Highly sensitive extraction-free saliva-based molecular assay for rapid diagnosis of SARS-CoV-2

Abstract

The COVID-19 pandemic highlighted the necessity of fast, sensitive, and efficient methods to test large populations for respiratory viruses. The "gold standard" molecular assays for detecting respiratory viruses, such as quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR), rely on invasive swab samples and require time-consuming and labor-intensive extraction processes. Moreover, the turnaround time for RT-qPCR-based assays is too lengthy for rapid screening. Extraction-free saliva-based methods provide a non-invasive sampling process with a fast turnaround time and are suitable for high-throughput applications. However, when used with a standard RT-qPCR system, the absence of extraction significantly reduces the assays' sensitivity. Here, using a novel optical modulation biosensing (OMB) platform, we developed a rapid and highly sensitive extraction-free saliva-based molecular assay. We blindly tested 364 paired nasopharyngeal swabs and saliva samples from suspected SARS-CoV-2 cases in Israel. Compared with the gold standard swab-based RT-qPCR assay, the sensitivity of the extraction-free saliva-based OMB assay is 90.7%, much higher than the sensitivity of extraction-free saliva-based RT-qPCR assay (77.8%) with similar specificity (95.3% and 97.6%, respectively). Moreover, out of 12 samples identified by the OMB-based assay as positive, 8 samples were collected from hospitalized patients in a COVID-19 ward and were verified to be SARS-CoV-2-positive upon admission, indicating that the actual clinical sensitivity and specificity of the OMB assay are higher. Considering its user-friendly saliva-based protocol, short and cost-effective extraction-free process, and high clinical accuracy, the OMB-based molecular assay is very suitable for high-throughput testing of large populations for respiratory viruses.

Importance: Three years after the SARS-CoV-2 outbreak, there are no molecular tests that combine low-cost and straightforward sample preparation, effective sample handling, minimal reagent and disposable requirements, high sensitivity, and high throughput required for mass screening. Existing rapid molecular techniques typically sacrifice certain requirements to meet others. Yet, localized outbreaks of novel viral diseases happen daily in different parts of the world. In this context, respiratory diseases are of specific importance, as they are frequently airborne and highly contagious, with the potential for a rapid global spread. The widely accepted opinion is that another pandemic is just a question of time. To ensure that the containment efforts for the upcoming "disease X" are successful, introducing rapid, high-throughput, and highly sensitive diagnostic methods for detecting and identifying pathogens is critical. A few months into the pandemic, saliva was suggested as a diagnostic matrix for SARS-CoV-2 detection. The collection of saliva does not require swabs and is minimally invasive. In particular, extraction-free saliva-based assays require fewer reagents and disposables, and therefore are faster and cheaper, offering an appealing alternative for low-income countries. Unfortunately, current extraction-free saliva-based detection methods, such as direct RT-qPCR or isothermal amplification, have either low sensitivity or low throughput. Therefore, we believe that the presented highly sensitive ht-OMBi platform and the extraction-free saliva-based molecular assay can become an essential tool in the infectious disease monitoring toolbox.

Keywords: COVID-19; MMB; SARS-CoV-2; assay; detection; diagnostics; extraction-free; high-throughput; highly sensitive; ht-OMBi; optical modulation biosensing; saliva-based.

Fig 1

(A) A double-quenched hydrolysis probe includes an additional quencher (Q2) at the 9th nucleotide from the reporter molecule, effectively reducing the non-specific fluorescent background of the qPCR assays. (B) A modified double-quenched hydrolysis probe includes a biotin molecule, covalently attached to the 5′ nucleotide, which also hosts the reporter molecule—making the probe compatible with MMB/ht-OMBi-based detection.

Fig 2

Schematic representation of the extraction-free saliva-based ht-OMBi molecular assay. Saliva samples are collected in proprietary collection tubes containing dry proteinase K enzyme. The tubes are incubated for 5 minutes at 56°C with constant mixing to ensure proper inactivation and degradation of the PCR inhibitors and nucleases in the saliva. Then, to inactivate the virus and the proteinase K enzyme, the temperature is elevated to 95°C for an additional 5 minutes. During the PCR, the modified double-quenched hydrolysis probe (SARS-CoV-2 specific) is degraded by the 5′−3′ exonuclease activity of Taq polymerase, and free fluorophores with attached biotin are released to the solution. The fluorophores are captured using streptavidin-coated magnetic beads, allowing the examination of the reaction products by a ht-OMBi detection system.

Fig 3

Probit regression analysis to determine the LoD of the ht-OMBi extraction-free saliva-based N1 molecular assay. 20 repetitions for each target concentration were tested. The calculated LoD is ~17 copies/reaction (CI₉₅ 14.1–23.6) or 3.4 copies/ µL.

Fig 4

Comparison of the results of clinical samples testing using the ht-OMBi assay against the gold-standard NP-swab RT-qPCR (reference method #1). The calculated sensitivity and specificity are 90.7% and 95.3%, respectively.

Fig 5

Comparison of the results for identical clinical samples tested using the ht-OMBi assay and the extraction-free saliva-based direct qPCR method (reference method #2). The calculated sensitivity and specificity are 98.9% and 93%, respectively.

Fig 6

Comparison of the results of clinical samples testing using the extraction-free saliva-based RT-qPCR (reference method #2) against the gold-standard NP-swab RT-qPCR (reference method #1). The calculated sensitivity and specificity are 77.8% and 97.6%, respectively.