Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp
Article first published online: 6 MAR 2013
© 2013 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 114, Issue 5, pages 1254–1263, May 2013
How to Cite
Suebsing, R., Prombun, P., Srisala, J. and Kiatpathomchai, W. (2013), Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp. Journal of Applied Microbiology, 114: 1254–1263. doi: 10.1111/jam.12160
- Issue published online: 15 APR 2013
- Article first published online: 6 MAR 2013
- Accepted manuscript online: 7 FEB 2013 06:03AM EST
- Manuscript Revised: 28 JAN 2013
- Manuscript Accepted: 28 JAN 2013
- Manuscript Received: 23 DEC 2012
- National Research Council of Thailand
- National Center for Genetic Engineering and Biotechnology (BIOTEC)
- Office of the Higher Education Commission (CHE)
- Mahidol University under the National Research University Initiatives
- Enterocytozoon hepatopenaei ;
- gold nanoparticle;
- loop-mediated isothermal amplification;
- penaeid shrimp;
- white faeces syndrome
Enterocytozoon 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 Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. 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 (AuNP) 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 ssDNA-labelled nanogold probe, followed by salt-induced AuNP 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-AuNP assay was specific for E. hepatopenaei.
Without sacrificing sensitivity or specificity, the new LAMP-AuNP 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.