Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen’s kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar’s test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.
【저자키워드】 viral infection, Molecular biology, 【초록키워드】 SARS-CoV-2, protocol, equipment, SARS-COV-2 infection, COVID-19 pandemic, Variation, diagnostic, severe acute respiratory syndrome coronavirus-2, severe acute respiratory syndrome Coronavirus, RT-PCR, Laboratory, Severe acute respiratory syndrome, RT-LAMP, specificity, PCR, Mass screening, SARS-CoV-2 genome, isothermal amplification, respiratory, turnaround time, Coronavirus-2, protocols, Coefficient of variation, clinical laboratory, reverse transcriptase, Chain Reaction, acute respiratory syndrome, acute respiratory syndrome coronavirus, reaction, individual, acute respiratory syndrome coronavirus-2, reproducibility, reagent, transcriptase, Cohen’s kappa, robust, flexible, classical, dependent on, demonstrated, 【제목키워드】 SARS-CoV-2, isothermal amplification, transcriptase,