Training
PIRSA animal health staff were given one 4-hour workshop on LAMP use in August 2017 by the primary user (NB). The workshop included background information on molecular diagnostics/LAMP and a run through of sample collection, processing, and a LAMP run with freshly collected samples. Materials provided included pre-made kits, a one-page LAMP workflow and a booklet containing detailed workflow instructions. Two PIRSA animal health staff were chosen as final secondary users, neither person has a significant background in laboratory techniques. These secondary users were provided with kits by the primary user and then performed VDN LAMP over the course of usual FR disease investigations.
Sample collection and VDN LAMP for aprV2 positive D. nodosus detection
The samples were collected by Department of Primary Industries and Regions, South Australia animal health officers from the interdigital skin of lame sheep as part of routine diagnostic testing. Current clinical foot scores of the sheep feet were recorded and the single highest scored foot was sampled. Two swabs per sampled sheep were collected simultaneously and used as biological duplicates. One swab was used for aprV2/aprB2 rtPCR and the second for immediate in-field processing with VDN LAMP.
Swabs for aprV2/aprB2 rtPCR were collected as previously described [3]. Swabs for in-field processing with VDN LAMP were placed into 500 µL alkaline polyethylene glycol, pH 13.0. Swab heads were snapped into the buffer tubes and left in, with collection and processing occurring at ambient temperature. Sample processing and VDN LAMP reactions were carried out as previously described Best et al, 2018 [7], with each kit containing the required reagents in volumes appropriate for the processing of 8 samples. Purified genomic gDNA from arpV2 positive D. nodosus strain A198 was used as a positive control for each run.
rtPCR for detection of D. nodosus
Samples for rtPCR had all nucleic acids present extracted and purified as described previously [3]. The presence of aprV2 and/or aprB2 in samples were identified using primers, probes and cycling conditions as described by Stäuble et al. (2014a). The AgPath-IDTM One-Step RT-PCR Kit (Ambion, Austin, USA) was used as master mix according to manufacturer’s instructions, adapted for 10 µL final volume. Reactions and analysis were carried out on the Mic rtPCR Cycler (Bio Molecular Systems, Queensland, Australia), using auto threshold detection and bulk analysis.
Statistical analysis
Sample results were defined as follows, and only samples with complete data collection were used for analysis;
VDN LAMP positive – sample has both a Tp (< 20 minutes) and Tm (87.7°C – 88.7°C).
VDN LAMP negative – sample has only a Tp or Tm, or the Tm does not fall within the above range, or no Tp and Tm.
rtPCR aprV2 positive – has an rtPCR result in the aprV2 channel with a Ct < 35
rtPCR aprV2 negative – has an aprV2 Ct ≥ 35, is aprB2 positive, or negative for D. nodosus.
Samples with Ct’s above 35 are considered negative due to a lack of clinical relevance (2). The sensitivity (Se) of VDN LAMP is defined as the percentage of VDN LAMP positive samples within rtPCR aprV2 positive samples, and the specificity (Sp) the percentage of VDN LAMP negative samples within rtPCR aprV2 negative samples. Se, Sp, NPV and PPV were calculated using GraphPad Prism 6. Run success is defined as the positive control amplifying before 20 minutes with a Tm between 87.7°C – 88.7°C and the negative control failing to amplify.