key: cord-1050521-fq11rhab
authors: Zhu, Xiong; Wang, Xiaoxia; Han, Limei; Chen, Ting; Wang, Licheng; Li, Huan; Li, Sha; He, Lvfen; Fu, Xiaoying; Chen, Shaojin; Mei, Xing; Chen, Hai; Wang, Yi
title: Reverse transcription loop-mediated isothermal amplification combined with nanoparticles-based biosensor for diagnosis of COVID-19
date: 2020-03-20
journal: nan
DOI: 10.1101/2020.03.17.20037796
sha: 094267d6c2306d5c87ed22e61b7a24b5e6d6dc4d
doc_id: 1050521
cord_uid: fq11rhab

Given the scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, known as 2019-nCov) infection (COVID-19), the ongoing global SARS-CoV-2 outbreak has become a huge public health issue. Rapid and precise diagnostic methods are thus immediately needed for diagnosing COVID-19, providing timely treatment and facilitating infection control. A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) coupled with nanoparticles-based biosensor (NBS) assay (RT-LAMP-NBS) was successfully established for rapidly and accurately diagnosing COVID-19. A simple equipment (such as heating block) was required for maintaining a constant temperature (63 C) for only 40 min. Using two designed LAMP primer sets, F1ab (opening reading frame 1a/b) and np (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified and detected in a one-step and single-tube reaction, and the detection results were easily interpreted by NBS. The sensitivity of SARS-CoV-2 RT-LAMP-NBS was 12 copies (each of detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. Among clinically diagnosed COVID-19 patients, the analytical sensitivity of SARS-CoV-2 was 100% (33/33) in the oropharynx swab samples, and the assay's specificity was also 100% (96/96) when analyzed the clinical samples collected from non-COVID-19 patients. The total diagnosis test from sample collection to result interpretation only takes approximately 1 h. In sum, the RT-LAMP-NBS is a promising tool for diagnosing the current SARS-CoV-2 infection in first line field, public health and clinical laboratories, especially for resource-challenged regions.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint INTRODUCTION 54 In late December 2019, an unexpected outbreak caused by severe acute respiratory 55 syndrome coronavirus 2 (SARS-CoV-2, known as 2019-nCov) emerged in Wuhan, 56 Hubei province, China, which had previously not been reported in animals or humans 57 (1). By press time, the novel coronavirus (SARS-CoV-2) has resulted in a huge Hence, there is an urgent requirement for easy-to-use, more rapid and simpler 81 detection techniques for diagnosis of COVID-19.

Loop-mediated isothermal amplification (LAMP), which is the most popular 83 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2020. . https://doi.org/10.1101/2020.03.17.20037796 doi: medRxiv preprint (Figure 1 and 2) . Two target sequences, including F1ab and nucleoprotein gene (np), 114 were simultaneously amplified in an isothermal reaction, and detected in a test step. 115 We will expound the basic COVID-19 RT-LAMP principle, optimize the reaction 116 parameters (e.g., amplification temperature), and demonstrate its feasibility. product also severed as the template for next amplification by LF* (forward loop 130 primer), which was modified at the 5' end with hapten (Step 5). As a result, a 131 double-labeled detectable product (LF*/LB* product) was formed, and one end of the 132 LF*/LB* product was labeled with hapten, and the other end with biotin (Step 6, 7). 133 One hapten is assigned to one primer set, which provide the possibility for multiplex 134 LAMP detection. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2020. . https://doi.org/10.1101/2020.03.17.20037796 doi: medRxiv preprint row). These results indicated that F1ab-and np-LAMP primer sets were available for 174 establish the COVID-19 RT-LAMP NBS assay for rapid and reliable detection of 175 SARS-CoV-2. The parameter of optimal temperature for COVID-19 RT-LAMP 176 technique also was tested, and reaction temperature of 63°C was used for performing 177 the COVID-19 RT-LAMP amplification (Figure S2 and S3) . The COVID-19 RT-LAMP-NBS was able to detect down 12 copies (each of 181 F1ab-plasmid and np-plasmid) (Figure 4) . Two target genes were detected and 182 identified in a one-tube reaction ( Figure 4A) . The COVID-19 RT-LAMP results 183 using NBS were in consistent with turbidity and VDR detection (Figure 4B and 4C) , 184 while traditional monitoring techniques (VDR and turbidity) could not facilitate 185 multiplex analysis. Furthermore, the sensitivity of COVID-19 RT-LAMP-NBS assay 186 was in conformity with F1ab-and np-RT-LAMP assay (Figure 4, S4 and S5) . 187 The optimal duration time of COVID-19 RT-LAMP-NBS assay at the isothermal 188 stage also was determined, and the template level at the detection limit appeared three indicating no cross-reaction with non-SARS-CoV-2 templates (Table S2) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

Of the total of 129 respiratory samples, which were initially analyzed using rRT-PCR China, is a huge public health concern (15). As of today (9 March 2020), 98 129 total is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2020. F1ab) (13, 14) . 250 The data of analytical sensitivity validated that RT-LAMP-NBS assay is 251 sufficiently sensitive for diagnosis of COVID-19. Us this protocol, detection limit of 252 COVID-19 RT-LAMP-NBS was 12 copies each of F1ab-plasmid and np-plasmid, 253 which is in conformity with assay's sensitivity generated from F1ab-RT-LAMP-NBS 254 and N-RT-LAMP-NBS detection (Figure 4, S4 and S5) . The COVID-19 255 RT-LAMP-NBS assay did not improve or decrease the analytical sensitivity when is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Two RT-LAMP primer sets (F1ab-RT-LAMP and np-RT-LAMP) were designed 294 according the LAMP mechanism using a specialized software (PrimerExplore V5), 295 which targeted F1ab and np gene of SARS-CoV-2 (GenBank MN908947, 296 Wuhan-Hu-1) (Figure 6) . Then, a Blast analysis of the GenBank nucleotide database 297 was performed for the F1ab-and np-LAMP primers to validate sequence specificity.

The more details of primer design, locations, sequences and modifications were 299 shown in Figure 6 and Table S2 . All of the oligomers were synthesized and purified is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2020. Table S1) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2020. . https://doi.org/10.1101/2020.03.17.20037796 doi: medRxiv preprint

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is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted March 20, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted March 20, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted March 20, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted March 20, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted March 20, 2020. . https://doi.org/10.1101/2020.03.17.20037796 doi: medRxiv preprint