Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

Jiang and Shi 2020).Similar to severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), the new coronavirus also belongs to Betacoronavirus, and shares highest sequence identity to three SARS-like CoVs of bat origin (bat_CoV_RaTG13: 96.0%, bat-SL-CoVZC45: 88.0% and bat-SL-CoVZXC21: 87.2%) (Zhou et al. 2020).Although only sharing about 79.5% genomic sequence identity to SARS-CoV, the new virus was demonstrate to use the same receptor angiotensin converting enzyme II (ACE2) for human infection as SARS-CoV (Lu et al. 2020; Wu 2020; Zhou et al. 2020) and is officially named as SARS-CoV-2 (also known as 2019-nCoV) (Gorbalenya et al. 2020).Epidemically data showed that the virus has strong human-to-human transmission ability, and it is spread by droplets produced by coughing and sneezing, infecting susceptible subjects through direct contacts and other possible transmission routes (e.g.fecal-mouth transmission) (Guan et al. 2020; Li et al. 2020).As of Mar. 27, 2020, the outbreak had resulted in 533,464 laboratory-confirmed cases, including 24,097 deaths around the world.Currently, there lacks suitable antiviral drugs or vaccines for SARS-CoV-2.Early, rapid, and reliable diagnostic assays for SARS-CoV-2 is of top priority to facilitate public health interventions that can reduce or avoid further spread of SARS-CoV-2 (Dennis Lo and Chiu 2020).Quantitative Real-time PCR (qPCR) is a robust technology for reliable laboratory diagnosis, and routinely used to detect causative pathogens.Since the release of genomic sequence of SARS-CoV-2 in Jan. 11, 2020, many TaqMan probe-based RT-qPCR assays had been developed and used in confirmation of large number of SARS-CoV-2 infections during the outbreak (Chu et al. 2020;Dennis Lo and Chiu 2020).Facing the on-going increase of suspected cases and potential contacts, however, the detection capacity of RT-qPCR assays have shown limitations in speed and volume because these assays require sophisticated equipment and highly trained personnel, and are relatively time-consuming (about 1.5-2 h).Therefore, there is an urgent demand for faster and sensitive Point-of-Care testing (POCT) assays to facilitate rapid detection of SARS-CoV-2 infection.However, there is no POCT assay available for SARS-CoV-2 yet.Here, we present a novel visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of SARS-CoV-2 using mismatch-tolerant technique.Based on SARS-CoV-2 genomic sequences, we designed six sets of LAMP primers (4, 1, and 1 in N, S and RdRp genes, respectively) using the open access Primer Explorer V.5 software tool (http://primerexplorer.jp/).We firstly excluded primer sets with non-specific amplification, Renfei Lu and Xiuming Wu have contributed equally to this work.