key: cord-0841992-6dc5oz66 authors: Takahashi, M.; Tehseen, M.; Salunke, R.; Takahashi, E.; Mfarrej, S.; Sobhy, M. A.; Alhamlan, F.; Hala, S.; Mandujano, G. R.; Al-Qahtani, A. A.; Alofi, F. S.; Alsomali, A.; Hashem, A. M.; Khogeer, A.; Almontashiri, N. A. M.; Lee, J. M.; Mon, H.; Sakashita, K.; Li, M.; Kusakabe, T.; Pain, A.; Hamdan, S. M. title: R3T (Rapid Research Response Team) One-step RT-qPCR kit for COVID-19 diagnostic using in-house enzymes date: 2020-08-04 journal: nan DOI: 10.1101/2020.07.31.20165704 sha: 0116d59e5be0d8e7b23d96fdadfd0088261120c9 doc_id: 841992 cord_uid: 6dc5oz66 One-step RT-qPCR is the most widely applied method for COVID-19 diagnostics. Designing in-house one-step RT-qPCR kits is restricted by the patent-rights for the production of enzymes and the lack of information about the components of the commercial kits. Here, we provide a simple, economical, and powerful one-step RT-qPCR kit based on patent-free, specifically-tailored versions of Moloney Murine Leukemia Virus Reverse Transcriptase and Thermus aquaticus DNA polymerase termed the R3T (Rapid Research Response Team) One-step RT-qPCR. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of the SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity as that of the Invitrogen SuperScript III Platinum One-step RT-qPCR and TaqPath 1-Step RT-qPCR kits. Overall, our kit has shown robust performance in both of laboratory settings and the Saudi Ministry of Health-approved testing facility. In December 2019, an outbreak of a new syndrome characterized by serious symptoms including fever, severe respiratory illness, and acute pneumonia eventually leading to respiratory failure and death was reported in Wuhan city of Hubei province in China. This disease swiftly spread out across the globe resulting in unprecedented preventive measures worldwide 1-2 . On January 7 th , 2020, the Chinese health authorities confirmed that this recently discovered syndrome was associated with a new member of the coronavirus (CoV) family closely related to a group of severe acute respiratory syndrome coronaviruses (SARS-CoV) [3] [4] . On February 11 th , the World Health Organization (WHO) designated the name "coronavirus disease 2019" abbreviated as (COVID-19) to this highly contagious disease and declared it as pandemic (http://www.euro.who.int/en/health-topics/health-emergencies/coronaviruscovid-19/novel-coronavirus-2019-ncov). As of July 2020, COVID-19 resulted in nearly 15.1 million confirmed cases including over 620,000 fatalities globally the appropriate thermal cycle program in the qPCR machine, and analyse the data. Because of the minimal experimental handling, this platform is the best suited for highthroughput screening environment within a fully automated work-flow. On the other hand, two-step RT-qPCR is a method that combines two separate RT and qPCR reactions. Because each reaction is spatially separated, two-step RT-qPCR gives better control over several experimental parameters; such as the size of RT reaction, The full-length gene of MMLV-RT with cleavable TEV-8xHis-Strep tag at the Cterminus was cloned into the pDEST8 plasmid as previously described 31 The CDC designed RT-qPCR assay primer and probe set (2019-nCoV CDC EUA kit, Cat. No. 10006606) was purchased from Integrated DNA Technologies (IDT). The kit contains research-use-only primer and probe sets based on the protocol released by All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20165704 doi: medRxiv preprint CDC (hereafter called CDC assay). The CDC assay includes three sets of the primers and probe labeled with 5´ FAM dye and 3´ Black Hole Quencher® (BHQ). perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . In order to determine the sensitivity of our R3T One-step RT-qPCR kit and detect SARS-CoV-2 in clinical samples, we performed one-step RT-qPCR using primers and perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. The expression and purification of the native form of Taq In contrast, we did not detect such enhancement for the expression of native Taq Pol (N-Taq Pol) under the control of pET vector system (data not shown). Tagging at the N-terminus with histidines also enabled us to eliminate the time-consuming polyethylenimine precipitation steps in the previous purification protocol 32 without affecting the purity of the final product. As shown in Figure 2C , the purity of the His-All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20165704 doi: medRxiv preprint To perform two distinct reactions within the same tube, the two enzymes, Taq perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20165704 doi: medRxiv preprint the mixing ratio to be 2 ng MMLV-RT to 1 unit Taq Pol for subsequent buffer optimization experiments. We also investigated the salt composition of the buffer to further improve the Taq Pol's activity in the PCR reaction with MMLV-RT. We opted for ammonium sulfate as additional salt and tested different concentrations in the PCR reactions ( Figure 3D ). We found that the intensity of the PCR product bands gradually increased up to 20 mM of ammonium sulfate followed by decrease at higher concentration of ammonium Determination of the enzyme amounts used in the one-step RT-qPCR reaction, its sensitivity and SARS-CoV-2 detection limit. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . https://doi.org/10.1101/2020.07. 31.20165704 doi: medRxiv preprint In order to successfully develop a one-step RT-qPCR platform for the detection of SARS-CoV-2, we formulated and evaluated the efficiency of purified Taq Pol and MMLV-RT in the one-step RT-qPCR scheme using N1 and N2 primer sets and synthetic SARS-CoV-2 RNA as a template. We found that a mixing ratio of 2 ng C-His/Strep MMLV-RT to 1 unit His-Taq Pol still sustained the Taq Pol's activity in PCR ( Figure 3C ). Therefore, in our initiail screening, we tried to determine the optimal amount of MMLV-RT in the RT-qPCR reaction while keeping its relative ratio to Taq RNAs ranging from 10 to 10 5 copies/µL as a template. The sensitivity of our one-step RT-qPCR system is estimated in terms of the limit of detection (LoD). The LoD assays demonstrated that our one-step RT-qPCR system can reliably detect 40 RNA copies per reaction in all of the three aforementioned combinations (data not shown). Furthermore, our data illustrated that the one-step RT-qPCR assays with 30 U: 60 ng/µL ratio was able to detect as low as 10 RNA copies per reaction from 9 out of 10 replicates as compared to 7 out of 10 replicates in case of 20 U: 40 ng/µL and 3 out of 10 replicates in case of 40 U: 80 ng/µL ratios, indicating the higher sensitivity of our system upon using 30 U: 60 ng/µL ratio. The slope and the R 2 values of each curve were used to evaluate the efficiency of individual assays (Figure 4) . The R 2 values provided an estimate of the goodness of the linear fit to the data points. In an efficient qPCR assay, R 2 should be very close or greater than 0.90. The amplification All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20165704 doi: medRxiv preprint efficiencies of all three His-Taq Pol/C-His/Strep MMLV-RT ratios were above 99% for both N1 and N2 primer sets ( Figure 4B, 4C, and 4D ). The standard curves showed high correlation coefficients of R 2 >0.99 for N1 primer set and R 2 >0.95 for N2 with the three His-Taq Pol/C-His/Strep MMLV-RT ratios. In order to validate the competency of our one-step RT-qPCR system to detect SARS-CoV-2 in clinical samples from patients, we used RNA samples extracted from the nasopharyngeal swabs of 20 different patients who tested positive besides three patients who tested negative for SARS-CoV-2 using Invitrogen SuperScript™ III Platinum™ One-step RT-qPCR kit ( Table 1 ). The positive samples had variable Ct values ranging from 16 to 38 applying the CDC qPCR N gene primer set from IDT. Based on the aformentioned LoD results, we used 30 U: 60 ng/µL ratio for our R3T One-step RT-qPCR kit on these clinical samples. The internal control human RNAseP gene (RP) was detected in all samples. As expected, the N1 and N2 primer pair of the viral N-gene was only detected in the positive samples not the negative ones (Table 1 ). In case of the three negative control samples (411, 429 and 440), Invitrogen SuperScript™ III Platinum™ One-Step RT-qPCR System gave some high Ct values upon applying N1 primer set but overall result undetermined. The overall Ct values demonstrated a narrow difference between our R3T Onestep RT-qPCR kit and Invitrogen SuperScript™ III Platinum™ One-Step RT-qPCR kit (Table 1) , demonstrating the sensitivity of our system for the detection of SARS-CoV-2 from clinical samples. