Saliva “Treat-and-Heat” Triplex Reverse Transcription Loop-Mediated Isothermal Amplification Assay for SARS-CoV-2

Marie Jennifer Y. Reolo, Ateneo de Manila University
Carlos Sebastian R. Eleazar, Ateneo de Manila University
Joseph P. Sonio, Ateneo de Manila University
Ryonne T. Solon, Ateneo de Manila University
Janika L B. Villamor, Ateneo de Manila University
Alexandra K. Loedin, Ateneo de Manila University
Keith J M. Moore, Ateneo de Manila University


The demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular diagnostics that are faster, cheaper, and simpler to run than nasopharyngeal-based reverse transcription quantitative PCR (RT-qPCR) tests remains unmet in many parts of the world. In the Philippines, geographical and economic access to quality diagnostic testing remains out of reach for many communities. We describe the preclinical development of a fluorescence-based reverse transcription loop-mediated isothermal amplification test that uses drooled saliva as the biospecimen. Six treat-and-heat (“direct”) procedures that inactivate the virus and release the target RNA were compared. Using duplexed As1e and E1 primers, protocols derived from Ben-Assa et al. (2020) using proteinase K or from Rabe and Cepko (2020) using TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)/EDTA provided reliable RNA amplification. The TCEP/EDTA-based method in particular showed improvement in robustness in duplex vs. singleplex format. Inclusion of human β-actin primers provided a triplex test with an internal amplification control that could be distinguished from SARS-CoV-2 amplicons based on melt curve analysis. After including the dUTP/uracil-DNA glycosylase system and implementing laboratory procedures to avoid cross-contamination, false positive amplification was acceptably rare. The duplex or triplex tests are predicted to reliably detect patient salivary viral loads >100 copies/μL and to yield equivocal results between 10 and 100 copies/μL. These viral loads, corresponding to RT-qPCR Ct ∼29–32, are expected to identify the majority of infected and, particularly, of infectious patients. If clinically validated, the test would provide additional testing capacity requiring only a fraction of the time, cost, and infrastructure of the current nasopharyngeal swab–based RT-qPCR test, thereby improving access to testing for more Filipinos.