Journal: Frontiers in Veterinary Science

Article Title: Development of a loop-mediated isothermal amplification detection assay for Dictyocaulus viviparus (Bloch, 1782) lungworm: DviLAMP

doi: 10.3389/fvets.2024.1454065

Figure Lengend Snippet: ITS2 with partial 5.8S rDNA sequence alignment of Dictyocaulus viviparus (clones 5, 6, 7, 9, and 14) highlighted with different DviLAMP primer DNA sequences; variable sites in the DNA sequence are presented as red letters.

Article Snippet: For conventional LAMP with LFD (without colorimetry), the 12.5 μL final volume contained 1.25 μL of 10X isothermal amplification buffer, 1.25 μL of 10X DviLAMP primer mix (a pair of biotin and FITC tagged primers), 1.75 μL of 10 mM/base dNTPs (N0447S, NEB), 0.75 μL of 100 mM MgSO 4 , 2.5 μL of 5 M Betaine, 0.5 μL of 8 U/μL Bst 2.0 WarmStart DNA Polymerase (NEB), 3.5 μL of DEPC H 2 O, and 1 μL of the DNA template (20 ng/μL–10 pg/μL DviITS2 plasmid) or H 2 O (for negative control).

Techniques: Sequencing, Clone Assay

Journal: Frontiers in Veterinary Science

Article Title: Development of a loop-mediated isothermal amplification detection assay for Dictyocaulus viviparus (Bloch, 1782) lungworm: DviLAMP

doi: 10.3389/fvets.2024.1454065

Figure Lengend Snippet: DviLAMP primers for Dictyocaulus viviparus lungworm detection: [BIO], biotin; [FITC], fluorescein-5-isothiocyanate; and bp, base pair.

Article Snippet: For conventional LAMP with LFD (without colorimetry), the 12.5 μL final volume contained 1.25 μL of 10X isothermal amplification buffer, 1.25 μL of 10X DviLAMP primer mix (a pair of biotin and FITC tagged primers), 1.75 μL of 10 mM/base dNTPs (N0447S, NEB), 0.75 μL of 100 mM MgSO 4 , 2.5 μL of 5 M Betaine, 0.5 μL of 8 U/μL Bst 2.0 WarmStart DNA Polymerase (NEB), 3.5 μL of DEPC H 2 O, and 1 μL of the DNA template (20 ng/μL–10 pg/μL DviITS2 plasmid) or H 2 O (for negative control).

Techniques: Sequencing

Journal: Frontiers in Veterinary Science

Article Title: Development of a loop-mediated isothermal amplification detection assay for Dictyocaulus viviparus (Bloch, 1782) lungworm: DviLAMP

doi: 10.3389/fvets.2024.1454065

Figure Lengend Snippet: Analytical sensitivity of the DviLAMP to detect DviITS2 in various DNA concentrations.

Article Snippet: For conventional LAMP with LFD (without colorimetry), the 12.5 μL final volume contained 1.25 μL of 10X isothermal amplification buffer, 1.25 μL of 10X DviLAMP primer mix (a pair of biotin and FITC tagged primers), 1.75 μL of 10 mM/base dNTPs (N0447S, NEB), 0.75 μL of 100 mM MgSO 4 , 2.5 μL of 5 M Betaine, 0.5 μL of 8 U/μL Bst 2.0 WarmStart DNA Polymerase (NEB), 3.5 μL of DEPC H 2 O, and 1 μL of the DNA template (20 ng/μL–10 pg/μL DviITS2 plasmid) or H 2 O (for negative control).

Techniques: Concentration Assay, Colorimetric Assay

Journal: Frontiers in Veterinary Science

Article Title: Development of a loop-mediated isothermal amplification detection assay for Dictyocaulus viviparus (Bloch, 1782) lungworm: DviLAMP

doi: 10.3389/fvets.2024.1454065

Figure Lengend Snippet: Analytical sensitivity test of the DviLAMP based on colorimetry with naked eye observation (A–C) and gel electrophoresis (D–F) with different incubation periods, including 45 min (A,D) , 60 min (B,E) , and 90 min (C,F) ; and various DNA concentrations (20 ng to 1 pg): L, DNA ladder; N, negative control.

Article Snippet: For conventional LAMP with LFD (without colorimetry), the 12.5 μL final volume contained 1.25 μL of 10X isothermal amplification buffer, 1.25 μL of 10X DviLAMP primer mix (a pair of biotin and FITC tagged primers), 1.75 μL of 10 mM/base dNTPs (N0447S, NEB), 0.75 μL of 100 mM MgSO 4 , 2.5 μL of 5 M Betaine, 0.5 μL of 8 U/μL Bst 2.0 WarmStart DNA Polymerase (NEB), 3.5 μL of DEPC H 2 O, and 1 μL of the DNA template (20 ng/μL–10 pg/μL DviITS2 plasmid) or H 2 O (for negative control).

