Abstract
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.
Highlights
Emerging viral infections continue to pose a major threat to global public health
In order to access absolute analytical sensitivity of the colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection, we calculated the limit of detection (LoD), which is the lowest detectable concentration of viral nucleic acid, here represented in viral copies per microliter (/μL), which was determined based on
The relative sensitivity was accessed in a panel of 367 clinical specimens from nasopharyngeal swab collected in viral transport medium (VTM), including 254 positive and 113 negative samples according to the colorimetric RTLAMP output that were previously characterized by Reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) (Table 1)
Summary
Different viral emergencies have been reported including the severe acute respiratory syndrome coronavirus (SARS-CoV), H1N1 influenza, Middle East respiratory syndrome coronavirus, Ebola vírus, Zika virus, and most recently, the new coronavirus has been described, which cause coronavirus disease 2019 (COVID-19; Wang et al, 2020; Zhu et al, 2020). People with COVID-19 have a wide range of symptoms reported such as fever, cough, anosmia, ageusia, headache, fatigue, muscle or body aches, sore throat, and shortness of breath or difficulty breathing. Some of these symptoms help spread the virus; human-tohuman transmission from infected individuals with no or mild symptoms has been extensively reported (Bai et al, 2020; Rothe et al, 2020). In contrast to antibody detection, which may take weeks after the onset of the infection, detection of viral RNA is the best way to confirm the acute infection phase, the most important phase for viral shedding, so that rationally managed social distancing and lockdown can be implemented (Long et al, 2020; Wang et al, 2020)
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