Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2.

Nawattanapaiboon, Kawin; Pasomsub, Ekawat; Prombun, Photchanathorn; Wongbunmak, Akanit; Jenjitwanich, Akarawit; Mahasupachai, Pantanat; Vetcho, Purichaya; Chayrach, Cholticha; Manatjaroenlap, Natthapon; Samphaongern, Chonchanok; Watthanachockchai, Treewat; Leedorkmai, Phonthanat; Manopwisedjaroen, Suwimon; Akkarawongsapat, Radeekorn; Thitithanyanont, Arunee; Phanchana, Matthew; Panbangred, Watanalai; Chauvatcharin, Somchai; Srikhirin, Toemsak

COVID-19, caused by the infection of SARS-CoV-2, has emerged as a rapidly spreading infection. The disease has now reached the level of a global pandemic and as a result a more rapid and simple detection method is imperative to curb the spread of the virus. We aimed to develop a visual diagnostic platform for SARS-CoV-2 based on colorimetric RT-LAMP with levels of sensitivity and specificity comparable to that of commercial qRT-PCR assays. In this work, the primers were designed to target a conserved region of the RNA-dependent RNA polymerase gene (RdRp). The assay was characterized for its sensitivity and specificity, and validated with clinical specimens collected in Thailand. The developed colorimetric RT-LAMP assay could amplify the target gene and enabled visual interpretation in 60 min at 65 °C. No cross-reactivity with six other common human respiratory viruses (influenza A virus subtypes H1 and H3, influenza B virus, respiratory syncytial virus types A and B, and human metapneumovirus) and five other human coronaviruses (MERS-CoV, HKU-1, OC43, 229E and NL63) was observed. The limit of detection was 25 copies per reaction when evaluated with contrived specimens. However, the detection rate at this concentration fell to 95.8% when the incubation time was reduced from 60 to 30 min. The diagnostic performance of the developed RT-LAMP assay was evaluated in 2120 clinical specimens and compared with the commercial qRT-PCR. The results revealed high sensitivity and specificity of 95.74% and 99.95%, respectively. The overall accuracy of the RT-LAMP assay was determined to be 99.86%. In summary, our results indicate that the developed colorimetric RT-LAMP provides a simple, sensitive and reliable approach for the detection of SARS-CoV-2 in clinical samples, implying its beneficial use as a diagnostic platform for COVID-19 screening.