A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal...

Area of Science:

• Molecular Biology
• Virology
• Clinical Diagnostics

Background:

• The COVID-19 pandemic highlighted critical shortages of reagents and equipment for reverse transcriptase (RT)-polymerase chain reaction (PCR) testing.
• This created a need for alternative, high-throughput methods for mass screening of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in clinical settings.

Purpose of the Study:

• To establish and evaluate a robust SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay for high-throughput clinical diagnostics.
• To compare the performance of the RT-LAMP assay against conventional RT-PCR protocols for SARS-CoV-2 detection.

Main Methods:

• Developed and validated an isolation-free SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay.
• Compared the RT-LAMP assay with two conventional RT-PCR protocols using 323 clinical samples from individuals with suspected SARS-CoV-2 infection.
• Determined the limit of detection (LoD) and reproducibility of the RT-LAMP assay.

Main Results:

• The RT-LAMP assay demonstrated almost perfect agreement with RT-PCR methods (Cohen's kappa > 0.8) with no systematic differences.
• Sensitivity ranged from 89.5% to 100%, and specificity ranged from 96.2% to 100%, depending on the reference RT-PCR protocol.
• The assay exhibited high reproducibility (intra-run CV = 0.4%, inter-run CV = 2.1%) and a LoD of 95 SARS-CoV-2 genome copies per reaction.

Conclusions:

• The developed SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR.
• This method is suitable for SARS-CoV-2 mass screening in clinical diagnostic laboratories utilizing existing infrastructure.
• The assay offers high throughput and short turnaround times, addressing limitations observed during the pandemic.