key: cord-0925370-eo2pcgix authors: Jiang, Minghua; Pan, Weihua; Arastehfar, Amir; Fang, Wenjie; ling, Liyan; Fang, Hua; Farnaz Daneshnia, Farnaz; Yu, Jian; Liao, Wanqing; Pei, Hao; Li, Xiaojing; Lass-Florl, Cornelia title: Development and validation of a rapid single-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and high-throughput screening of COVID-19 date: 2020-03-20 journal: nan DOI: 10.1101/2020.03.15.20036376 sha: c56e83855676999341988c6873ad6568f0bde1f2 doc_id: 925370 cord_uid: eo2pcgix The recent pandemic of COVID-19 has involved tens of thousands of patients in numerous countries and the causative virus, SARS COV-2 is highly transmissible. Molecular diagnostic tools are central to containment of the virus and initiating proper clinical care. Rapidity, user-friendliness, and high degree of sensitivity and specificity are desirable features of diagnostic assays for screening purposes. Herein, we present a single step reverse transcriptase LAMP assay (RT-LAMP), which can detect up to 500 viral copies in 30 minutes. We challenged our assay with a large number of clinical samples collected from 47 confirmed cases and 213 negative patients. Our LAMP assay showed a high degree of sensitivity and specificity compared to two commercialized qRT-PCR assay as gold standard. We present a rapid RT-LAMP assay that could extend the capacity of laboratories to process 2.5 more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes. A new virus causing pneumonia-like infection, COVID-19, which was found in Wuhan, Hubei Province, China, and to be linked to a seafood market has caused a serious crisis worldwide (1) . Almost two months after the first report, COVID-19 severe outbreaks were reported in numerous countries and became a public health priority in the world (World Health Organization, Situation Report 48). As of March 14, 2020, COVID-19 cases are found in 122 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 preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The virulent nature of this virus and its high rate of transmissibility warrants robust, rapid, sensitive, specific, and quantitative diagnostic tools to supplement clinical symptoms aiding clinicians to confidently rule in and rule out patients. Moreover, such a diagnostic tool will help with preventing spread of virus by identifying the infected cases and can monitor the health status of infected patients by quantifying the viral load. Center for Disease Control was the first to develop a quantitative reverse transcriptase real-time PCR (RT-PCR), which later became the gold standard technique (https://www.cdc.gov/coronavirus/2019-ncov/about/testing.html). Subsequently, a Chinese group used a RNA-based metagenomics next generation sequencing (mNGS) to diagnose the viral RNA from the clinical samples of two patients (3) . However, the requirement for advanced technology and skilled personnel and long turn-around time (24 hours) are not feasible for local and referral laboratories. Therefore, a colorimetric loop mediated isothermal amplification, also known as LAMP, was developed to obviate the need for expensive technologies, e.g. real-time PCR and NGS, as well as to shorten the turn-around time to up to 40 minutes (4). However, this assay was a qualitative one and also only the swab samples from limited number of patients (n=7) were included for testing (4) . Most recently a newer generation of single step RT-LAMP were developed to detect SARS COV-2, but these assays were not challenged with real clinical samples obtained from COVID-19 positive patients (5, 6). Therefore, we developed a sensitive, specific, and rapid RT-LAMP assay and its performance was challenged by an extensive number of confirmed COVID-19 (n=47) and negative patients (n=213) relative to qRT-PCR assays approved by two Chinese Food and Drug Administration (qRT-PCR NMPA). Altogether, we present a rapid and reliable diagnostic tool 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 downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) to select the most specific target region. Genenious v11.1.14 was used for alignment analysis and to find the most specific region for designing LAMP primers. LAMP Designer (PREMIER Biosoft International, San Francisco, CA) was used for primer design. Designed primers were subjected to BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the specific candidates were used for analytical sensitivity and specificity testing (Table 1 ). Primers were synthesized by Sangon Biotech Co., Analytical sensitivity and specificity testing was performed in a P2 lab and in order to mimic the real viral particles we purchased pseudotyped SARS-CoV-2 assay system containing ORF1ab part sequence, N gene and E gene (DAAN gene Co. Ltd, Guangzhou, China). RNA of pseudotyped virus were extracted using EZ-10 Spin Column Viral Total RNA Extraction Kit 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 LAMP incubation time was set to 60 minutes to observe both limit of detection and crossreactivity (LAMP conditions are mentioned in clinical evaluation section). The reaction endpoint time was set in a way to detect the lowest possible copy number of virus without any cross-reaction. Clinical samples obtained from patients contain a wide range