key: cord-0702129-okmowdem authors: Asprino, P.; Bettoni, F.; Camargo, A.; Coelho, D.; Coppini, G.; Correa, I.; Freitas, E.; Inoue, L.; Kitajima, J. P.; Kuroki, M.; Masotti, C.; Marques, T.; Reis, A.; Reis, L. F.; Santos, B.; dos Santos, E.; Schlesinger, D.; Sena, C.; Spadaccini, T.; Taniguti, L. title: A Scalable Saliva-based, Extraction-free RT-LAMP Protocol for SARS-Cov-2 Diagnosis date: 2020-10-27 journal: nan DOI: 10.1101/2020.10.27.20220541 sha: 67e372afdfc8fdfaf8cbdfc98bf54ad192cf15bd doc_id: 702129 cord_uid: okmowdem Scalable, cost-effective screening methods are an essential tool to control SARS-CoV-2 spread. We have developed a straight saliva-based, RNA extraction-free, RT-LAMP test that is comparable to current nasopharyngeal swab RT-PCR tests in both sensitivity and specificity. Using a 2-step readout of fluorescence and melting-point curve analysis, the test is scalable to more than 30,000 tests per day with average turnaround time of less than 3 hours. The test was validated using samples from 244 symptomatic patients, and showed sensitivity of 78.9% (vs. 85.5% for nasopharyngeal swabs RT-PCR) and specificity of 100% (vs. 100% for nasopharyngeal swabs RT-PCR). Our method is therefore accurate, robust, time and cost effective and therefore can be used for screening of SARS-CoV-2. The corona virus disease 2019 caused by the SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) has become a major public health emergency worldwide. Accurate diagnosis of COVID-19 is crucial to control disease transmission and manage infected patients. Reverse transcription followed by real-time Polymerase chain reaction (RT-PCR) assays using RNA extracted from nasopharyngeal swabs are the gold standard for SARS-CoV-2 molecular diagnostic and are currently the screening strategy used worldwide. These methods are cost and labor-intensive, including several steps that require sample handling, and have been restrained by lack of real-time PCR-specific instruments and reagents. Sample collection through nasopharyngeal swabs itself is also a major barrier to high-scale, low-cost testing due to limited availability of swabs and necessity of a trained professional, themselves at risk for infection. In (Notomi et al. 2000) LAMP is faster than PCR, utilizes a different DNA polymerase, and can be read out through multiple methods, ranging from simple colorimetric , to direct fluorescence (Lu et al. 2020 ) , Crispr-based assays (Joung et al. 2020) , and sequencing (James et al., n.d.) . LAMP methodology has been used for diagnosis of influenza virus (Poon et al. . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 2005) , Ebola virus (Kurosaki, Magassouba, and Oloniniyi 2016) , and a variety of other pathogens (Bartolone et al. 2018 ) . Several groups have published LAMP protocols for SARS-CoV-2 diagnosis, including FDA-approved protocols. However, the majority of these protocols are based on direct nasopharyngeal swab samples followed by RNA extraction which are still associated with some limitations, as has been discussed previously. To contribute towards solutions for Covid-19 diagnosis, we combined and optimized previously published protocols to establish a simplified, saliva-based, RNA extraction-free RT-LAMP test that centralized labs can execute at large-scale population levels. ) By focusing on simplicity as the primary goal, we have added to this body of scientific literature by creating a test that is cheap, fast, scalable, and accurate. IRB approval (HSL 2020-43) was obtained prior to clinical studies. Subjects included in the study presented to the Hospital Sírio-Libanês in São Paulo, Brazil with 1-7 days of symptoms suggestive of Covid-19. All subjects were diagnosed by routine nasopharyngeal swab RT-PCR collected at the same time as the saliva samples. Saliva samples were collected by patients themselves in sterile 50 ml conical tubes with no additives . Approximately 1 ml of saliva was collected and the pre-collection instruction was to refrain from eating, drinking or smoking for 60 minutes. Samples were then stored at room temperature for a period ranging from 1 to 3 days. The RNA-extraction free protocol for RT-LAMP was adapted from Lamb et al. 2020) . Saliva aliquots (50 ul) were pre-heated at . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10.1101/2020.10.27.20220541 doi: medRxiv preprint 98oC for 10 minutes to release RNA from cells and 8ul of the supernatant were transferred to 42ul of RT-LAMP reaction mix. The reaction was incubated for 5 minutes at room temperature, for Uracil-DNA-Glycosylase (UDG) to eliminate eventual carryover contaminants. Using a conventional thermocycler, RT-LAMP reaction (table 1) was heated to 63oC for 30 minutes and then reaction was stopped by heating to 80oC for an additional 5 minutes. Primer set (table 3) consists of six primers designed by Joung and colleagues. (Joung et al. 2020) to target the Nucleocapsid gene, which has higher copy numbers than other segments of the SARS-CoV-2 genome. Synthetic SARS-CoV-2 RNA from Twist Biosciences was used as positive control and nuclease-free water and saliva from healthy subjects as negative controls. RT-LAMP products were diluted 40x with EvaGreen fluorescent dye (CellCo, 100x diluted in TAE1x) and read in a fluorescence spectrophotometer. RT-LAMP products from positive samples were also analyzed by Agilent 2100 Bioanalyzer and real time PCR equipment. Readings in the fluorescence spectrophotometer (Promega Glomax) were considered negative if the diluted reaction had less than 1500 RFU/ul. The fluorescence in each well was measured once, with excitation modules and emission at 475nm and 500-550nm respectively. Resulting RT-LAMP products were also analyzed using Bioanalyzer, for characteristic banding patterns (Lamb et al. 2020 ) and confirmed by dissociation curve analysis in a real time PCR equipment (CFX96 Touch Real-Time PCR Thermal Cycler Biorad or 7300-Applied Biosystems), using cycling parameters as follows: 63°C for 1:00 min, 95°C for 15 seconds, melt analysis performed from 63°C to 95°C , in a method broadly similar to (Rolando et al. 2020) . The incorporation of dissociation curves allows the confirmation of low viral load positives from negatives samples, as well as increases the specificity for SARS-CoV-2 detection. . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10.1101/2020.10.27.20220541 doi: medRxiv preprint RT-LAMP analytical sensitivity was determined by limiting dilution of synthetic SARS-CoV-2 RNA (Twist Bioscience). Ten replicates of six different concentrations ranging from 10,000 copies to 10 copies per reaction were processed through the assay. Limit of detection (LoD) was defined as the lowest concentration at which >90% of the replicates were detected. LoD was determined as 20 copies per reaction (cutoff -d(RFU)/dT = 20). ( Figure 1 ). Considering the use of 8 μl of saliva as input, the estimated detection limit is 2.5 copies per μl. Inter-plate replicability was determined using 122 consecutive plates containing 1 positive control and 1 negative control. (Figure 2 ). All 122 positive controls scored above the 1,900 RFU stringent cutoff (minimum positive control value = 2,066 RFU). All 122 negative controls scored below the more sensitive cutoff of 1,500 used for subsequent melting point evaluation (maximum negative control value = 817 RFU). In Figure 3 , 50,000 consecutive samples, positive and negative subjects show a similar fluorescence readout distribution to corresponding controls. Saliva RT-LAMP results were compared to nasopharyngeal RT-PCR results from an independent lab. Validation was independently carried out by Mendelics and Hospital Sírio-Libanês, and the results were combined. RT-LAMP and RT-PCR results were blinded to the complementary results. As shown in Table 4 , a total of 244 symptomatic patients, ranging from days 1 through 7 of symptoms, were tested. SARS-CoV-2 RNA was detected in 31% (76/244) of patients by at least one method. RT-PCR identified 65 of 76 patients (sensitivity of 85.5%) while RT-LAMP identified 60 of 76 patients . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. Efforts to control SARS-CoV-2 depend on sensitive (>70%), highly specific (>98%), fast turnaround time diagnostic tests. (Larremore et al. 2020 ) In this study we describe a scalable method that is accurate, time and cost effective, and displays a sensitivity and specificity similar to the gold standard RT-qPCR. As a result, it has enormous potential in helping to identify infected individuals to control disease spread. 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 October 27, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 efficiency of the methods. The protocol described and validated here focuses on scalability and short turnaround times. Variations on this protocol, such as the addition of standard RNA extraction and concentration can be used to improve sensitivity in detriment of speed and cost. Alternatively, increase in the fluorescence detection cutoff, with removal of the dissociation curve, can be used to speed and decrease costs in detriment of sensitivity and specificity. Each variation can serve distinct purposes (screening or diagnosis), fit different geographic location socio-economic constraints, and moment of the pandemic. In conclusion, we contribute efforts to contain the pandemic by describing and validating a scalable, fast, and low cost SARS-CoV-2 test that can be easily collected. The ease of implementation should allow this to be implemented in both developed and developing countries. CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10. 1101 ACAAACTGTTGGTCAACAAGAC . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10. 1101 . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10.1101/2020.10.27.20220541 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10.1101/2020.10.27.20220541 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a 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 October 27, 2020. ; https://doi.org/10. 1101 … of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in … Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva with Colorimetric LAMP Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification Rapid Detection of Novel Coronavirus (COVID19) by Reverse Transcription-Loop-Mediated Isothermal Amplification Test Sensitivity Is Secondary to Frequency and Turnaround Time for COVID-19 Surveillance A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Loop-Mediated Isothermal Amplification of DNA Detection of Human Influenza A Viruses by Loop-Mediated Isothermal Amplification Real-Time Kinetics and High-Resolution Melt Curves in Single-Molecule Digital LAMP to Differentiate and Study Specific and Non-Specific Amplification Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-CoV-2 Saliva Is More Sensitive for SARS-CoV-2 Detection in COVID-19 Patients than Nasopharyngeal Swabs Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity