Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp
Authors: J Srisala
P Prombun
W Kiatpathomchai
R Suebsing
Journal: Journal of Applied Microbiology (2013)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Enterocytozoon hepatopenaei; LAMP assay; colorimetric nanogold; shrimp production; detection
Affiliations: Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp); Faculty of Science; Mahidol University; Bangkok Thailand, National Center for Genetic Engineering and Biotechnology (BIOTEC); National Science and Technology Development Agency; Pathumthani Thailand

Abstract:  Aims: 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 in 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. Conclusions: 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. 

Retracted: No

PubPeer comments:
 I would like to rise a question to the authors. In their published paper there seems to be a manipulated figure 7, in the lanes where controls should be, showing a repeated pattern in the lanes 5 - 12. This raises serious concerns about the specificity of the method if the unedited photographs had bands in them. Can the authors provide unedited gel photographs? [![jam12160-fig-0007-m-edited.jpg] ] Also another concern is in the figure 6, which also seems post-processed in a questionable way, with an artificially produced band. [![jam12160-fig-0006-m-edited.jpg] ] An explanation and original data are necessary. , 
 #3. Morover, looking at the ladder in the figure 6 and figure 7 - these look identical. #4. Lane U from the figure 6 looks identical to lane 1 in figure 7. ,