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. Table 1 SARS-CoV-2 has originated the present international outbreak of a severe respiratory syndrome termed as COVID-19. The outburst of this viral infection has become a pandemic and severe public health challenge worldwide. Owing to the absence of effective medicines and vaccines, quick identification of the infected individuals and imposing self-quarantine measurements are the only effective containment strategies to avoid widespread community transmission. Therefore, the rapid development of low-cost, easy-to-make, yet sensitive and reliable diagnostic tests are crucial. In this study, we devised a sensitive, cost-effective, in-house one-step RT-qPCR system based on patent-free Taq Pol and MMLV-RT. These enzymes were discovered in the 1970s [33] [34] ; however, they still play a key role as molecular biology tools owing to their robustness and unique enzymatic properties. To avoid legitimate concerns regarding the intellectual property rights, we opted to purify those patentfree enzymes and optimize their use for the one-step RT-qPCR platform. To simplify All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. In the scheme of COVID-19 detection, the RT primers are explicitly designed to be gene-specific, besides the RNA regions annealed by the RT primers are carefully selected to avoid erroneous interference in cDNA synthesis. We assume that the welldesigned RT primers for cDNA synthesis conceals any possible enzymatic All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20165704 doi: medRxiv preprint incompetency of our MMLV-RT and enables the detection of COVID-19 RNAs even at low threshold (see discussion below). Our His-Taq Pol is also not a hot-start polymerase like Platinum Taq Pol that we used as a benchmark for performance comparison during this study. However, the advantages of the hot-start PCR polymerase, such as the prevention of primer-dimer formation and non-specific amplification, were not pronounced for the TaqMan based RT-qPCR platform. We also found the use of the relatively short fragments for amplification to be in favor for our His-Taq Pol in the one-step RT-qPCR scheme. We believe that the synergy between using TaqMan based detection system and the short size of amplicons makes the performance of our non-hot-start His-Taq Pol equally robust to that of hot-start Platinum Taq Pol in the one-step RT-qPCR reaction. Our aim from the current study was to establish the one-step RT-qPCR kit with in-house enzymes. The advantages of a one-step RT-qPCR platform over a two-step process in the work-flow at the point-of-care diagnostics include minimal sample handling, reduced bench time, and less chances for pipetting errors and crosscontamination ( Figure 1 ). This makes the one-step platform the first choice for highthroughput mass screening in the diagnostic point-of-care tests. In the one-step platform, it is crucial to carefully choose the optimal conditions for both MMLV-RT and Taq Pol to work together in the same reaction (1, 2). A major obstacle in assembling our one-step RT-qPCR system was the inhibition of the Taq Pol's activity in the Finally, we assessed our R3T One-step RT-qPCR kit with actual patient samples isolated from individuals infected by SARS-CoV2. We screened 23 different patients under laboratory settings, including three who were suspected but tested negative for SARS-CoV2. All the tests performed with our kit showed the expected true positive and negative results with a slight difference in the Ct values obtained from Invitrogen SuperScript™ III Platinum™ One-Step RT-qPCR. Furthermore, we evaluated the performance of our R3T One-step RT-qPCR kit in a Saudi Ministry of Health-approved testing facility using 192 patient samples who were screened for SARS-CoV-2 infection. Our kit matched their endorsed TaqPath™ 1-Step RT-qPCR kit by identifying the same 20 positive and 172 negative cases, demonstrating that our kit is ready for deployment in the actual testing facilities. Here, we provide a guide on how to assemble an in-house one-step RT-qPCR kit based on patent-free enzymes within a few weeks of conceptualizing the project. We believe that our one-step RT-qPCR system can be successfully applied for the routine SARS-CoV2 diagnostics and extended to detect other pathogens. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20165704 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20165704 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . https://doi.org/10.1101/2020.07.31.20165704 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. . Extraction/Purificatoin of RNAs Figure 2 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 4, 2020. 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