Journal: Frontiers in microbiology

Article Title: 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.

doi: 10.3389/fmicb.2021.713713

Figure Lengend Snippet: FIGURE 5 | Microbial cross-reactivity assay to test SARS-CoV-2 RT-LAMP analytical sensitivity. The test was performed using potentially cross-reacting respiratory viruses or local occurring arboviruses. RT-LAMP reaction was performed at 65◦C during 30 min, with additional 10 min, to confirm the absence of cross-reactivity when targeting SARS-CoV-2 E and N genes. The assay was performed using the WarmStart R⃝colorimetric LAMP 2× master mix (NEB #M1800). Yellow (positive) reaction is observed only when the template is SARS-CoV-2 viral RNA. hRSV, human respiratory syncytial virus; NTC, nontemplate control; M, molecular size marker. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed R⃝(Biotium #41003) to confirm DNA amplification. DENV3, dengue virus serotype 3; ZIKV, Zika virus; CHIKV, Chikungunya virus; YFV, yellow fever virus; Influenza A (H1N1/H3N2); and influenza B (Yamagata/Victoria).

Article Snippet: RT-LAMP reactions were performed according to NEB recommendations, containing the following components: 10 μL of WarmStart R© Colorimetric LAMP 2× Master Mix [NEB #M1800 or #M1804, the latter contains dUTP UDG (uracil-DNA-glycosylase) to avoid carryover contamination; composition of both are NEB’s proprietary]—ready-to-use mixture of WarmStart R© Bst 2.0 DNA polymerase and WarmStart R© RTx (reverse transcriptase for onestep transcription/amplification reaction) in presence of a pH sensor that turns from fuchsia (pink) to yellow in presence of increased proton (acid pH) during DNA polymerization on isothermal amplification, 1.6 μmol/L forward inner/backward inner primers (FIP/BIP); 0.2 μmol/L forward and backward 4https://primerexplorer.jp/e/ 5https://lamp.neb.com Frontiers in Microbiology | www.frontiersin.org 15 November 2021 | Volume 12 | Article 713713 fmicb-12-713713 November 12, 2021 Time: 14:43 # 16 Alves et al. Colorimetric RT-LAMP for SARS-CoV-2 Detection

Techniques: Virus, Control, Marker, Agarose Gel Electrophoresis, Staining

Journal: Frontiers in microbiology

Article Title: 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.

doi: 10.3389/fmicb.2021.713713

Figure Lengend Snippet: FIGURE 6 | Colorimetric RT-LAMP for SARS-CoV-2 detection using genes N, E, and RdRp as target. Selected SARS-CoV-2–positive clinical samples by RT-qPCR were classified as low (Ct 18.9 and 21.7), medium (Ct 26.6 and 28.4), and high (Ct 31.6 and 35.2) Ct values for E gene. They were included as input for colorimetric RT-LAMP reaction using primers targeting N, RdRp (A), and E genes (B). RT-LAMP SARS-CoV-2 false-negative samples were more frequent when using E and RdRp genes as target (C). RT-LAMP reaction was performed at 65◦C during 30 min, using the WarmStart R⃝colorimetric LAMP 2× master mix (NEB #M1800). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed R⃝(Biotium #41003) to confirm DNA amplification. +C, positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC, nontemplate control.

Article Snippet: RT-LAMP reactions were performed according to NEB recommendations, containing the following components: 10 μL of WarmStart R© Colorimetric LAMP 2× Master Mix [NEB #M1800 or #M1804, the latter contains dUTP UDG (uracil-DNA-glycosylase) to avoid carryover contamination; composition of both are NEB’s proprietary]—ready-to-use mixture of WarmStart R© Bst 2.0 DNA polymerase and WarmStart R© RTx (reverse transcriptase for onestep transcription/amplification reaction) in presence of a pH sensor that turns from fuchsia (pink) to yellow in presence of increased proton (acid pH) during DNA polymerization on isothermal amplification, 1.6 μmol/L forward inner/backward inner primers (FIP/BIP); 0.2 μmol/L forward and backward 4https://primerexplorer.jp/e/ 5https://lamp.neb.com Frontiers in Microbiology | www.frontiersin.org 15 November 2021 | Volume 12 | Article 713713 fmicb-12-713713 November 12, 2021 Time: 14:43 # 16 Alves et al. Colorimetric RT-LAMP for SARS-CoV-2 Detection

Techniques: Quantitative RT-PCR, Agarose Gel Electrophoresis, Staining, Positive Control, Cell Culture, Control

Journal: Frontiers in microbiology

Article Title: 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.

doi: 10.3389/fmicb.2021.713713

Figure Lengend Snippet: FIGURE 8 | Colorimetric RT-LAMP allows the detection of SARS-CoV-2 VOCs and VOIs. RT-LAMP reaction was performed at 65◦C for 30 min, using the WarmStart R⃝colorimetric LAMP 2× master mix (NEB #M1804), using multiplex N2/E1 primer sets. The amplicons were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed R⃝staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control. The top panel shows a schematic representation of SARS-CoV-2 spike protein (upper) and where the main mutations are highlighted and represented in SARS-CoV-2 virions (right hand side) present in VOC gamma (B.1), delta (B.1.167.2), and VOI zeta (P.2). The VOCs alpha (B.1.1.7) and beta (B.1.3.51), first reported in the United Kingdom and South Africa, respectively, are also represented. K417N: lysine-to-asparagine substitution at position 417 of spike protein at the receptor biding domain (RBD); V445A: valine-to-alanine substitution at position 445 and so on. L, leucine; Q, glutamine; E, glutamic acid; Y, tyrosine; T, threonine; P, proline; H, histidine; D, aspartic acid; S, serine; F, phenylalanine. del, deletion. Segments of SARS-CoV-2 protein NTD, N-terminal domain; CTD2, C-terminal domain 2 or C terminus of S1 fragment after furin cleavage; FP, fusion peptide; HR1, heptad repeat region 1. SARS-CoV-2 variants were previously sequenced. Variants of interest B.1.1.371 and B.1.1.374 were first reported in Saudi Arabia and Finland, respectively, (https://cov-lineages.org/). Created with biorender.com.

Article Snippet: RT-LAMP reactions were performed according to NEB recommendations, containing the following components: 10 μL of WarmStart R© Colorimetric LAMP 2× Master Mix [NEB #M1800 or #M1804, the latter contains dUTP UDG (uracil-DNA-glycosylase) to avoid carryover contamination; composition of both are NEB’s proprietary]—ready-to-use mixture of WarmStart R© Bst 2.0 DNA polymerase and WarmStart R© RTx (reverse transcriptase for onestep transcription/amplification reaction) in presence of a pH sensor that turns from fuchsia (pink) to yellow in presence of increased proton (acid pH) during DNA polymerization on isothermal amplification, 1.6 μmol/L forward inner/backward inner primers (FIP/BIP); 0.2 μmol/L forward and backward 4https://primerexplorer.jp/e/ 5https://lamp.neb.com Frontiers in Microbiology | www.frontiersin.org 15 November 2021 | Volume 12 | Article 713713 fmicb-12-713713 November 12, 2021 Time: 14:43 # 16 Alves et al. Colorimetric RT-LAMP for SARS-CoV-2 Detection

Techniques: Multiplex Assay, Agarose Gel Electrophoresis, Control, Positive Control