Background

Fast and sensitive detection of SARS-CoV-2, the etiologic agent of COVID-19, is important to control current pandemic situation as it enables early detection, isolation and treatment as well as monitoring screening. Current standard methods for detection of SARS-CoV-2 utilize RT-qPCR as World Health Organization recommends (WHO, 2020) However, proper performance of RT-qPCR diagnosis requires high profile facilities and specialists, often not available in the hospital/sampling places, thus, lacking POCT suitability.

Isothermal amplification methods are developed to accomplish diagnosis by nucleic acid detection in various point-of-care as it can be performed with relatively simple instruments (Niemz et al., 2011). Loop-mediated isothermal amplification (LAMP) is one of such isothermal NAAT (Notomi et al., 2000). Amplification by LAMP reaction can be observed through fluorescent dye with real-time PCR instruments (Oscorbin et al., 2016), or through cost effective colorimetric detection methods (Goto et al., 2009; Miyamoto et al., 2015; Tanner et al., 2015). Indeed reverse transcription LAMP (RT-LAMP) may be considered a promising tool as several other groups are employing this technique for SARS-CoV-2 detection (Yan et al., 2020; Zhang et al., 2020).

Here, we present our protocol used to develop RT-LAMP assays targeting SARS-CoV-2. Candidate primer sets are designed using web based tool PrimerExplorerV5 (https://primerexplorer.jp/e/) targeting SARS-CoV-2 specific regions compare to SARS-CoV and SARS-CoV-2 Nucleocapsid. The primer sets are screened for limit of detection and threshold time measured through real time fluorescent method. Below illustrated protocol includes RNA standard preparation, titration of cultured viral RNA and LAMP reaction which are used during primer screening (Figure 1). Optimized RT-LAMP condition for finally selected two primer sets, namely “Nsp3_1-61” and “Nsp3_2-24”, are representatively provided. We also adapted leuco crystal violet (LCV) colorimetric detection method optimized for Bst 3.0 buffer system (Miyamoto et al., 2015). A commercially available colorimetric RT-LAMP premix uses pH sensitive dye so that the premix is not compatible with viral genome extraction methods which affect pH of weakly buffered reaction solution (Lalli et al., 2020). However, LCV method is compatible with such viral genome extraction methods because the color change depends on dsDNA product.


Figure 1. Protocol overview