Figure 2.

LAMP-Based COVID-19 Test

(A) LAMP mechanism. (i) The reaction mix contains dNTPs, DNA polymerase with high displacement activity, and pairs of primers (F1-F1c; F2-F2c; F3-F3c; B1-B1c; B2-B2c; B3-B3c). Forward inner primer (FIP) binds to the F2c region in the target and is extended. F3 primer also hybridizes to the F3c region and is extended, displacing the FIP-linked complementary strand. (ii) The displaced single-stranded DNA serves as a template for extension reactions by backward inner primer (BIP) and B3 primer. (iii) The BIP-linked DNA self-anneals and forms a dumbbell-like structure that initiates subsequent rounds of amplification. FIP binds and opens up the loop at the 3′-end converting the dumbbell shape into a stem-loop structure through its extension toward 5′ end. (iv) The extended strand from (iii) forms a new loop and serves as a template for BIP binding and extension. Both these products become the seed for the exponential amplification.

(B) RT-LAMP-based detection of ORF1ab gene. The amplification process monitored via turbidimeter readings at 650 nm or visual observation of calcein-mediated color change from orange to green. The target concentration is inversely proportional to detection time and the color change.

Reproduced with permission from Ref (Yan et al., 2020a). Copyright 2020 European Society of Clinical Microbiology and Infectious Diseases.