While public health efforts to curtail the spread of COVID-19 have continuously yielded promising results, continued diagnostic testing constitutes a foremost requirement in the prevention, and early detection of viral infection. Here, we report a new, non-invasive RT-LAMP-based assay for the rapid detection of SARS-CoV-2 in saliva, encompassing novel components for enhanced test accuracy. Following comparative analyses of the SaliVISION assay, in conjunction with both SalivaDirect and Thermo Fisher Scientific TaqPath FDA EUA-approved diagnostic testing platforms, the SaliVISION assay offers 94.29% and 98.28% accuracy, respectively. Moreover, we report 100% specificity for SARS-CoV-2, with no presentable cross-reactivity with other pathogens. Additionally, careful primer design for the targeted N-gene of the SARS-CoV-2 genome, with the inclusion of novel spacer-loop elements, have yielded greater amplification fidelity, and have contributed to the robustness of our test, in spite of a myriad of pervasive SARS variants. Furthermore, the versatility and scalability for which this assay presents allows for increased point-of-care and high-throughput testing capabilities, with a robustness that allows for consistent detection of SARS-CoV-244.
Thus far, molecular diagnostics that have been designed for the diagnosis of COVID-19 have presented an array of challenges which have been further compounded by a myriad of different factors. The newly developed SaliVISION test is not an exception. The primary challenge in this pH-based assay is the original pH of the saliva samples that can significantly impact to the stability of the reporter system and thus affects the specificity of the result. The normal range human saliva’s pH is 6.2–7.4, with an average at 6.7. Additionally, approximately 10% of human saliva is acidic41. The pH in these samples could reduce the pH of the WarmStart master mix and cause a color change in the final reaction, despite the absence or presence of the SARS-CoV-2 DNA, leading to a false positive result. To solve this problem, we diluted the inactivated saliva samples with a defined concentration of NaOH to neutralize the (1) natural acidity of saliva sample, and (2) stabilize the original pH of the reaction after sample input, without interfering with the sensitivity of the assay. In addition, a precise dilution factor was optimized to provide an adequate sample input to the final reaction, preventing inhibitory effects due to overwhelming concentrations, thus enhancing both efficiency and efficacy of the assay. In this test, the assay is designed to tolerate saliva sample with pH ≥ 4.9 while still ensuring adequate stability and reliability for on-site performance. In the interest of preventing cross-contamination, we have incorporated Uracil-DNA Glycosylase (UDG) into our assay as a means of preventing amplicon contamination from antecedent reactions45,46. Moreover, each reaction strip is completely closed prior to amplification and remains so as visualization of results occurs simultaneously with amplification. Lastly, because the success of RT-LAMP reactions is largely contingent upon successful primer design, several amendments to our primer sets, over the course of test development, were made necessary to maximize test specificity. As it stands, the SaliVISION assay provides 100% specificity for SARS-CoV-2, utilizing a two-primer set for distal locations on the N-gene. The specificity for which this test presents is also, in part, attributed to a novel spacer-loop element which is purposed for increased amplification fidelity for viral SARS-CoV-2 RNA.
Given the projected prevalence of asymptomatic persons carrying COVID-19, designated as asymptomatic spreaders, as well as the increase of new variants with more contagious characteristics, high testing sensitivity in molecular diagnostics for COVID-19 is of paramount importance. Moreover, as some studies have suggested high false-negative rates among RT-PCR diagnostics for COVID-1947,48,49,50, the importance of high-sensitivity testing is further underscored as the continuation of false-negative diagnoses perpetuate the spread of COVID-19. RT-LAMP has since served as a popular alternative to traditional laboratory diagnostics due to its relative ease-of-use and overall versatility. However, current RT-LAMP tests typically provide a 75%-91% clinical sensitivity28,51,52 and offer a limit of detection-in congruence with the reported sensitivity-corresponding to a range of Ct values from ~ 30–33.553,54,55. We report that the SaliVISION assay has an overall clinical sensitivity of 99.46% for saliva specimens with a Ct value of ≤ 36, as confirmed on both SalivaDirect and TaqPath testing platforms. Moreover, when specifically compared to the TaqPath NP swab testing platform using 98 paired patient samples, collected on site, the SaliVISION assay displayed an overall specificity and sensitivity of 97.62% and 85.71%, respectively; however, among samples with a Ct value of ≤ 36, assay sensitivity was 100%. Although this is not ideal-as the standard convention for ruling out the possibility of COVID-19 infection is confirmed when a Ct value of viral RNA is > 40,-the relative increase in sensitivity