Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction

Abstract

Convenient, repeatable, large-scale molecular testing for SARS-CoV-2 would be a key weapon to help control the COVID-19 pandemic. Unfortunately, standard SARS-CoV-2 testing protocols are invasive and rely on numerous items that can be subject to supply chain bottlenecks, and as such are not suitable for frequent repeat testing. Specifically, personal protective equipment (PPE), nasopharyngeal (NP) swabs, the associated viral transport media (VTM), and kits for RNA isolation and purification have all been in short supply at various times during the COVID-19 pandemic. Moreover, SARS-CoV-2 is spread through droplets and aerosols transmitted through person-to-person contact, and thus saliva may be a relevant medium for diagnosing SARS-CoV-2 infection status. Here we describe a saliva-based testing method that bypasses the need for RNA isolation/purification. In experiments with inactivated SARS-CoV-2 virus spiked into saliva, this method has a limit of detection of 500-1000 viral particles per mL, rivalling the standard NP swab method, and initial studies also show excellent performance with 100 clinical samples. This saliva-based process is operationally simple, utilizes readily available materials, and can be easily implemented by existing testing sites, thus allowing for high-throughput, rapid, and repeat testing of large populations. Graphical Abstract3 BackgroundThe slow roll-out and inconsistent availability of diagnostic testing for SARS-CoV-2 has hobbled efforts to control the COVID-19 pandemic in many countries. Testing protocols based on the use of nasopharyngeal (NP) swabs as the collection agent, placed in a tube containing viral transport media (VTM), followed by RNA isolation/purification and subsequent analysis by RT-qPCR is currently the most common method ( Figure 1A). 1,2 While some variant of this process has been implemented worldwide, there are multiple challenges with this workflow. Sample collection using NP swabs requires healthcare workers wearing personal protective equipment (PPE) to collect samples, the swabs can be uncomfortable for the patients during collection, and the swabs and the associated VTM have been in short supply at many times and in most locations. In addition, RNA isolation/purification is another significant bottleneck, both in the time and labor required for this process, and in the availability of the equipment and reagents. All of these components also add to the cost of the testing process.There is emerging consensus that widespread, frequently repeated testing is necessary for a safer return to activities that are important for society. Given the data suggesting that SARS-CoV-2 can be spread by pre-symptomatic/asymptomatic carriers, 3-6 localized outbreaks could be dramatically reduced or prevented if individuals shedding SARS-CoV-2 could be readily identified and isolated. For example, imagine a testing bubble placed over a group that desires face-to-face interaction -employees of a company, members of a sports team, extended family networks, etc. If all members of...

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

“…Boiling samples at 95ºC has been used as a means to both inactivate infectious samples and to liberate SARS-CoV-2 genomic RNA from virions (Batejat et al, 2020;Pastorino et al, 2020;Ranoa et al, 2020). However, the use of in vitro transcribed SARS-CoV-2 RNA as a standard requires that it be spiked into saliva samples after boiling.…”

Comparison of media and standards for SARS-CoV-2 RT-qPCR without prior RNA preparation

“…As a prelude to understanding sample preparation and controls for assay development, we similarly assessed the sensitivity of an extraction-free RT-qPCR assay for naked genomic SARS-CoV-2 RNA (ATCC). Naked RNA was serially diluted in saline and directly subjected to RT-qPCR without RNA extraction, and could be detected at a concentration of 50 copies per 20 microliter qPCR reaction (approximately 2.5 copies per microliter) with the N1 primer set, which amplifies the nucleocapsid (N) gene of SARS-CoV-2 (Figure 1), confirming extraction-free RT-qPCR as a plausible diagnostic tool [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] .…”

“…In particular, while RT-qPCR remains the gold standard for analysis, the requirement of RNA purification prior to enzymatic amplification slows the overall throughput of the assay and limits the number of samples that can be tested. In consequence, many groups have reported on "direct RT-PCR" assays for CoV-2 RNA that bypass the RNA purification [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] . As a prelude to understanding sample preparation and controls for assay development, we similarly assessed the sensitivity of an extraction-free RT-qPCR assay for naked genomic SARS-CoV-2 RNA (ATCC).…”

SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations

“…A review by Esbin et al describes alternative COVID-19 diagnostic protocols involving lysis reagents and direct addition of samples (extraction-free protocols) into the amplification reaction mix [22]. There have been recent advances in extraction-free protocols for COVID-19 amplification [154][155][156][157][158][159] and sequencing-based tests [160]. However, such protocols involve trade-offs between lower sensitivity and simplicity [22,161,162].…”

“…Hi-throughput pooled tests [23][24][25][26][27][28] Pooled sequencing using barcodes [93] Commercial platforms-Hi-throughput RT-PCR [220] Alternative reagents and protocols. for diagnostic testing to augment shortages and supply chain constraints during pandemic peak [22] Extraction-free protocols to augment shortage of RNA extraction kits for amplification-based tests [154][155][156][157][158][159] for sequencing-based tests [160] Drive-through collection of respiratory samples [142] At-home self-collection of clinical samples to augment conventional sample collection formats [147,149] Antibody tests to measure seroprevalence of SARS-CoV-2 antibodies and understand the extent of community transmission [133,134] Large scale population-wide screening for identification of asymptomatic cases in low-prevalence regions [23,25,26] Screening for infections at clinics/doctor's offices, airports, borders, etc.…”