Abstract
Neoparamoeba perurans is the causative agent of amoebic gill disease (AGD). Two loop‐mediated isothermal amplification (LAMP) assays targeting the parasite 18S rRNA and the Atlantic salmon EF1α, used as internal control, were designed. The N. perurans LAMP assay did not amplify close relatives N. pemaquidensis and N. branchiphila, or the host DNA. This assay detected 106 copies of the parasite 18S rRNA gene under 13 min and 103 copies under 35 min. Five “fast‐and‐dirty” DNA extraction methods were compared with a reference method and further validated by TaqMan™ qPCR. Of those, the QuickExtract buffer was selected for field tests. Seventy‐one non‐lethal gill swabs were analysed from AGD‐clinically infected Atlantic salmon. The pathogen was detected under 23 min in fish of gill score >2 and under 39 min for lower gill scores. About 1.6% of the tests were invalid (no amplification of the internal control). 100% of positives were obtained from swabs taken from fish showing gill score ˃3, but only ~50% of positives for lower gill scores. The present LAMP assay could be implemented as a point‐of‐care test for the on‐site identification of N. perurans; however, further work is required to improve its performance for lower scores.
Highlights
Neoparamoeba perurans is the causative agent of amoebic gill disease (AGD)
DNA was extracted from a total of 60-gill swabs using the QuickExtract protocol, and the presence of N. perurans was analysed by loopmediated isothermal amplification (LAMP) assay
DNA was extracted from 10-fold serial dilutions of amoebic cells, containing 103 to 1 cell, and the CT values were correlated with the number of copies of the 18S rRNA gene in a standard curve
Summary
Neoparamoeba perurans trophozoites were isolated from the gills of naturally infected Scottish farmed sea-cage Atlantic salmon showing typical AGD lesions as described previously (Morrison, Crosbie, & Nowak, 2004). A second LAMP assay to amplify a region of 295 bp on the Atlantic salmon elongation factor 1 alpha (EF1α) (NM_001123629.1) was designed as described above and used as an internal control (Table 1). TA B L E 1 Sequences of primers designed for the Neoparamoeba perurans 18S rRNA gene and the Atlantic salmon elongation factor 1 alpha LAMP assays. For testing the KAPA extraction kit, swabs were placed in 472 μl of water, 25 μl of KAPA buffer and 3 μl of KAPA enzyme, and incubated for 10 min at 75°C, followed by 5-min incubation at 95°C as recommended by the manufacturers. DNA was extracted from a total of 60-gill swabs using the QuickExtract protocol, and the presence of N. perurans was analysed by LAMP assay. An additional 12 swabs, taken from the specific-pathogen-free Atlantic salmon, were used as non-infected control samples
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