A One-Pot RT-LAMP Diagnostic Assay for SARS-CoV‑2 from Saliva Samples

Area of Science:

• Molecular Diagnostics
• Virology
• Point-of-Care Testing

Background:

• Nasopharyngeal swabs are invasive; anterior nasal swabs and saliva offer less invasive alternatives for SARS-CoV-2 detection.
• Saliva's complexity (mucins, RNases) historically hindered its use in point-of-care diagnostics, potentially causing false results in molecular assays.
• Addressing saliva matrix effects is crucial for enabling its application in rapid molecular diagnostics.

Purpose of the Study:

• To develop a one-pot reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for colorimetric detection of SARS-CoV-2 in saliva.
• To overcome the challenges posed by saliva's heterogeneity for reliable point-of-care testing.
• To evaluate the clinical performance of the developed saliva-based assay.

Main Methods:

• A five-step protocol involving passive saliva collection, heat inactivation, addition to RT-LAMP reagents, isothermal amplification at 65°C, and colorimetric output observation.
• Utilized a one-pot RT-LAMP reaction for direct detection from undiluted saliva samples.
• Clinical validation performed on 127 patient samples, comparing results against the gold standard RT-PCR.

Main Results:

• The developed assay achieved 98% overall accuracy, 88% sensitivity, and 100% specificity.
• Demonstrated excellent diagnostic agreement with RT-PCR, the established gold standard.
• Colorimetric output provided immediate, observable results post-amplification.

Conclusions:

• The one-pot RT-LAMP assay effectively detects SARS-CoV-2 in saliva with high accuracy.
• This method shows significant potential for improving the clinical utility of saliva for point-of-care diagnostics.
• The assay is suitable for testing SARS-CoV-2 and potentially other respiratory viruses in diverse settings, including mobile clinics.