Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP) for the visual detection of Enterocytozoon hepatopenaei (EHP).

Autor: Sathish Kumar T; ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome High Road, MRC Nagar, Chennai, India. Electronic address: sathishkumar.t@icar.gov.in., Radhika K; ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome High Road, MRC Nagar, Chennai, India., Joseph Sahaya Rajan J; ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome High Road, MRC Nagar, Chennai, India., Makesh M; ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome High Road, MRC Nagar, Chennai, India., Alavandi SV; ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome High Road, MRC Nagar, Chennai, India., Vijayan KK; ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome High Road, MRC Nagar, Chennai, India.
Jazyk: angličtina
Zdroj: Journal of invertebrate pathology [J Invertebr Pathol] 2021 Jul; Vol. 183, pp. 107624. Date of Electronic Publication: 2021 May 30.
DOI: 10.1016/j.jip.2021.107624
Abstrakt: Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.
(Copyright © 2021 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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