key: cord-0916576-kftffes0 authors: Mohon, Abu Naser; Oberding, Lisa; Hundt, Jana; van Marle, Guido; Pabbaraju, Kanti; Berenger, Byron; Lisboa, Luiz; Griener, Thomas; Czub, Markus; Doolan, Cody; Servelitta, Venice; Chiu, Charles; Greninger, Alexander; Jerome, Keith; Pillai, Dylan R. title: Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2 date: 2020-09-15 journal: J Virol Methods DOI: 10.1016/j.jviromet.2020.113972 sha: 73e0fc04d24332f5ff4884ed0439096a88ec3ff2 doc_id: 916576 cord_uid: kftffes0 A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25 copies per reaction) as commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48% (95% CI 91.84% to 99.96%) and NPA 100.00% (95% CI 93.84% to 100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification. Over the last several decades, we have witnessed the rise of both known and novel viruses, areas. In December 2019 and early January 2020, a cluster of pneumonia cases from a novel coronavirus, SARS-CoV-2, was reported in Wuhan, China (2) (3) (4) . SARS-CoV-2 has now resulted in a global pandemic with the epicentre at the time of writing in Europe and North America (5) . A common theme in the public health response to COVID19 and similar threats is the lack of rapidly deployable testing in the field to screen large numbers of individuals in exposed areas, international ports of entry, and testing in quarantine locations such as the home residences, as well as low-resourced areas (6) . This hampers case finding and increases the number of individuals at risk of exposure and infection. With the ease of travel across continents, delayed testing and lack of screening programs in the field, global human-tohuman transmission will continue at high rates. These factors make a pandemic very difficult to contain. Early identification of the virus and rapid deployment of a targeted point of care test (POCT) can stem the spread through immediate quarantine of infected persons (7) . We used existing viral genome sequences to develop a SARS-CoV-2 loop mediated amplification (LAMP) assay for clinical use (8, 9) . LAMP relies on an alternate set of reagent chemistry that does not depend on or hinder critical elements of the RT-PCR supply chain which is now under duress (10) . Our group has previously demonstrated the utility of LAMP for other infectious agents like malaria and dengue (11) (12) (13) . Clinical samples used in this study were standard archived nasopharyngeal (NP) in viral transport medium (VTM) stored at -80 o C at Alberta Precision Laboratories (Calgary, Canada), J o u r n a l P r e -p r o o f University of Washington, and University of California, San Francisco. Ethical approval for use of the archived samples was obtained from the Conjoint Health Research Ethics Board (CHREB) of the University of Calgary (REB20-0402). This study was approved by the institutional review board (IRB) at University of California, San Francisco (UCSF IRB #10-02598) as a no subject contact study with waiver of consent. Remnant NP swab after clinical testing and PCR cycle threshold results were collected for analysis. The use of de-identified specimens were deemed non-human subject work by the University of Washington Institutional Review Board (IRB). Genomic sequences (cDNA) of the SARS-CoV-2 were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/) and multiple sequence alignment analysis (https://www.ebi.ac.uk/Tools/msa/clustalo/) was conducted with other related viruses. From the multiple sequence alignment, several regions unique to the SARS CoV-2 were identified. The primers were designed using the Primer Explorer V5 software (http://primerexplorer.jp/lampv5e/) by uploading the sequences of the S and RdRP gene. Initially, primers were designed with the default settings of the software and then screened based on the optimal self-dimer, 5' end, and 3'end G values. Additionally, the intervening sequence between F2/F1 and B1/B2 primer binding sites was manually set at a minimum of 18 nucleotides. Other LAMP primer design software (eg. http://www.optigene.co.uk/lampdesigner/) are available commercially but were not used in this study. Initial screening experiments were performed on several potential primer sets and two chosen based on amplification efficiency (data not shown). LAMP primer sets were designed targeting unique regions of the Spike (S) protein gene and RNA-dependent RNA Polymerase gene (RdRP) were ultimately used (Table 1) . For the external LAMP amplification control, primers were used against bacteriophage MS2 as previously described (14). In silico analysis of primer combinations to determine cross-reactivity and inclusivity J o u r n a l P r e -p r o o f A blast search alignment (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for primers in set 2 (Spike gene) and set 3 (RdRP gene) were performed against a critical list of infectious agents that cause upper respiratory tract infections. A nucleotide local alignment using BLASTn with the default parameters was performed against the National Center of Biotechnology Information (NCBI) Nucleotide database (see Supplementary Data). The twelve RT-LAMP primers were aligned against 16,247 SARS-CoV-2 (taxid: 2697049) viral sequences in the NCBI nucleotide database available on August 20th, 2020. The output of the "discontiguous megablast" algorithm showed no divergence available between the 12 primers and the SARS-CoV-2 database and all of the query sequences showed a 100% identity against the expected sequences from the SARS-CoV-2 virus (92 genomes). Four fragments of specific SARS-Co-V2 regions (ORF1ab (nsp 3,10-11), RdRP (nsp 12), and spike (S)) were synthesized by SGI-DNA Inc. (San Diego, CA). Fragments were ligated to make one large concatenated DNA template using the BioXP3200 (SGI-DNA, San Diego, CA) automated Gibson assembly system. The final template was 1097 base pairs long containing concatenated single artificial construct together with flanking plasmid sequence in that order ( Figure 1 ). To create a recombinant virus expressing the relevant RNA, the template containing the targeted sequences of interest was cloned into Sindbis Virus (SV) viral vector system (SINrep5) containing green fluorescent protein (EGFP) and then transfected into BHK21 cell lines (18, 19) . The number of RNA genome copies was based on the number of fluorescent focus forming units generated by the recombinant SV vector. The dual-target LAMP reaction was conducted using a combination of Warmstart Rtx Reverse Limit of detection of the LAMP assay was evaluated by using a nasopharyngeal (NP) swab sample infected with SARS-CoV-2 for which the viral load was quantified using digital droplet PCR (see Supplementary Methods). The NP sample was serially diluted to achieve the described copies of virus per LAMP reaction. Calculation of viral copy number using digital droplet PCR is J o u r n a l P r e -p r o o f 8 The workflow used to conduct RT-LAMP is depicted in Figure 2 . The primer sequences used to target the Spike gene (set S2) and RdRP gene (Set 3) are listed in Table 1 . The limit of detection was evaluated using a patient sample (NP swab in VTM viral load confirmed by digital droplet PCR). The quantified sample was serially diluted to achieve a range from 100 to 12.5 copies per reaction. The limit of detection was confirmed at 25 copies per reaction when using 40mM Guanidine hydrochloride (pH8.0) in the reaction mix ( Table 2 ). Twenty four replicates from a serial dilution containing 25-50 copies of SARS-CoV-2 which equates to 1X LOD (patient sample NP swab in VTM viral load confirmed by digital droplet PCR) per reaction were tested using dual-target RT-LAMP (Table 3) . Twenty-three of 24 samples (95.8%) were positive. In silico analysis confirmed that no significant cross-reactivity that affects LAMP reactions that rely on six primers per reaction were present (Supplementary Table 1) . Finally, further studies were performed for precision on a daily basis (2 replicates for 20 days twice day and 5 replicates on 3 instruments daily for 5 days) that demonstrated 100% concordance (Supplementary Table 2 and 3). A clinical validation sample set of nasopharyngeal swabs were used in the analysis. Given no gold standard exists, percent positive agreement (PPA) and negative percent agreement (NPA) were calculated. Reference methods included RT-PCR (E gene and N2 gene) methods employed by reference laboratories. Dual-target RT-LAMP dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48% (95% CI 91.84% to 99.96%) and NPA 100.00% (95% CI 93.84% to 100.00%) based on the E gene and N2 gene reference RT-PCR methods (Table 4) . One false negative sample by RT-LAMP was positive by both E gene and N2 gene. One sample out of 124 J o u r n a l P r e -p r o o f was strongly positive by the N2 gene and negative by E gene and considered a true positive. No cross-reactivity was observed with known circulating respiratory