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

Abstract

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.