Simple RNA extraction method for reverse transcription loop-mediated amplification detection of SARS-CoV-2

• Main Authors: Zulkefli, Nur Zulaikha, Bukhari, Fatma Diyana Mohd, Lai, Meng Yee, Suppiah, Jeyanthi, Thayan, Ravindran, Mat Isa, Mohd Khairi, Lau, Yee Ling
• Format: Article
• Published: Seameo Regional Tropical Medicine & Public Health 2022
• Subjects: R Medicine
• Online Access: http://eprints.um.edu.my/41466/
• Institution: Universiti Malaya

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a robust and cost-effective assay for rapid diagnosis of SARS-CoV-2 compared to reverse transcription quantitative (RT-q) PCR. The study evaluated the performance of RT-LAMP technique that incorporated a simple Chelex 100 resin-based RNA extraction step for SARS-CoV-2 detection targeting virus E (encoding envelope protein) and RdRP (encoding RNA-dependent RNA polymerase). Using primer sets for E and RdRP, the developed RT-LAMP assay had a limit of detection (LOD) of 1 copy/mu l transcribed RNA. For nasopharyngeal and oropharyngeal swab samples (n = 58), in comparison to the gold standard RT-qPCR (amplifying E and RdRP) sensitivity, specificity, positive predictive value, and negative predictive value of SARS-CoV-2 RT-LAMP assay targeting E gene was 88% (95% confidence interval (CI): 75-96%), 87% (95% CI: 59-98%), 99% (95% CI: 97-100%), and 28% (95% CI: 14-48%), respectively and for RdRP gene was 67% (95% CI: 51-98%), 87% (95% CI: 59-98%), 100% (95% CI: 96-100%), and 12% (95% CI : 8-18%), respectively. The whole process of RT-LAMP assay was completed within similar to 60 minutes. This developed RT-LAMP method for on-site COVID-19 detection should be useful in resource limited settings.