Rapid visual detection of Enterocytozoon hepatopenaei by recombinase
polymerase amplification combined with a lateral flow dipstick
Enterocytozoon hepatopenaei (EHP) is a high-impact pathogen in
shrimp farming, causing huge economic losses to the global shrimp
farming industry every year. However, current EHP detection methods are
primarily based on the development of polymerase chain reaction (PCR)
techniques that rely on sophisticated and expensive instruments.
Consequently, a rapid, practical, and sensitive protocol for the
detection of EHP is necessary. Recombinase polymerase amplification
combined with a lateral flow dipstick (LFD-RPA) assay was developed
using a pair of primers and nfo-probe targeting the conserved region of
the spore wall protein gene. Under optimized reaction conditions, the
LFD-RPA assay can detect 10 copies/μL of standard plasmid within 20 min
at 40°C. Furthermore, this assay cannot amplify DNA from other shrimp
pathogens. Thirty-nine samples of Litopenaeus vannamei were
collected in shrimp farms and detected using LFD-RPA and nested PCR.
Thirty-two positive samples were detected by LFD-RPA. Compared with
those of nested PCR (32/39), the diagnostic sensitivity and specificity
of LFD-RPA were 100% and 100%, respectively. These results indicated
the great application potential of the newly developed LFD-RPA assay for
point-of-care diagnosis, epidemic surveillance, and epidemiological
investigation of EHP.