Retracted: Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidianEnterocytozoon hepatopenaeiin penaeid shrimp

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 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 (0Á02 fg total DNA) than a conventional nested PCR (0Á2 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 LAMPAuNP 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.

A Nested PCR Assay to Avoid False Positive Detection of the Microsporidian Enterocytozoon hepatopenaei (EHP) in Environmental Samples in Shrimp Farms

“…In the nested PCR step, both methods had identical sensitivity at 10 copies per reaction vial (Fig 3B), similar to what had previously been reported for the SSU-PCR method [23]. …”

“…However, for those without equipment to carry out the process, an isothermal loop mediated (LAMP) method with a sensitivity of 2 EHP copies/reaction vial of total DNA from EHP-infected shrimp has been reported [23]. In this study, we found that the SSU-PCR and SWP-PCR methods could detect DNA plasmids containing respective target sequences at 10 copies per reaction vial, although the SWP-PCR method had better sensitivity than the SSU-PCR method for the first PCR step.…”

“…They include conventional PCR, nested PCR, isothermal loop-mediated amplification (LAMP) and in situ hybridization [18,22,23]. Thus, to explore the real possibility of obtaining false positive test results by using the SSU-PCR methods, we tested DNA extracted from microsporidia that are closely related to EHP and available to us.…”

Rapid detection and typing of live bacteria from human joint fluid samples by utilizing an integrated microfluidic system

“…Once there is bacteria universal gene, the oligonucleotides conjugated with gold nanoparticles will hybridize with its target sequence that brings the gold nanoparticles together. Thus, the color of the liquid will change after hydrochloric acid addition due to the light absorption properties of nanoparticles, which is usually referred as the redshift effect (Aslan et al, 2004;Bakthavathsalam and Rajendran, 2012;Ploschner et al, 2012;Suebsing et al, 2013). For this purpose, thiol chemically modified 16S oligonucleotides conjugated with gold nanoparticles was prepared according to a protocol reported in a previous study (Hill and Mirkin, 2006) before the onchip assay.…”

“…Then, the 20-nm gold nanoparticle conjugated 16S probes were added to hybridize the singlestrand DNA at 58°C. After 0.01 N hydrochloric acid was added into the test tubes, the colors of samples with bacterial genome DNA turned into red due to the red shift effect of gold nanoparticle particles (Suebsing et al, 2013) while the color of negative control stayed in purple, which was the original color of labeled gold nanoparticle probes. Therefore, by observing the colors of samples and comparing it with that of the negative control, the existence of bacteria could be confirmed.…”