SARS-CoV-2 detection by fluorescence loop-mediated isothermal amplification with and without RNA extraction

Area of Science:

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

Background:

• Rapid point-of-care detection of SARS-CoV-2 is essential for pandemic control.
• Reverse transcription loop-mediated isothermal amplification (RT-LAMP) offers faster detection than RT-PCR.
• Saliva is a non-invasive specimen for mass screening, but its utility with RT-LAMP, especially without RNA extraction, needs clarification compared to nasopharyngeal swabs (NPS).

Purpose of the Study:

• To compare the diagnostic performance of a novel RT-fluorescence LAMP (RT-fLAMP) assay using saliva and NPS samples.
• To evaluate the necessity of RNA extraction for SARS-CoV-2 detection by RT-fLAMP in different sample types.

Main Methods:

• A novel RT-fluorescence LAMP (RT-fLAMP) assay was developed.
• Saliva and NPS samples were tested with and without prior RNA extraction.
• Assay performance was evaluated using sensitivity and specificity metrics.

Main Results:

• With RNA extraction, RT-fLAMP demonstrated high sensitivity (97%) and specificity (100%), with equivalent performance for both NPS and saliva samples.
• Without RNA extraction, sensitivity significantly decreased to 71% for NPS and 47% for saliva.
• These findings indicate RNA extraction is critical for optimal SARS-CoV-2 detection by RT-fLAMP.

Conclusions:

• The RT-fLAMP assay is a potentially rapid and effective method for SARS-CoV-2 detection.
• RNA extraction is a critical step for achieving high sensitivity and reliability in RT-fLAMP assays, regardless of sample type (NPS or saliva).
• Further optimization may be needed for direct sample processing in RT-LAMP assays.