Rapid and sensitive colorimetric detection of microsporidian Enterocytozoon hepatopenaei (EHP) based on spore wall protein (SWP) gene using loop-mediated isothermal amplification combined with DNA functionalized gold nanoparticles as probes

Enterocytozoon hepatopenaei (EHP) is a newly emerging microsporidian parasite that causes growth retardation in cultured shrimp. Currently, EHP infection can be detected by standard polymerase chain reaction (PCR)-based methods, as well as loop-mediated isothermal amplification (LAMP) methods that target EHP's small subunit (SSU) ribosomal RNA (rRNA) gene (LAMP-SSU). However, this gene target has a propensity for generating false positive test results due to cross-reactivity of LAMP primers with SSU-rRNA genes from closely related microsporidia that infect other aquatic organisms. Herein, we describe the development of a simple, specific and sensitive method for detection of EHP by the naked eye. The method is a LAMP technique targeting the spore wall protein (SWP) gene combined with DNA-functionalized gold nanoparticle probes (LAMP-SWP). It has analytical sensitivity comparable to that of nested-PCR (10 copies of plasmid DNA; 1 pg of total DNA) and other reported LAMP methods. Furthermore, no cross-reactivity was observed with other pathogens known to infect shrimp. LAMP-SWP had a sensitivity of 97% (95% CI: 88.2–99.9%) and specificity of 100% (95% CI: 88.4–100%)for EHP detection, while the corresponding sensitivity and specificity of the LAMP-SSU we used for comparison were 100% (95% CI: 91.9–100%) and 56% (95% CI: 37.4–74.5%), respectively. Hence, our novel colorimetric LAMP-SWP combines high specificity for EHP (due to the SWP target), high sensitivity for 10 target copies (similar to conventional nested-PCR), confirmation of PCR amplicons by DNA hybridization and colorimetric visualization of PCR amplicons by the naked-eye (i.e., no electrophoresis or fluorometric equipment needed). It is a rapid method that could allow for real-time management decisions at shrimp hatcheries and farms without the need for sophisticated and expensive laboratory equipment. •The first report of LAMP targeting spore wall protein (SWP) gene combined with colorimetric AuNPs assay established to detect EHP in shrimp.•The selection of SWP gene as a specific target for EHP detection offers a more satisfactory performance relative to targeting the SSU rRNA gene.•The combination of LAMP-SWP and colorimetric AuNPs assay described in this work can be used as a reliable method for identification of EHP in shrimp.•The system developed in this work is practically suitable for use especially in settings with the limited testing infrastructure.