Colorimetric ReadiLAMP Kit for Optimized SARS-CoV-2 Detection and Surveillance Using LAMP Assay

Figure 1. The Colorimetric ReadiLAMP Kit enables highly specific SARS-CoV-2 detection.

Samples from 12 unique nasopharyngeal donors (D1 – D12) were supplemented with γ-irradiated SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources), Influenza A H1N1, or Influenza A H3N2 (ATCC) or 5.0 μL NATtrol™ Respiratory Pathogen Panel-1 or Panel-2 (RP1/RP2, Zeptometrix). RNA was purified using the MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit and the Thermo Scientific KingFisher Flex Magnetic Particle Processor. The 25 μL Colorimetric ReadiLAMP reactions were incubated at 65 °C for 30 min. In all samples supplemented with SARS-CoV-2, a positive blue color change was observed, while all other virally supplemented samples and controls remained a negative purple color. [RP1 contains the following viral particles and bacteria: Influenza A H3N2 (Brisbane/10/07), Influenza A H1N1 (NY/02/2009), Rhinovirus (Type 1A), Adenovirus (Type 3), Parainfluenza (Type 1), Parainfluenza (Type 4), Metapneumovirus (Peru 6-2003), Chlamydia pneumoniae (CWL-029), Mycoplasma pneumoniae (M129), and Coxsackievirus (Type A1). Whereas RP2 contains the following viral particles and bacteria: from Influenza A H1 (New Caledonia/20/99), Influenza B (Florida/02/06), RSV (Type A), Parainfluenza (Type 2), Parainfluenza (Type 3), Coronavirus (HKU-1 recombinant), Coronavirus (OC43), Coronavirus (NL63), Coronavirus (229E), Bordetella pertussis (A639).]

Figure 2. The Colorimetric ReadiLAMP Kit efficiently detects multiple SARS-CoV-2 strains.

Fifteen strains of SARS-CoV-2 RNA were obtained and evaluated in the Colorimetric ReadiLAMP assay. Each 25 μL reaction included 2.5 x 101 – 1.0 x 10⁴ copies of SAR-CoV-2 in a background of 50 ng human genomic DNA. After 65 °C incubation for 30 min, 80% of strains were detected at a sensitivity of 25 copies/ reaction with all strains being detected at 50 copies/ reaction. These detectable strains, obtained from ATCC, were Hong Kong (NR-52388), Italy (NR-52498), England (NR-52499), Singapore (NR-52501), Germany (NR-52502),  USA-IL (NR-52503), USA-CA1 (NR-52504), USA-AZ (NR-52505), USA-WI (NR-52506), USA-CA3 (NR-52507), USA-CA4 (NR-52508), USA-CA2 (NR-52509), Chile-Santiago (NR-52510), USA-New York (NR-53518), and USA-WA (NR-52285).

Figure 3. Using saliva-derived samples, the Colorimetric ReadiLAMP kit detections as few as 20 copies SARS-CoV-2 per reaction.

Samples from 12 unique saliva donors (D1 - D12) were supplemented with γ-irradiated SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources). RNA was purified using the MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit and the Thermo Scientific KingFisher Flex Magnetic Particle Processor. The 25 μL Colorimetric ReadiLAMP reactions were assembled using 1.0 – 12.5 μL input sample containing 0 – 250 copies of SARS-CoV-2 per reaction. After a 65 °C incubation for 30 min, all samples containing SARS-CoV-2 RNA transitioned to blue, positive reactions, while non-templated reactions remained purple. To increase sensitivity, sample input volume may be increased to 50 % of the total reaction volume (12.5/ 25 μL) with the caveat that individual reaction sensitivity is dependent on sample type, donor, and RNA purity. For saliva, the minimum range that can be robustly detected varies from 20 – 100 copies/ reaction for uncomplicated to challenging samples, respectively.