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. 2020 Jul;43(7):779-790.
doi: 10.1111/jfd.13175. Epub 2020 May 4.

Non-lethal loop-mediated isothermal amplification assay as a point-of-care diagnostics tool for Neoparamoeba perurans, the causative agent of amoebic gill disease

Irene Cano  1 , Robin McCullough  1 , Brian Mulhearn  1 , Susie Gunning  1 , Ava Waine  1 , Claire Joiner  1 , Richard Paley  1
Affiliations

Non-lethal loop-mediated isothermal amplification assay as a point-of-care diagnostics tool for Neoparamoeba perurans, the causative agent of amoebic gill disease

Irene Cano et al. J Fish Dis. 2020 Jul.

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.

Keywords: Neoparamoeba perurans; amoebic gill disease; loop-mediated isothermal amplification; point-of-care test.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Neoparamoeba perurans 18S rRNA gene and Atlantic salmon EF1α LAMP assays. (a) Isothermal amplification. (b) Anneal derivative of isothermal amplified products [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
The specificity of the Neoparamoeba perurans LAMP assay. Isothermal amplification of DNA extracted from Neoparamoeba perurans, N. pemaquidensins, N. branchiphila and Atlantic salmon, each species in duplicate. Water was used as a negative control [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
The sensitivity of the Neoparamoeba perurans LAMP assay. (a) Amplification graph of serial dilutions ranging from 106 to 1 copy of a recombinant plasmid. Each dilution was run in duplicate. (b) Anneal derivative of isothermal amplified products [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
Linear correlation of the Neoparamoeba perurans LAMP assay between the plasmid copy number (expressed as Log10(x)) and the time of positivity (Tp). Mean data of three independent assays
Figure 5
Figure 5
Isothermal amplification of Neoparamoeba perurans from Isohelix swabs. DNA was extracted either with: NaOH, QuickExtract, KOH, KAPA Express Extract or Buccalyse DNA release, and compared with a reference laboratory method (EZ1 Biorobot) [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 6
Figure 6
Summary of the number of Atlantic salmon gill swabs analysed (bars) and the percentage of LAMP‐positive tests (line) per gill score range. Bars include swabs taken both from naturally infected and from challenged fish
See this image and copyright information in PMC

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