-qPCR platform we failed to determine samples 4 and 5 as positive. However, using the CDC N2 assay resulted in 100% correct calls. ( Figure 6A ) The CDC N1 and N2 assays on NEXTGENPCR resulted in 100% correlation with diagnostic laboratory results (Fig. 6 C, D, E) . The discordant samples may have failed to be detected since only 20% of the recommended samples volume was available for the testing here. These samples could not be re-tested due the limited amounts donated for this study. 16 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We aimed to analyse a potential correlation of Cq values measured by RT-qPCR with grey levels measured in a scanner after endpoint PCR using NEXTGENPCR. Therefore, we plotted Cq values on the y-axis and grey levels on the x-axis. Strikingly, we could see a good correlation of the endpoint measurement with the Cq values with a R² of 0.94 for the Drosten panel tested with a synthetic RNA dilution series (Fig. 7 A) . We could only detect minimal change of the function from linear regression between the dilution assay of the synthetic RNA and the clinical samples For the Drosten panel endpoint assay, a trend of saturation can be observed for fluorescence values above 185 (Fig 7 C) . This either results from optical limitations of the scanner or from chemical limitations as a component of the chemical reaction might be spent. We hypothesized, that for the CDC protocol saturation effects might have biochemical reasons as it appears earlier than in the Drosten protocol. 18 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. To determine whether the saturation effect in the Drosten panel results from optical limitations, a dilution experiment was measured at different scanner exposure time settings. As the spread of the data is maximal between 2x and 3x exposure time and decreases in higher exposure time settings, the observed saturation effect is of optical nature. Of note, different exposure time settings are needed to reach an optimal distinguishability of data points on the lower or higher ends of the spectrum. 19 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. As mentioned above, the adaptation of other RT-qPCR based protocols should work in a similar fashion, with the possibility of further cost reduction. At the time of publication, the three other SARS-CoV-2 assays shown in Table 5 have also been optimized on the NEXTGENPCR (GDV, PVH personal communication). Table 5 shows five different RT-qPCR based SARS-CoV-2 test panels with the associated costs. While reagent costs vary between 1,59 and 1,93 € per reaction, the main difference is the performed number of reactions in the protocols. However, the less genes are included in the panel, the higher the possibility of false results. 20 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. . https://doi.org/10.1101/2020.06.25.20137398 doi: medRxiv preprint The sensitivity was considered using RNA or plasmid standards and eluates of clinical samples. Another important aspect is the preparation of PCR templates (e.g. isolation of nucleic acids), which is not considered in this study. The most obvious limitation of the endpoint PCR is a need for an additional analysis step using the fluorescence scanner. We noted before that the sensitivity of the Drosten panel in the NEXTGENPCR is reduced in low copy number clinical samples (Fig. 4) . As shown in Figure 8 , the scanner has some optical limitations. Adjusting the scanning settings for low fluorescence and high fluorescence wells separately might optimize sensitivity, while also allowing good correlation with corresponding Cq values. Future effort could be concentrated on the development of better fluorescence scanners and or the development of a real time extension for NGPCR. In its current version NEXTGENPCR is fast and easy to use, and hands on time is reduced when integrated into a robotic workflow. Although usually qPCR is used to score and quantify the amount of virus in a sample, it is not clear that the amount of sample is related to the severity of the disease. Rather certain genetic properties could be linked to the severity of the disease. Furthermore, RT-qPCR platforms have higher costs and rely on highly skilled personnel. Therefore, it is only logical to reduce the complexity of the method of detection and rely on endpoint PCR. Using a NEXTGENPCR machine and RNA extraction rather than isolation brings a complete test from sample to result in less than 30 minutes into view. The method described here would facilitate both population screening or point-of-care testing. 21 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 26, 2020. . Coronavirus disease 2019 (COVID-19): current status and future perspectives WHO Coronavirus Disease (COVID-19) Dashboard. Accessed The socio-economic implications of the coronavirus pandemic (COVID-19): A review Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV-2) Am 8:00. Coronavirus cases have dropped sharply in South Korea Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front Microbiol Pm 12:15. The standard coronavirus test, if available, works well-but can new diagnostics help in this pandemic? Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources Super fast using sufficiently less time and energy Information for Laboratories about Coronavirus (COVID-19) We acknowledge funding of the University of Bielefeld and a supply of reagents and consumables from Molecular Biology Systems B.V., Goes, The Netherlands. Is not required. All samples were collected by the authors in accordance with the AH, ML, EA, JS, AK, CKn, CKa, BK, TV have declared no competing interest. GDV and PVH are employees of Molecular Biology Systems B.V., Goes, The Netherlands. We acknowledge funding by the University of Bielefeld. Some test-kits and PCR plates were kindly provided by Molecular Biology Systems B.V., Goes, The Netherlands. Funding for equipment was partially provided by Evangelisches Klinikum Bethel (EvKB).