We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-μL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8–94.7%) and specificity of 92.4% (95% CI 83.2–97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.
【저자키워드】 Molecular medicine, clinical microbiology, 【초록키워드】 SARS-CoV-2, SARS-CoV2, reliability, Local, outcome, RNA, amplification, colorimetric detection, RT-LAMP, sensitivity, specificity, stability, Surveillance, Patient, Greece, isothermal amplification, Swab, 95% CI, measure, fluorescent, National, extracted RNA, positive sample, objective, human coronavirus, detect, reported, addition, applied, less, demonstrated, reduce, reactivity, 【제목키워드】 Infection, RT-LAMP, clinical, diagnosis of SARS-CoV-2,