Premium
Loop‐mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian E nterocytozoon hepatopenaei in penaeid shrimp
Author(s) -
Suebsing R.,
Prombun P.,
Srisala J.,
Kiatpathomchai W.
Publication year - 2013
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12160
Subject(s) - loop mediated isothermal amplification , shrimp , penaeus monodon , biology , litopenaeus , detection limit , broodstock , decapoda , microbiology and biotechnology , polymerase chain reaction , penaeidae , chromatography , dna , fishery , chemistry , gene , aquaculture , genetics , crustacean , fish <actinopterygii>
Abstract Aims E nterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome ( WFS ) in cultivated giant or black tiger shrimp P enaeus ( P enaeus) monodon and whiteleg shrimp P enaeus ( L itopenaeus) vannamei in A sia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop‐mediated isothermal amplification ( LAMP ) assay combined with colorimetric nanogold ( A u NP ) for rapid, sensitive and inexpensive detection of this parasite. Methods and Results A set of six specific primers was designed to successfully detect the SSU rRNA gene of E . hepatopenaei by a LAMP reaction of 45 min at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ss DNA ‐labelled nanogold probe, followed by salt‐induced A u NP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0·02 fg total DNA ) than a conventional nested PCR (0·2 fg total DNA ). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP ‐ A u NP assay was specific for E . hepatopenaei . Conclusions Without sacrificing sensitivity or specificity, the new LAMP ‐ A u NP assay significantly reduced the time, ease and cost for molecular detection of E . hepatopenaei in shrimp. Significance and Impact of the study The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.