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

Journal: PloS one

Article Title: Kinetic analysis of mouse brain proteome alterations following Chikungunya virus infection before and after appearance of clinical symptoms.

doi: 10.1371/journal.pone.0091397

Figure Lengend Snippet: Figure 5. Western blot validations of differentially regulated proteins identified by 2D-DIGE and/or iTRAQ analyses. (A) Protein samples from each group used for proteomic analysis were minimally labeled with cyanine-3 dye. At the top, a representative protein profile of three biological replicates from brain lysates of mock (M), early (E), late paralytic (LP) and late tetanus-like (LT), separated by 10% SDS-PAGE is shown. WB with fluorescence-based methods was used to detect an overlaid fluorescent scan of the general protein patterns (Cy3 dye; green) and the specific immunoreactive proteins (FITC or Cy5 dye; red). To better visualize protein detection signals observed with each specific antibody used, corresponding cropped WB images are presented in grey levels. (B) The graphs correspond to the mean 6 S.D. of protein quantity measured by densitometry of the antigenic bands. Densitometry analyses were performed using TotalLab Quant v12.2 software (Nonlinear Dynamics), and data were normalized to levels of global protein pattern intensity. The values indicated under each graph correspond to fold changes from paired comparisons. The significance of the differential protein expression are indicated *, p,0.05; **, p,0.01; ***, p,0.001. A.U., arbitrary units. ANXA2: annexin A2; ARRB1: b-arrestin; GABRA1: c-aminobutyric acid receptor subunit alpha-1; GRASP1: GRIP-associated protein; ITGAV: integrin aV; MYPT1: myosin phosphatase target subunit 1; N-Ras: N-Ras; RABEP1: rabaptin-5; SYNGR3: synaptogyrin-3. doi:10.1371/journal.pone.0091397.g005

Article Snippet: Blots were saturated with 5% nonfat dried milk in PBS containing 0.05% (v/v) Tween 20 (PBS-T-milk) for 1 h. Western blot (WB) analyses were carried out with rabbit mono- or polyclonal antibodies directed against b-arrestin (1:5000, ARRB1, no. 4674, Cell Signaling Technology, Danvers, MA), GRIP-associated protein (1:500, GRASP1, no. sc-135681, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), annexin A2 (1:100, ANXA2, no. sc-9061, Santa Cruz), integrin aV (1:100, ITGAV, no. 10179, Santa Cruz), myosin phosphatase target subunit 1 (1:100, MYPT1, no. sc25618, Santa Cruz), rabaptin-5 (1:1000, RABEP1, no. sc-15351, Santa Cruz), N-Ras (1:500, N-Ras, no. sc-519, Santa Cruz), synaptogyrin-3 (1:1000, SYNGR3, no. sc-68936, Santa Cruz), or with a goat polyclonal antibody directed against c-aminobutyric acid receptor subunit alpha-1 (1:100, GABAARa1 or GABRA1, no. sc-31045, Santa Cruz), diluted in PBS-T-milk and incubated overnight at 4uC.

Techniques: Western Blot, Multiplex sample analysis, Labeling, SDS Page, Fluorescence, Software, Expressing