of inhibitors impairing the efficacy of diagnostic assay. Therefore, the tolerance of our LAMP assay was assessed when 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 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 March 20, 2020. . https://doi.org/10.1101/2020.03.15.20036376 doi: medRxiv preprint florescent data were collected each minute. RT-PCR and RT-LAMP tested separately by two assessors, final results were recorded and were compared with one another. The whole workflow of our study from in-silico analysis to analytical evaluation and clinical validation is depicted in Figure 1 . Nine LAMP primer systems were designed and evaluated insilico, among them six primers showed the highest sensitivity and specificity and used in the next steps (Table 1 and Figure 1 ). Primarily, our assay was meant to be quantitative and it showed an optimal reproducibility when tested in analytical evaluation step using armored viral particle diluted in water (R 2 value ˷0.99) and sputum sample (R 2 value ˷0.83). Analytical sensitivity yielded reliable LOD of 500 copies/ml less than 30 minutes regardless of matrix used for serial dilution (Figure 2 ). Of note, our assay could detect 50 copies/ml, but some replicates showed unstable amplification. Therefore, we considered the LOD of 500 copies/ml. Analytical specificity was 100% when used a wide range of closely-and distantly-related viral species, prominent fungal and bacterial species, and human DNA. Moreover, analytical evaluation included a wide range of inhibitors and 500 copies of the simulated viral particles were successfully detected below 30 minutes ( Figure 3 ). In order to evaluate the performance of our assay in clinic, we provided our assay and respective instructions to two clinical centers ( Figure 3 ). In total, 168 patients from center 1, among which 35 confirmed COVID-19 cases, and 92 patients from center 2, among which 12 patients were confirmed COVID-19 cases, were recruited. One asymptomatic patient tested positive by qRT-PCR (Ct values 37) and by our RT-LAMP was categorized suspected by in-charge physician and few days later became positive. Four patients tested positive by qRT-PCR were negative by our RT-LAMP and one patient tested negative by qRT-PCR was positive by our assay (Figure 3 and Supplementary Table 1 ). Subsequently, our RT-LAMP assay showed the sensitivity, specificity, negative predictive value, and positive predictive value of 91.4%, 99.5%, 98.1%, and 97.7%, respectively 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 Table 2 and 3). The fact that our assay could not detect four positive patients was owing to using 2.5 less RNA input (2 µl) relative to qRT-PCR (5 µl). In the future, we will try to use various RNA input volume (5, 8 , and 10 µl) to observe if we could obtain a higher sensitivity. Although our RT-LAMP assay was developed to be quantitative, we could not find any pattern and association between the time to positivity by our RT-LAMP assay and the Ct values reported by qRT-PCR when using clinical samples. Therefore, we considered our assay a qualitative one. This fact will show that the analytical valuation should be always accompanied by clinical validation to observe the real capabilities of a given assay and that the results obtained in analytical evaluation step are not always reflected in real-life. Our assay has several advantageous compared to qRT-PCR. First, our RT-LAMP assay is 2.5 times faster relative to qRT-PCR and given the optimal diagnostic features could be used as a reliable screening method in local and referral laboratory to keep up with the increasing demand of suspected patients in critical situations. Secondly, our assay does not need the clod chain and could be shipped at room temperature. In conclusion, we present a rapid RT-LAMP assay that allows processing 2 to 2.5 more clinical samples relative to CDC RT-PCR, which is indicative of its capacity to be deployed for highthroughput screening applications in local and referral laboratories. We admit that our assay does not has the quantitative aspect of qRT-PCR and its sensitivity requires improvement. These two limitations will be the subject of future investigation. Moreover, we will try to use simple and fast nucleic acid extraction procedures (7) that only uses heat that will further decrease the turn-around-time. Not applicable Disclaimers 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 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 March 20, 2020. . https://doi.org/10.1101/2020.03.15.20036376 doi: medRxiv preprint A Novel Coronavirus from Patients with Pneumonia in China SARS-CoV-2 is an appropriate name for the new coronavirus RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak Rapid Molecular Detection of SARS-CoV-2 ( COVID-19 ) Virus RNA 2 Development of Reverse Transcription Loop-mediated Isothermal Amplification ( RT-LAMP ) Assays Targeting SARS-CoV-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 The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated. Dr. Minghua Jiang, director of the Department of Laboratory Medicine, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University. His current interest is in development and validation of rapid diagnostic tools for infectious diseases.