Abstract
Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4–100.0%) and 98.6% (95% CI: 94.9–99.8%), respectively, showing high consistency (Cohen’s kappa, 0.986; 95% CI: 0.966–1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.
Highlights
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral etiological agent of a novel severe acute respiratory infection called coronavirus disease 2019 (COVID-19) that was declared a pandemic by the World Health Organization (WHO) on March 11, 2020 (Li et al, 2020; Wu et al, 2020)
To select the final primer sets, the candidate sets were evaluated by multiplex reverse transcription loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) using a panel of SARS-CoV-2 RNA obtained from clinical samples (n=7) with a viral load gradient
We developed and evaluated the suitability of a multiplex RT-LAMP assay in a lyophilized format for detecting SARS-CoV-2 infection
Summary
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral etiological agent of a novel severe acute respiratory infection called coronavirus disease 2019 (COVID-19) that was declared a pandemic by the World Health Organization (WHO) on March 11, 2020 (Li et al, 2020; Wu et al, 2020). Since the outbreak that originated in December 2019 in Wuhan city, China (Adhikari et al, 2020), the new b-coronavirus rapidly spread globally, being responsible for more than 160 million confirmed cases and 3,339,002 deaths in over 200 countries, as of May 14, 2021 [World Health Organization (2020a), COVID-19 situation dashboard]. A similar epidemiological situation is taking place in Peru, which has one of the highest mortality rates in the current COVID-19 pandemic, with Lima city being one of the major epicenters of SARS-CoV-2 infections in Peru, as of September 11, 2020 (Juscamayta-Ló pez et al, 2020; Ministry of Health, Peru, 2020). COVID-19 clinical features have some resemblance to other previously reported coronavirus infections (SARS-CoV and MERS-CoV) and to other types of infections (such as influenza or the common cold) (Huang et al, 2020)
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