Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse-transcription loop-mediated isothermal amplification without the need for RNA extraction

Anahtar, Melis N; McGrath, Graham E G; Rabe, Brian A; Tanner, Nathan A; White, Benjamin A; Lennerz, Jochen K M; Branda, John A; Cepko, Constance L; Rosenberg, Eric S

BACKGROUND: Amid the enduring pandemic, there is an urgent need for expanded access to rapid, sensitive, and inexpensive COVID-19 testing worldwide without specialized equipment. We developed a simple test that uses colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 in forty minutes from sample collection to result. METHODS: We tested 135 nasopharyngeal specimens from patients evaluated for COVID-19 infection at Massachusetts General Hospital. Specimens were either added directly to RT-LAMP reactions, inactivated by a combined chemical and heat treatment step, or inactivated then purified with a silica-particle based concentration method. Amplification was performed with two SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted. RESULTS: Direct RT-LAMP testing of unprocessed specimens could only reliably detect samples with abundant SARS-CoV-2 (>3,000,000 copies/mL), with sensitivities of 50% (95% CI, 28 to 72) and 59% (95% CI, 43 to 73) in samples collected in universal transport medium and saline, respectively, compared to qPCR. Adding an up-front RNase inactivation step markedly improved the limit of detection to at least 25,000 copies/mL, with 87.5% (95% CI, 72 to 95) sensitivity and 100% specificity (95% CI, 87 to 100). Using both inactivation and purification increased the assay sensitivity by ten-fold, achieving a limit of detection comparable to commercial real-time PCR-based diagnostics. CONCLUSION: By incorporating a fast and inexpensive sample preparation step, RT-LAMP accurately detects SARS-CoV-2 with limited equipment for about US$6 per sample, making this a potentially ideal assay to increase testing capacity especially in resource-limited settings.