key: cord-336237-hmczy0am
authors: Kitagawa, Yutaro; Orihara, Yuta; Kawamura, Rieko; Imai, Kazuo; Sakai, Jun; Tarumoto, Norihito; Matsuoka, Masaru; Takeuchi, Shinichi; Maesaki, Shigefumi; Maeda, Takuya
title: Evaluation of rapid diagnosis of novel coronavirus disease (COVID-19) using loop-mediated isothermal amplification
date: 2020-05-21
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104446
sha: 
doc_id: 336237
cord_uid: hmczy0am

With the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for more rapid and simple detection technologies at the forefront of medical care worldwide. In this study, we evaluated the effectiveness of the Loopamp® 2019-SARSCoV-2 Detection Reagent Kit, which uses loop-mediated isothermal amplification (LAMP) technology. In this protocol, cDNA is synthesized from SARS-CoV-2 RNA using reverse transcriptase, followed by DNA amplification under isothermal conditions in one step. The RT-LAMP test kit amplified the targeted RNA of a SARS-CoV-2 isolate with a detection limit of 1.0 × 101 copies/μL, which was comparable to the detection sensitivity of quantitative reverse transcription PCR (RT-qPCR). Comparison with the results of RT-qPCR for 76 nasopharyngeal swab samples from patients with suspected COVID-19 showed a sensitivity of 100% and a specificity of 97.6%. In the 24 RNA specimens derived from febrile Japanese patients with or without influenza A, no amplification was observed using RT-LAMP. RT-LAMP could be a simple and easy-to-use diagnostic tool for the detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has emerged as a serious threat to human health worldwide [1, 2] .

With the rapid increase in the number of patients, a reliable, rapid, and simple detection system for SARS-CoV-2 is needed that can be used in all medical institutions as quickly as possible [3] .

Loop-mediated isothermal amplification (LAMP)-based analysis, which can be performed without a thermal cycler, is suitable for the diagnosis of infectious diseases as a point-of-care test in resource-limited settings [4, 5] . In particular, the use of bst DNA polymerase with high strand displacement activity, to which reverse transcriptase (RT) activity has also been added, makes amplification of specific viral RNA possible in one step at a constant temperature.

This study examined the usefulness of a commercially available RT-LAMP-based diagnostics kit for COVID-19 (Loopamp ®︎ 2019-SARS-CoV-2 Detection Reagent Kit; http://loopamp.eiken.co.jp/), with the view that if the approach proves feasible, it could support the rapid detection of SARS-CoV-2.

To evaluate the analytical sensitivity of the RT-LAMP method, we used purified and quantified total RNA of SARS-CoV-2, which was provided by the National Institute of Infectious Diseases, Japan, as a standard specimen for the molecular diagnosis of COVID-19. Analytical sensitivity was determined using 10-fold serially diluted standard RNA ranging from 1.0 × 10 3 copies/μL to 1.0 copy/µL and stored at -30 °C until required.

The specificity of the RT-LAMP reaction was evaluated using 12 RNA samples extracted from nasopharyngeal swabs taken from patients with influenza A at Saitama Medical University Hospital, Japan, from November to December 2019. In addition, 12 RNA samples negative for influenza A from febrile Japanese patients during the same period were also used in this study.

During this sampling period, there were no cases of COVID-19 in Japan. The RT-LAMP reaction was performed using the same kit and conditions described above.

A 10-fold serial dilution of SARS-CoV-2 RNA was amplified to determine the lower limit of detection with the RT-LAMP kit. Figure 1 shows the results for the detection of real-time turbidity with the LA-200 turbidimeter. The minimum amount of RNA detected was 1.0 × 10 1 copies/µL, which was achieved within 35 min in this procedure. After measurement, turbidity of the reaction solution could be observed visually under natural light (Fig. 2) .

Based on the analysis of conventional RT-qPCR, 30 of the 76 patients were identified as positive for SARS-CoV-2; the median Ct value obtained was 30.85 (interquartile range, 25.31 to 36.08). Of the 76 patients who underwent conventional RT-qPCR, 32 were positive and 44 were negative by RT-LAMP. As shown in Table 1 , the agreement between RT-qPCR and RT-LAMP was 97.4% (74/76). Among them, 2 patients were found to be negative with RT-qPCR but positive with RT-LAMP. In the 24 RNA specimens derived from febrile Japanese patients with or without influenza A, no amplification was observed using RT-LAMP.

RT-qPCR is a sensitive and specific nucleic acid amplification method that can be used to diagnose emerging viral infections, including COVID-19. However, it requires trained personnel, expensive equipment, and an extended period of time to generate test results. Conversely, RT-LAMP is extremely convenient to use (the isothermal reaction requires a simple heating device) and produces rapid results (within 30-60 min). In addition to these advantages, the amplification products generated by the RT-LAMP test kit can be detected by turbidity under natural light.

In this study, we found that the detection limit of the RT-LAMP test kit was 1.0 × 10 1 copies/µL within 35 min with real-time detection of its amplification products. Previous studies using the same molecular diagnostic strategy reported sensitivity ranging from 2.0 × 10 1 to 1.0 × 10 2 copies/reaction, indicating that this RT-LAMP test kit has extremely high sensitivity and is also valuable for diagnosis, in terms of not only its convenience but also its detection sensitivity [7, 8] . It is highly specific because it uses a set of four primers that recognize at least six different sequences in SARS-CoV-2 RNA. In the present study, it was considered that there was no nonspecific amplification with the RT-LAMP test kit. The results for two samples, re-collected at more than 1 week after the initial SARS-CoV-2-positive result, were inconsistent with those for RT-qPCR (RT-LAMP was positive, whereas RT-qPCR was negative). This phenomenon may depend on the concentration gradient or the aspiration rate in the RNA extracts because the amount of sample used for RT-qPCR was 5 μL, while that used for RT-LAMP was 10 μL. 

Masaru Matsuoka: Prepared the manuscript, Providing scientific guidance. Shinichi Takeuchi: Provided scientific guidance, Reviewed and Edited Shigefumi Maesaki: Provided scientific guidance

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Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia

Exploring the reasons for healthcare workers infected with novel coronavirus disease 2019 (COVID-19) in China

Operational feasibility of using loop mediated isothermal amplification (LAMP) for the diagnosis of pulmonary TB in microscopy centers of developing countries

Evaluation of a simple loop mediated isothermal amplification test kit for the diagnosis of tuberculosis

Manual for the Detection of Pathogen 2019-nCoV Ver.2.6

Development of Reverse Transcription Loopmediated Isothermal Amplification (RT-LAMP) Assays Targeting SARS-CoV-2

Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay

None.J o u r n a l P r e -p r o o f

All authors have no conflicts of interest to declare.

The authors declare that they have no competing interests.