Techniques: Colorimetric Assay, Nucleic Acid Electrophoresis, Incubation, Negative Control

Journal: Frontiers in Veterinary Science

Article Title: Development of a loop-mediated isothermal amplification detection assay for Dictyocaulus viviparus (Bloch, 1782) lungworm: DviLAMP

doi: 10.3389/fvets.2024.1454065

Figure Lengend Snippet: DviLAMP with real-time detection (A) , melting curve plot (B) and lateral flow (C,D) . The real-time fluorescence signal was calculated from three replicates. Ct values and time of the highest change in the fluorescence were calculated and indicated in the amplification plot. Numbering in the amplification plot indicates the DviITS2 plasmid DNA concentrations as follows: 1 = 20 ng; 2 = 10 ng; 3 = 5 ng; 4 = 1 ng; 5 = 0.5 ng; 6 = 0.1 ng; 7 = 50 pg; 8 = 10 pg; 9 = 5 pg; and 10 = 1 pg.

Article Snippet: For conventional LAMP with LFD (without colorimetry), the 12.5 μL final volume contained 1.25 μL of 10X isothermal amplification buffer, 1.25 μL of 10X DviLAMP primer mix (a pair of biotin and FITC tagged primers), 1.75 μL of 10 mM/base dNTPs (N0447S, NEB), 0.75 μL of 100 mM MgSO 4 , 2.5 μL of 5 M Betaine, 0.5 μL of 8 U/μL Bst 2.0 WarmStart DNA Polymerase (NEB), 3.5 μL of DEPC H 2 O, and 1 μL of the DNA template (20 ng/μL–10 pg/μL DviITS2 plasmid) or H 2 O (for negative control).

Techniques: Fluorescence, Amplification, Plasmid Preparation

Journal: Frontiers in Veterinary Science

Article Title: Development of a loop-mediated isothermal amplification detection assay for Dictyocaulus viviparus (Bloch, 1782) lungworm: DviLAMP

doi: 10.3389/fvets.2024.1454065

Figure Lengend Snippet: Specificity test of the DviLAMP with two biotin-FITC tagged position schemes, (II) [FITC]-LFL and [BIO]-LBL; and (III) [BIO]-FIPL and [FITC]-LFL, showing the results by LFD (A) and gel electrophoresis (B) for Dictyocaulus viviparus (Dvi), Ostertagia ostertagi (Oos), and Cooperia oncophora (Con) after amplification for 60 min.

Article Snippet: For conventional LAMP with LFD (without colorimetry), the 12.5 μL final volume contained 1.25 μL of 10X isothermal amplification buffer, 1.25 μL of 10X DviLAMP primer mix (a pair of biotin and FITC tagged primers), 1.75 μL of 10 mM/base dNTPs (N0447S, NEB), 0.75 μL of 100 mM MgSO 4 , 2.5 μL of 5 M Betaine, 0.5 μL of 8 U/μL Bst 2.0 WarmStart DNA Polymerase (NEB), 3.5 μL of DEPC H 2 O, and 1 μL of the DNA template (20 ng/μL–10 pg/μL DviITS2 plasmid) or H 2 O (for negative control).

Techniques: Nucleic Acid Electrophoresis, Amplification

Journal: Frontiers in Veterinary Science

Article Title: Development of a loop-mediated isothermal amplification detection assay for Dictyocaulus viviparus (Bloch, 1782) lungworm: DviLAMP

doi: 10.3389/fvets.2024.1454065

Figure Lengend Snippet: Analytical specificity test of the DviLAMP by LFD (A) , gel electrophoresis (B) , and colorimetry (C) ; the amplification plot (D) and the melting plot (E) showing the fluorescence increasing for Dictyocaulus viviparus (Dvi), Ostertagia ostertagi (Oos), and Cooperia oncophora (Con) for 60 min of the incubation. The amplification plot and melting plot show the results for each species individually from three technical replicates.

Article Snippet: For conventional LAMP with LFD (without colorimetry), the 12.5 μL final volume contained 1.25 μL of 10X isothermal amplification buffer, 1.25 μL of 10X DviLAMP primer mix (a pair of biotin and FITC tagged primers), 1.75 μL of 10 mM/base dNTPs (N0447S, NEB), 0.75 μL of 100 mM MgSO 4 , 2.5 μL of 5 M Betaine, 0.5 μL of 8 U/μL Bst 2.0 WarmStart DNA Polymerase (NEB), 3.5 μL of DEPC H 2 O, and 1 μL of the DNA template (20 ng/μL–10 pg/μL DviITS2 plasmid) or H 2 O (for negative control).

Techniques: Nucleic Acid Electrophoresis, Colorimetric Assay, Amplification, Fluorescence, Incubation