A One-Pot RT-LAMP Diagnostic Assay for SARS-CoV‑2 from Saliva Samples.

Results of efforts to diagnose infections with SARS-CoV-2 using a sampling method that was less invasive than the nasopharyngeal swab led to the rapid adoption of anterior nasal swabs. Saliva was also shown to have potential as a sample matrix and, like anterior nasal swabs, could be obtained noninvasively (e.g., passive drool). However, due to its inherent complexity and heterogeneity across patient populations (e.g., presence of mucins and RNases), saliva was largely disregarded as point-of-care diagnostics were being developed and broadly implemented. For molecular diagnostic approaches (e.g., RT-PCR or RT-LAMP), these matrix effects from saliva could lead to undesirable false positives or false negatives. The opportunity to address these challenges by normalizing the performance of saliva could enable important applications of molecular tests, particularly at the point-of-care. Toward these goals, we developed a one-pot RT-LAMP assay for the colorimetric detection of SARS-CoV-2 from saliva samples. The assay is performed in five steps: (i) a patient collects a passive saliva sample, (ii) the sample is placed on a heat block for 10 min at 95 °C, (iii) the undiluted sample is added to the one-pot RT-LAMP assay, (iv) the RT-LAMP reaction tube is place on a heat block for 40 min at 65 °C, and, (v) immediately postamplification, the reaction tube is inverted to observe the colorimetric output. We demonstrated the clinical performance of our assay using a panel of 127 patient samples. Our assay had an overall accuracy of 98%, with a sensitivity of 88% and a specificity of 100%. These results indicate excellent diagnostic agreement with the gold standard, RT-PCR, and highlight the potential to improve the clinical utility of saliva for point-of-care (e.g., mobile clinics) testing of SARS-CoV-2 and other upper respiratory viruses and emerging pathogens.