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: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: Overall schematic of multiplex nucleic acid diagnostic tests for combating SARS-CoV-2 variants and other respiratory viruses.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: Multiplex Assay, Diagnostic Assay

Journal: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: PCR techniques and applications. ( a ) Basic schematic of the core PCR amplification steps. The dsDNA containing the targets of interest are denatured into two separate ssDNA strands. Hybridization of primers complementary to target sequences of interest signals DNA polymerase to amplify ssDNA targets through thermocycling. ( b ) Adaptation of a standard laboratory centrifuge into a fully customizable disc that allows for PCR into 64 independent direct RT-qPCR across four independent loading units. Adapted with permission from Ref. . Copyright 2020, Royal Society of Chemistry. ( c ) The utilization of fluorescent microspheres (MPA enhanced ARMS-PCR platform) for the amplification of fluorophores on primers targeting genes of interest in the detection of SARS-CoV-2. Adapted with permission from Ref. . Copyright 2022, Elsevier. ( d ) Decreasing the signal-to-noise ratio of genomic target to background interference using the ddPCR platform. As shown in this platform, a series of microfluidic channels create individual droplets, which can then be separated into individual bioreactor units on a silicon chip. Adapted with permission from Ref. . Copyright 2021, Elsevier.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: Amplification, Hybridization, Quantitative RT-PCR

Journal: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: CRISPR/Cas technologies and multiplexing applications. ( a ) Demonstration of cis-cleavage and trans-cleavage of CRISPR/Cas mechanism, as crRNA-M recognizes the mutant DNA (MT-DNA). Adapted with permission from Ref. . Copyright 2022, Elsevier. ( b ) Multiplex colorimetric assay triggered by LAMP (ON signal) and eliminating false positives by using a CRISPR/Cas9 system (OFF signal) to detect VOCs. Adapted with permission from Ref. . Copyright 2022, American Chemical Society. ( c ) The combination of nested RPA and CRISPR/Cas12a in the same reaction pot. Adapted with permission from Ref. . Copyright 2022, Wiley-VCH. ( d ) Multiple crRNAs targeting different regions of SARS-CoV-2 RNA with single reporter RNA type to improve the sensitivity of the assay. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( e ) The magnetic beads conjugated with CRISPR/Cas complex triggered by target RNA and pulled down by the magnetic force into the microchamber, concentrating the reactions. Adapted with permission from Ref. . Copyright 2022, Springer Nature. ( f ) RT-PCR CRISPR-based diagnostic and microfluidic panel, mCARMEN, for parallelizing nucleic acid detection, utilizing a commercial chip Fluidigm, which can identify up to 21 viruses. Adapted with permission from Ref. . Copyright 2022, Springer Nature.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: CRISPR, Multiplexing, Mutagenesis, Multiplex Assay, Colorimetric Assay, Magnetic Beads, Reverse Transcription Polymerase Chain Reaction, Diagnostic Assay

Journal: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: Fluorescence readout and applications. ( a ) Illustration of how fluorescent signals are generated to detect SARS-CoV-2 nucleic acids of interest. Fluorescent optical detection involves a fluorophore emitting a signal at a particular wavelength from an initiating, excitation wavelength. ( b ) Intercalation of dyes sandwiched between the hydrogen bonds of complementary nucleic acid sequences. ( c ) Analysis of melting curve profiles to detect and distinguish two targets in a multiplex assay utilizing intercalating dyes as a fluorescent signal. Adapted with permission from Ref. . Copyright 2021, the Authors. ( d ) FRET-based fluorescent detection has two distinct mechanisms: increasing the distance between a fluorophore and the quencher or decreasing the distance between two fluorophores to allow for energy transfer from one fluorophore to another. ( e ) A MoS 2 nanosheet-modified DMA platform designed to detect SARS-CoV-2 genomic material on the nM scale using a FRET-based optical detection method. This multiplex test enabled the detection of SARS-CoV-2 genetic material alongside HIV. Adapted with permission from Ref. . Copyright 2020, American Chemical Society. ( f ) A one-pot OPIPE assay utilizing pre-coated tubes for the detection of RdRp and E genes. The TaqMan probes used have different fluorophores that enable fluorescent detection after cleavage during the RT-PCR amplification process. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( g ) Utilization of the Proofman enzyme, a protein that recognizes SNVs, which can cleave fluorophores away from restrictive quenchers on a primer specifically targeting mutations. This set-up is performed in a singular pot for multiple targets of interest. Adapted with permission from Ref. . Copyright 2021, Elsevier.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: Fluorescence, Generated, Multiplex Assay, Modification, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: Point-of-care technologies. ( a ) POCT for improving diagnostic testing accessibility to medical deserts, especially in lower income communities around the world. The use of smartphone technology, microfluidic integration, and/or continued LFA development is pertinent to empowering individuals without high-resource clinicians and hospitals nearby. ( b ) Smartphone-based detection for amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( c ) Smartphone-based detection of VOCs by PAM-targeting mutations. Adapted with permission from Ref. . ( d ) Lateral flow assay using nanoparticles. Adapted with permission from Ref. . Copyright 2020, Elsevier. ( e ) Lateral flow assay with RPA for low resource setting. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( f ) 3D-printed integrated microfluidic chip for colorimetric detection. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( g ) Microfluidic chip with LAMP-based portable assay. Adapted with permission from Ref. . Copyright 2020, the Authors.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: Diagnostic Assay, Amplification, CRISPR, Lateral Flow Assay

Journal: Journal of Biomolecular Techniques : JBT

Article Title: RT qLAMP—Direct Detection of SARS-CoV-2 in Raw Sewage

doi: 10.7171/jbt.21-32-03-016

Figure Lengend Snippet: Run B: September 23, 2020. Run using NEB E2019 reaction mix. Left to right, a) E-gene amplification, b) E-gene melt, c) E-gene standard curve, d) N-gene amplification, e) N-gene melt, and f) N-gene standard curve.

Article Snippet: It is important to note that although the NEB E2019 kit is supplied with E1-gene and N2-gene primers (both sets differ from the E- and N-gene primers used in our work; ), except for run 16 (October 1, 2020; d-f ), we did not use the NEB primers.

Techniques: Amplification