Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
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.
Article activity feed
-
-
SciScore for 10.1101/2021.05.26.21257488: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: All procedures involving human participants, collection and use of clinical samples and data were in accordance with ethical standards and approved by the human research ethics committee under license protocol number: 4051614; CAAE (certificate of presentation for ethical appreciation): 31984720300005091. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Linear regression was performed using Prism software, version 9 (GraphPad Software, San Diego, CA, USA) leading to the equation: Y = -3.6383X + 38.771 and coefficient of correlation R2 = 0.9938 (Supplementary … SciScore for 10.1101/2021.05.26.21257488: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: All procedures involving human participants, collection and use of clinical samples and data were in accordance with ethical standards and approved by the human research ethics committee under license protocol number: 4051614; CAAE (certificate of presentation for ethical appreciation): 31984720300005091. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Software and Algorithms Sentences Resources Linear regression was performed using Prism software, version 9 (GraphPad Software, San Diego, CA, USA) leading to the equation: Y = -3.6383X + 38.771 and coefficient of correlation R2 = 0.9938 (Supplementary Figure S2). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:One of the main limitations for direct sample test by colorimetric RT-LAMP based on pH-sensing is the false positive results upon input sample addition (previous to amplification), due to naturally acidic samples (30,52). To prevent spurious amplification due to the presence of DNA from oral microbiome, food or host cells on primary samples, Bokelmann and cols (2021) treated samples with λ exonuclease that acts by preferentially digesting 5’-phosphorylated DNA leaving non-phosphorylated primers or LAMP products intact (30). Here we shown preliminary results on RNA extraction-free, pre-treatment free, primary 10x diluted hydrochloride guanidine-containing VTM nasopharyngeal samples directly accessed to compared colorimetric results. Three out of five RT-qPCR true positives directly accessed samples returned positive yellow output on colorimetric RT-LAMP for SARS-CoV-2 detection. This provides clues on the use of unextracted samples for massive COVID-19 testing campaigns with a trade-off on cost-benefits for limit of detection and test sensitivity. Most high and medium viral load samples will be detected on unextracted protocols. However, to meet RT-qPCR detection sensitivity levels, this requires some type of purification step and RNA concentration (11,24,31). We are currently observing rapid converging evolution of SARS-CoV-2 during COVID-19 pandemics worldwide. Several reports alert for the emergence of variants of concern and interests such the B.1.1.7, first detected in En...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
-
