Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: (A) Overall operation of the cDDLAMP assay for the detection of STEC strains. Culture free enrichment: E. coli was enriched from milk samples and lettuce surface rinse water using magnetic beads functionalized with anti- E. coli (α- E. coli ) antibodies. cDDLAMP: gDNA was extracted and amplified by LAMP in heat block to produce DNAzyme-containing LAMP amplicons. Colorimetric detection: The colorimetric signal produced by DNAzyme reaction was captured for analysis by using SpotXel Reader app on a smartphone. A micro-USB-powered light pad and a light-proof enclosure were used to eliminate the impact of ambient light. (B) cDDLAMP: Top panel shows a schematic illustration of target stx1 , stx2 , and eae genes of E. coli O157:H7. LAMP Dumbbell: The forward inner primer (FIP) and backward inner primer (BIP) were modified with reverse-complement sequence of DNAzyme (rcDNAzyme), rcEAD2 or rcDz-00. LAMP product: LAMP reaction generates amplicons containing two types of DNAzymes (EAD2, and Dz-00) in each amplification unit. Upon adding hemin, the DNAzymes produce a colorimetric signal in the presence of H 2 O 2 and TMB. The color change is captured by smartphone camera, and analyzed with SpotXel, a smartphone-based microplate reader application.

Article Snippet: Furthermore, three non-pathogenic reference E. coli strains (ATCC 43745, 25922, and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were provided by Department of Animal Science, University of Tennessee.

Techniques: Magnetic Beads, Amplification, Blocking Assay, Produced, Modification, Sequencing

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: The following bacterial species were tested: O157:H7 EDL932 (ATCC 43894), six clinical STEC strains (O111:H8, O26:H11, O103:H11, O45:H2, O145:NT, and O121:H19), three non-pathogenic E. coli strains(ATCC 43745, 25922,and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ). Means from three independent experiments are shown with error bars representing standard error of the means. a–c; Asterisks above means denote significant differences and means carrying different superscripts are significantly different at p < 0.05 (****P < 0.0001). The cut-off value of stx1 indicated as dotted line = 1.45. The stx2 and eae genes were 1.37and 1.18, respectively.

Article Snippet: Furthermore, three non-pathogenic reference E. coli strains (ATCC 43745, 25922, and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were provided by Department of Animal Science, University of Tennessee.

Techniques: Bacteria

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: ClustalW alignment of the stx1 (A), stx2 (B), and eae (C) genes from STEC strains is presented. Target sequences for stx1 , stx2 , and eae from E. coli O157:H7 (strain EDL933*) were retrieved from the GenBank database (accession number CP008957.1). Annotation labels indicate the binding regions for the primers, including outer primers (F3, B3), loop primers (LF, LB), forward inner primer (FIP; F1c, F2), and backward inner primer (BIP; B1c, B2). Shading indicates sequence similarity as follows: black (100%), dark gray (80–100%), light gray (60–80%), and white (<60%). Sequence logos, generated using Geneious 2019.2.1 software, display the consensus across the sequences.

Article Snippet: Furthermore, three non-pathogenic reference E. coli strains (ATCC 43745, 25922, and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were provided by Department of Animal Science, University of Tennessee.

Techniques: Binding Assay, Sequencing, Generated, Software

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: gDNA was extracted from recovered E. coli , amplified with cDDLAMP, and analyzed colorimetrically (A). Results represent means from three independent experiments, with error bars representing standard error. Significant differences (p < 0.05) are denoted by asterisks (****P < 0.0001) and different superscripts (a–d). The cut off value of stx1 indicated as dotted line = 1.18. The stx2 and eae genes were 0.96 and 1.06, respectively. A photograph of representative plates is shown in (B). NTC: no template control; PC: positive control.

Article Snippet: Furthermore, three non-pathogenic reference E. coli strains (ATCC 43745, 25922, and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were provided by Department of Animal Science, University of Tennessee.

Techniques: Amplification, Control, Positive Control

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: gDNA of the recovered E. coli without SMB-PAb enrichment (A) or with SMB-PAb enrichment (B) was amplified with cDDLAMP. In (A), the cut off value of stx1 , and eae genes indicated as dotted line = 1.44. In (B), the cut off value of stx1/2 and eae genes (dotted line) were 2.08 and 1.12, respectively. Means from three independent experiments are shown with error bars representing standard error. Significant differences (p < 0.05) denoted by different superscripts (a–h). n.s.: Not significant (ns p > 0.05).

Article Snippet: Furthermore, three non-pathogenic reference E. coli strains (ATCC 43745, 25922, and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were provided by Department of Animal Science, University of Tennessee.

Techniques: Amplification

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: gDNA of E. coli was extracted and amplified with cDDLAMP. The cDDLAMP amplicons were reacted with TMB, and the signal to noise ratio (S/N) was calculated (A). A representative photograph of well plate is shown in (B). Means from three independent experiments are shown with error bars representing standard error. Significant differences (p < 0.05) are denoted by asterisks (****P < 0.0001) and different superscripts (a–d). The cut off value of stx1 indicated as dotted line = 1.29. The stx2 and eae genes were 1.05 and 1.10, respectively.

Article Snippet: Furthermore, three non-pathogenic reference E. coli strains (ATCC 43745, 25922, and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were provided by Department of Animal Science, University of Tennessee.

Techniques: Amplification

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: The food matrices (milk and lettuce) artificially inoculated with E. coli O157:H7 at 5.2 x 10 5,3,0 CFU/mL or g. Red and blue data points represent SpotXel reader and microplate reader (OD 655 nm), respectively. Each dot indicates a single data point. The data for the plots are appended in .

Article Snippet: Furthermore, three non-pathogenic reference E. coli strains (ATCC 43745, 25922, and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were provided by Department of Animal Science, University of Tennessee.

Techniques:

Journal: PLOS One

Article Title: Colorimetric dual DNAzyme reaction triggered by loop-mediated isothermal amplification for the visual detection of Shiga toxin-producing Escherichia coli in food matrices

doi: 10.1371/journal.pone.0320393

Figure Lengend Snippet: Lettuce was artificially inoculated with E. coli O157:H710 9-3 CFU/25g. E. coli were recovered from the lettuce with IMS (IMS) or without IMS (No IMS). Recovered E. coli was subjected to cDDLAMP. Red and blue data points represent SpotXel reader and microplate reader (OD 655 nm), respectively. Each dot indicates a single data point. The data for the plots are appended in .

Article Snippet: Furthermore, three non-pathogenic reference E. coli strains (ATCC 43745, 25922, and K12), and four non- E. coli bacteria ( Salmonella cholerasuis ATCC BAA 664 , Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa , and Staphylococcus aureus ) were provided by Department of Animal Science, University of Tennessee.

Techniques: