Development and Validation of a Rapid, Single-Step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) System Potentially to Be Used for Reliable and High-Throughput Screening of COVID-19

• Published in: Frontiers in cellular and infection microbiology 2020-06, Vol.10, p.331-331
• Main Authors: Jiang, Minghua, Pan, Weihua, Arasthfer, Amir, Fang, Wenjie, Ling, Liyan, Fang, Hua, Daneshnia, Farnaz, Yu, Jian, Liao, Wanqing, Pei, Hao, Li, Xiaojing, Lass-Flörl, Cornelia
• Format: Article
• Language: English
• Subjects: Betacoronavirus - genetics; Cellular and Infection Microbiology; Coronavirus Infections - diagnosis; COVID-19; diagnostic test; Humans; Mass Screening - methods; Nucleic Acid Amplification Techniques; Pandemics; Pneumonia, Viral - diagnosis; qRT-PCR; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RT-LAMP; SARS-CoV-2; Sensitivity and Specificity
• ISSN: 2235-2988; 2235-2988
• DOI: 10.3389/fcimb.2020.00331

Development and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS-CoV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread. Primers showing optimal features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays. The analytical specificity and sensitivity of our assay was 100% and 500 copies/ml when serial dilution was performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes. We present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.