In this study, we have developed a rapid, near-patient, saliva test for COVID-19 using lyophilized LAMP reagents with fluorometric detection by the naked-eye. Experiments were designed to satisfy regulatory standards. Saliva-Dry LAMP showed no cross-reactivity or interference from any tested respiratory pathogens or medicines, respectively (Table S1, S2). In silico analysis by Mohon et al. did not identify any primer cross-reactivity in 13 relevant respiratory pathogens either14. As an RT-LAMP test, this method uses different reagents than RT-PCR, thus averting some supply chain bottlenecks and export restrictions2,3,19. Saliva-Dry LAMP detects SARS-CoV-2 from saliva, instead of the specimens from the standard nasopharyngeal swab, as there is a higher likelihood of detecting virus in saliva than detecting virus in nasopharyngeal swab specimens during the early phase of infection when diagnostic testing is most useful20. Saliva can be collected without a healthcare worker20,21 and self-collection does not induce coughing, sneezing or bleeding21,22. Therefore, saliva collection avoids depleting critical supplies of PPE and swabs while reducing healthcare worker demand and exposure20,21. Saliva is also favourable for testing children as NP swabs are invasive21.
An important area of ongoing development for point-of-care nucleic acid tests is rapid RNA extraction. Standard laboratory RNA extractions are very time-consuming; however, replacing an RNA purification step with a simple inactivation step can compromise assay sensitivity 14,23,24 . RNA purifications result in the concentration of viral RNA and the removal of amplification inhibitors, both of which increase sensitivity. Some rapid RNA extraction methods exist, but many of them require a cold-chain 25–28 . The streamlined, column-based RNA extraction developed for Saliva-Dry LAMP purifies RNA in under 30 minutes while concentrating RNA 2.8-fold and costing only $3.15 (CAD) per preparation. Using commercially-available equipment, this test has a throughput of 10 samples per batch. Manufacture of the Biobox instrument reduced the capital equipment cost 5-fold from US $1977.44 to US $386.72 (Table S5 and S6), while still achieving an excellent limit of detection. Saliva-Dry LAMP has a capital equipment cost an order of magnitude less than RT-PCR when using commercially-available instruments, and a capital equipment cost nearly two orders of magnitude less than RT-PCR when using the Biobox (S. Rudgar, personal communication). Either of these options enables the deployment of Saliva-Dry LAMP in resource-limited settings.
The performance characteristics, limit of detection, and ease-of-use of Saliva-Dry LAMP is comparable to other commercial near-patient nucleic acid COVID-19 tests. STOPCovid is a rapid diagnostic test that employs LAMP and CRISPR technology but relies on nasopharyngeal swabs25. Saliva-Dry LAMP achieved similar sensitivity as STOPCovid (93.1% sensitivity, 188/202) and specificity (98.5% specificity, 197/200)25. Lalli et al. developed an extraction-free RT-LAMP test for COVID-19 using saliva with colorimetric detection28. Lalli et al. achieved a slightly higher limit of detection than Saliva-Dry LAMP (59 copies/reaction or 21.6 copies/µL of saliva) but the clinical validation achieved a sensitivity of 85% (17/20) and a specificity of 90% (9/10) The test performed well compared to an internationally recognized reference method. The one false positive was likely a sampling error for the NP swab as the concomitant saliva sample was positive (CDC N2 RT-PCR positive, Ct 25.97). However, the clinical validation set did not contain more samples with very late Ct values in the 35–40 range which in our experience fail to amplify by LAMP. Due to the paucity of in vivo studies, the clinical and epidemiological significance of high Ct value (> 35), low viral copy, individuals remains unclear29,30. In vitro studies suggest individuals with low viral loads are rarely infectious or not infectious29,30. The analytical sensitivity of a diagnostic test is not the only measure by which it should be judged8. In terms of limiting the transmission of SARS-CoV-2, the advantages of Saliva-Dry LAMP are its superior sample-to-result time (~ 105 minutes) compared to RT-PCR, near-patient deployment, ease of application, and cost8.
This method has its limitations. Firstly, the positive controls used in this study are not room-temperature stable reagents and require cold chain. Second, while the sample-to-result time near the patient is useful, the time required to perform the test is in approximately 105 minutes with a minimal throughput of four samples per run. Finally, the equipment developed still requires electricity and further refinements to increase the portability. A second prototype of the Biobox relying on a lithium-ion battery is feasible and is currently being evaluated. Future studies will aim to port Saliva-Dry LAMP onto a microfluidic cartridge, improving speed, and point-of-care feasibility.