Volume 28, 2021
Special Issue – Combatting Anthelmintic resistance in ruminants. Invited Editors: Johannes Charlier, Hervé Hoste, and Smaragda Sotiraki
|Number of page(s)||12|
|Published online||15 December 2021|
Supplementary Figure 1: Optimisation of Colourimetric LAMP Assay with positive control gDNA of H. contortus. 1: 60 °C. 2: 61 °C. 3: 62 °C. 4: Non-template control. 30–45 min reaction time.
Supplementary Figure 2: Colourimetric LAMP assay validation on suspected H. contortus field samples. 1: Positive Control (PEK). 2: Farm I. 3: Farm III. 4, 9 & 14: Positive control H. contortus gDNA (PEB). 5 & 6: Suspected adult H. contortus worms from deer (Farm DF). 7 & 8: Unidentified adult worms from deer large intestine (Farm DF). 10: Farm II. 12 & 13: Goat faeces sample (Farm GF). Yellow/Orange = Positive/Weak Positive, Pink/Purple = Negative.
Supplementary Figure 3: Colourimetric LAMP assay validation on suspected H. contortus field samples using the individual as well as pooled faecal samples. A: Farm VI. 1-5: Individual sheep samples (random). 6: Positive control H. contortus gDNA. 7: No Template Control. B: Farm IV. 1-5: Individual sheep samples (random). 6: Positive control H. contortus gDNA. 7: Non-template control. C: Farm IV. 1-10: Pooled samples. 11: Positive control H. contortus gDNA. 12: No Template Control. D: Farm VIII. 1-3: Pooled samples. 4 & 5: Individual sheep samples (random). 6: Positive control H. contortus gDNA. 7: No Template Control. Yellow/Orange = Positive/Weak Positive, Pink/Purple = Negative.
Supplementary Figure 4: LAMP Assay with H. contortus egg DNA extracted by genesig® DNA/RNA Easy Magnetic Bead Extraction (Primerdesign Ltd, UK). 1 & 2: Positive control (PEK). 4: Farm IV. 5 & 6: Farm VI. 7: Non-template Control. 8: Positive control H. contortus gDNA. Yellow/Orange = Positive/Weak Positive, Pink/Purple = Negative.
Supplementary Figure 5: Bead-beating lysis and DNA extraction. Farm I samples. A: Direct Fill-FLOTAC® solution. B: Fill-FLOTAC® solution after low g centrifugation egg harvesting steps. PC: Positive control. NTC: Non-template control.
Supplementary Figure 6: LF-LAMP optimisation results using diluted tagged amplicons. 1-3: Positive control H. contortus gDNA. 4-6: Non-template control. Positive detection of H. contortus presents as a band in both control and test lines. Negative detection presents as a band in only the control line. Failed assay presents as no bands on LF strip.
Supplementary Figure 7: Peanut Agglutination (PNA) fluorescence microscopy result of Farm I (Khangembam et al, unpublished) performed as per Jurasek et al (2010). 1 & 2: Different fields of view of H. contortus eggs seen as bright-green stained eggs. 10x magnification.(Access here)
© R. Khangembam et al., published by EDP Sciences, 2021
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