viruses, namely (HCoV) OC43, 229E, NL63, and HKU1 or influenza virus A (H1N1) pdm09 (data not shown). The global pandemic with SARS-CoV-2 has resulted in the need for diagnostic test development at a scale never seen before. Rapid deployment of validated laboratory-developed diagnostic tests or commercial tests is essential to the containment of the virus as it allows for selfquarantine measures to be imposed in a strategic fashion before widespread community transmission occurs (6, 7) . Diagnostic tests have to be analytically sensitive in order to not to miss any cases in the acute phase of viremia (11) . As such, nucleic acid amplification tests serve this purpose. In particular, RT-PCR has been employed as the primary diagnostic countermeasure (18) . However, reagent supply chains for key items are under immense pressure. Local solutions to reagent sources have become paramount because barriers to trade of these selected items have been a concern. The dual-target RT-LAMP test for SARS-CoV-2 developed in this study has comparable analytical sensitivity and specificity, limit of detection, precision, and achieved excellent agreement compared to the reference RT-PCR methods used internationally. The addition of guanidine hydrochloride (pH8.0) at 40mM in the LAMP reaction improves the limit of detection (50 copies per reaction) to a level comparable to RT-PCR methods. LAMP does not rely on the same reagents as RT-PCR and thus alleviates pressure on key supply chain items. The LAMP method is amenable to high throughput testing in either 96-well or 384-well. Other groups have J o u r n a l P r e -p r o o f presented RT-LAMP solutions in the literature (19) (20) (21) (22) (23) (24) . The LAMP solutions differ in several ways: first, the target genes of choice vary between studies as do the specific primer sequences chosen; second, the limits of detection reported vary in terms of SARS-CoV-2 copies detected per reaction; third, the detection systems vary from thermocycler based detection for laboratory developed test (LDT) solutions to near-patient solutions based on visual detection of dyes or fluorophores; fourth, the extent which data reflect requirements for clinical validation. The RT-LAMP assay described is unique in that it offers the most thorough clinical validation to date meeting regulatory standards which include precision studies on several instruments, reproducibility studies over 20 days, a robust clinical validation sample set, and a limit of detection equal or superior to other LAMP studies (50 copies per reaction). These data should enable a clinical laboratory to perform this assay as a LDT. Additionally, the LAMP assay chemistry presented in this work is able to detect SARS-CoV-2 in VTM without the need for a kit-based RNA extraction method using lyophilized reagents and visual detection (manuscript in preparation). This format may be of particular interest to resource-limited settings. Limitations of the study include not testing other sample types such as alternate swabs, nasal washes, saliva, sputum, or stool. This work is ongoing with a special emphasis on swab-free testing and direct visualization. LAMP presents a much needed alternative approach to SARS-CoV-2 diagnostic testing that is available for deployment immediately in a laboratory-developed test format as it relies on other key reagents that do not cannibalize RT-PCR reagents. In the future, the LAMP chemistry has potential to be adapted to a microfluidic device POCT to be deployed in the community, either at ports of entry, homes, pharmacies, or workplaces. CD is an employee of Illucidx Inc. (a start-up company of the University of Calgary) which retains patents related to LAMP technology. DRP is a scientific advisor to Illucidx Inc. nasopharyngeal and oropharyngeal swabs at Hampshire Hospitals NHS Foundation Trust. preprint, Infectious Diseases (except HIV/AIDS). Figure 1 : Map of the gene fragments from SARS-Co-V2 (Genbank ID MT2078.1) that were used for synthesizing the genetic construct template. Four fragments of specific SARS-Co-V2 regions (ORF1ab (nsp 3,10-11), RdRP (nsp 12), and spike (S)) were concatenated into a single artificial construct 1097 base pairs long. J o u r n a l P r e -p r o o f Figure 2 : Workflow used to analyze samples in this study. Images were obtained from the Centers for Disease Control (www.cdc.gov) and Bio-Rad Laboratories (www.bio-rad.com). 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