FQ-LAMP Assay for Detection of CoV2 in Clinical Nasal Swabs
Les Jones, Hemant K. Naikare, Yung-Yi C. Mosley, and Ralph A. Tripp
Abstract
COVID-19 is a public health challenge requiring rapid testing for detecting infections and transmission. Nucleic acid amplification tests (NAAT) targeting SARS-CoV-2 (CoV2) are used to detect CoV2 in clinical samples.Real-time reverse transcription-quantitative PCR (RT-qPCR) is the standard NAAT for CoV2, although Reverse Transcription Loop-mediated isothermal amplification (RT-LAMP) is used in diagnostics. We show a sequence-specific RT-LAMP-based NAAT assay that is finished within 30 min using minimally processed clinical nasal swab samples and describe a fluorescent-quenched RT-LAMP assay (FQ-LAMP) using labeled primers and a quencher oligo. This assay can achieve rapid (30 min) and sensitive (1000 PFU/ml) fluorescent detection of CoV2 (WA1/2020), B.1.1.7 (Alpha), and variants of concern Delta (B.1.617.2) and Omicron (B.1.1.529) in nasal samples.
Steps
FQ-LAMP Sample Preparation
mix 20ul of clinical swab material with 20 ul of SPS buffer containing Proteinase K.
Incubate at 37C for 15 minutes, then 95C for 5 minutes
Sample is ready to use in FQ-LAMP at 2ul / 25ul reaction
Prepare FQ-LAMP Master Mix
Determine the number of 25ul FQ-LAMP reactions needed and add 10%
Mix the following in clean tube per 25ul FQ-LAMP reaction
2X WarmStart LAMP reagent 12.5ul
10X Detection Oligo/Primer Mix 2.5ul
deionized water 8ul
Dispense 23ul of FQ-LAMP master mix to each required wells of a 96 well PCR plate
Add 2ul of prepared sample in SPS buffer per assay
Centrifuge the plate to settle all contents and seal the plate with appropriate cover material.
Run FQ-LAMP assay on real time PCR machine
Set up the real time machine to measure fluorescence in the FAM channel.
Set up the real time machine amplification program:
HOLD 65C / 30 minutes
HOLD 25C / 30 seconds
measure fluorescence
Record results
