Rapid and Real-Time Viral Detection in a Drop of Minimally Processed Saliva

A Case Study by Bal Ram Adhikari

Current Challenge

Current rapid antigen tests still fail to meet the ongoing demand for viral diagnostic kits that are fast, sensitive, and user-friendly. Traditional nucleic acid amplification tests, such as PCR and LAMP, are accurate but costly, time-consuming, and operationally complex, making them unsuitable for onsite testing. Lateral flow assays, though simpler and with onsite testing ability, often lack sensitivity and specificity, particularly in unprocessed clinical matrices such as saliva.

Innovative Solution

To bridge this gap, Adhikari et.al. (2025) developed a Real-Time Enzyme-Linked Aptamer Assay (RT-eELAA), a rapid aptamer-based diagnostic tool capable of detecting SARS-CoV-2 and Influenza A virus in saliva within 10 minutes. RT-eELAA uniquely integrates two innovations:

• RT-eELAA detection format
• Internal referencing using a non-functional mutant aptamer, allowing each clinical sample to be normalized against its matrix interference.

Technical Highlights

1. Sandwich-format aptamer design: Uses aptamers that have very strong binding affinity, like an antibody, to the specific virus. In this study, we have used a primary aptamer attached to the chip, which captures a virus, and a secondary aptamer conjugated with horseradish peroxidase (HRP), which generates a readable signal upon binding with the virus.
2. Limit of Detection (LOD): The developed diagnostic kit achieved 301 copies/mL for SARS-CoV-2 and 743 copies/mL for Influenza A, outperforming typical antigen tests.
3. Assay time: The sample response time is as quick as 30 seconds, which means the diagnostic kit can deliver results within 30 seconds of reagent addition.
4. No need for amplification or extensive sample processing.

 Clinical Validation

When tested with 20 real clinical saliva samples collected from symptomatic SARS-CoV-2 patients, the assay, at first, struggled with sample-to-sample variability. To overcome this, the revised RT-eELAA protocol employed paired testing on:

• A functional aptamer chip (FA), which utilizes a functional aptamer that has a specific binding affinity with COVID-19
• A mutant aptamer chip (MA)- which utilizes a non-functional aptamer that has no specific binding affinity with SARS-CoV-2

By calculating the FA/MA signal ratio per sample, the assay achieved:

• 100% sensitivity
• 100% specificity
• 100% concordance at the 30-second readout window.

Impact

RT-eELAA delivers laboratory-grade diagnostic performance in a format suitable for point-of-care settings. Its adaptability to multiple viruses and reliance on synthetic aptamers rather than antibodies also improve supply chain resilience, cost efficiency and stability. This assay is a promising candidate for integration into handheld diagnostic platforms or rapid screening tools during a public health crisis.