The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/μL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3–99.5%] sensitivity and 100% (95% CI = 94.5–100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non–SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction–free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.
【저자키워드】 COVID-19, SARS-CoV-2, diagnostic test, RT-LAMP, respiratory virus, molecular test, 【초록키워드】 coronavirus disease, Brazil, coronavirus, pandemic, equipment, variant, SARS-CoV-2 variant, diagnostic, B.1.617.2, Transmission, virus, RNA, cross-reactivity, Nasopharyngeal swab, point-of-care, SARS-CoV-2 detection, sensitivity, specificity, RT-qPCR, COVID-19 testing, P.1, limit of detection, Pandemics, P.2, variants of interest, reverse transcription, isothermal amplification, molecular, genomic, SARS-CoV-2 RNA detection, alternative method, confidence interval, Viral transport medium, acute respiratory syndrome, 95% CI, reaction, gold standard, effort, N genes, widespread, country, polymerase chain, performed, detect, collected, demonstrated, meet, Typical, 【제목키워드】 validation, clinical, sensitive, concern, optimization, Specific,