for which the SaliVISION assay provides, coupled with its ease-of-use, make it a reliable alternative to traditional laboratory diagnostics, which may be less cost and time efficient.While saliva has been established as a less reliable source of bodily excretion for COVID-19 molecular diagnostics, as opposed to nasopharyngeal excretions, it has become a promising alternative due to less invasive sampling procedures. Further, it has been reported that while saliva typically carries a lighter viral load, the SARS-CoV-2 virus is capable of infecting, and replicating, within cells lining the oral mucosa19,20. In addition to infected cells of the lining of oral mucosa, it has also been observed that even in asymptomatic individuals, an acellular fraction of SARS-CoV-2 from infected glands is capable of making de novo virus19, therein contributing to infectiousness as well as providing a source of live virus for saliva-based COVID-19 testing. What’s more, the viral longevity in saliva has been reported to be, on average, 18–20 days10,56,57, which allows for saliva-based testing to be a valid diagnostic modality for the entire duration of an infected individual’s contagious window. Therefore, saliva has become an appealing source for molecular diagnostic testing; coupled with the associated non-invasive sampling procedures, as well as array of saliva-based rapid testing that currently exists, the use of saliva in COVID-19 testing is rapidly becoming an attractive alternative to other forms of testing.
In the interest of expanding the scope of COVID-19 rapid saliva-based testing, it is imperative to establish a feasible methodology that encompasses stringent safety measures to prevent further spread of the virus. Currently, the body of rapid molecular diagnostics for COVID-19 is primarily comprised of antibody testing; however, such testing is markedly inadequate as the production COVID-19 specific antibodies may take up to several weeks, and thus will not inform an individual of active infection. Other rapid testing modalities include nucleic acid-based testing and antigen testing; the latter has yet to prove reliable in individuals present with low viral loads, while the former, albeit reliable, may still take up to a couple days for results, and is considerably more expensive. Given the foregoing, testing efforts, on all scales, would greatly benefit from an expanded repertoire of readily available rapid testing. Our SaliVISION assay provides enhanced specificity and sensitivity when compared to other modes of rapid diagnostic testing, while proving more time efficient, providing test results within 45 min. In congruence with these benefits, RT-LAMP provides a cheaper alternative testing method due to its minimalistic approach with regards to equipment and reagents. Comparatively, both SalivaDirect and TaqPath testing platforms require a minimum of several hours to process samples, and often take up to 24–48 h for the results to made available. Additionally, the necessity for specialized equipment and instrumentation (e.g. biological safety cabinet, qPCR machine, qPCR consumables/reagents, etc.) can easily triple sample processing costs when compared to the SaliVISION assay. Moreover, we utilize an intuitive, convenient self-sample collection process that allows for increased throughput, while mitigating health-exposure risks (Fig. 6). Further, self-collection accommodation with pre-loaded lysis buffer inside the collection tube allows for specimens to be lysed and inactivated in a closed tube after sample collection, thus circumventing the need for expensive biological safety cabinets and prolonged safety procedures. Therefore, expansion of the RT-LAMP testing platform for the rapid diagnosis of COVID-19 is a promising avenue by which large-scale testing efforts can achieved, more efficiently.
As ongoing efforts to curb the spread of the COVID-19 global pandemic-such as mass vaccination-continue, the demand for readily accessible diagnostic testing remains to be of unequivocal importance, until the efficacy and accessibility of existing vaccines is better established. The early detection, and isolation of pre-symptomatic and asymptomatic individuals with COVID-19 is critical in mitigating the transmission of SARS-CoV-2. With its combined ease-of-use, accuracy, and rapid turn-around time, the SaliVISION assay has the potential to serve as a means of curbing the spread of the COVID-19 pandemic, particularly in stemming asymptomatic transmission. Moreover, when used in conjunction with other easily accessible technologies, such as smartphone-based microfluidic systems of detection, the beneficial impact of the RT-LAMP platform is magnified due to its ease-of-use, analytical robustness, and potential role as a test result database58. To this end, the capability to increase testing throughput and accessibility, in addition to another means of monitoring the spread of COVID-19, provide yet another tool in the fight against the COVID-19 pandemic. Therefore, given the versatility of our RT-LAMP assay, we aim to eventually modify this technology to allow for the rapid diagnostic testing of other viral pathogens, for which detection is possible through salivary extracts.