Rapid visual detection of SARS-CoV-2 by colorimetric loop-mediated isothermal amplification

Abstract

Evaluation of the performance of a new set of primers defined from the ORF1ab sequence, and its combination with a previously published set of primers from the N sequence, to detect SARS-CoV-2 RNA by the loop-mediated isothermal amplification technique is presented. The ORF1ab primer set enables visual detection of SARS-CoV-2 RNA in 16 min. In addition, a simultaneous reaction with both ORF1ab and N primers allows for higher sensitivity of detection, particularly when low numbers of copies are present (250 viral RNA copies). Further, the protocol is able to detect viral RNA in saliva samples. The procedure reported could be easily implemented in the generation of a new and sensitive rapid point-of care device for SARS-CoV-2 RNA visual detection.

Keywords: COVID-19; ORF1ab primers; SARS-CoV-2 detection; nucleocapsid primers; virus LAMP amplification.

Figure 1.. SARS-CoV-2 visual detection.

(A & B) Percentage of visually positive replicates (seven replicates) for each primer set (ORF1ab and N) and their combination (ORF1ab + N) after (A) 16 or (B) 22 min of LAMP reaction with different amounts of synthetic SARS-CoV-2 RNA (10,000, 5000, 1000, 500, 250, 100 and 50 copies). (C) Color shown in the test tube of nontemplate control for each primer set (ORF1ab and N) and their combination (ORF1ab + N) after 22 min of LAMP reaction (seven replicates).

Figure 2.. Spectroscopy and visual positive SARS-CoV-2 detection.

Ratio of absorbance at 440/560 nm ratio (mean ± SEM, seven replicates), visual detection and positive well ratio (X/7) after 30 min of LAMP reaction with different amounts of synthetic SARS-CoV-2 RNA (10,000, 5000, 1000, 500, 250, 100 and 50 copies) for each set of primers. (A) ORF1ab primers. (B) N primers. (C) Combination of both primer sets (ORF1ab + N). (D) Ratio of absorbance at 440/560 nm ratio (mean ± SEM, seven replicates), visual detection and the maximum detected ratio (440/560 nm) after 30 min of LAMP reaction of nontemplate control. Samples with a ratio <1.5 for the ORF1ab and ORF1ab + N primers sets, and <1.3 for the N primer set, were excluded and considered negative for the positive well ratio. The assay was conducted in 96-well PCR plates and the image has been taken from the bottom. LAMP: Loop-mediated isothermal amplification; SEM: Standard error of the mean.

Figure 3.. SARS-CoV-2 visual detection in saliva samples.

Visual detection at 16, 22 and 30 min of LAMP reaction with 1 × 10⁴ copies of synthetic SARS-CoV-2 RNA (five replicates) or nontemplate control (one replicate), for each set of N and ORF1ab primers, with or without a pretreated saliva sample. The assay was conducted in eight-well PCR strips and the image has been taken from the front of the strips.