Abstract:

Enterocytozoon hepatopenaei (EHP) is an intracellular parasite that can infect various shrimp species. To develop an on-site rapid detection method of EHP, recombinant polymerase chain reaction (RPA) and lateral flow strip (LFS) techniques were combined in this study. 3 pairs of primers and probes were designed targeting the highly conserved region of EHP spore wall protein 1(SWP) gene according to primer design principle of RPA. By optimizing reaction conditions and screening the best primers and probes, an RPA with lateral flow dipstick (RPA-LFS) detection method for EHP was developed in this study. Then, detection limit, specificity and reliability were detected. After optimization, the target gene was successfully amplified in reaction temperature of 37℃ for only 15 minutes. With LFS detection, the overall detection time was within 20 minutes. The detection limit of RPA-LFS was 10 copies of EHP DNA. The results of specificity test showed that this method just specifically reacted with the EHP DNA, and had no cross-reaction with other aquatic animal virus such as white spot syndrome virus (WSSV). The clinical samples detection results of the RPA-LFS method were the same as the results of real time PCR method recommended by WOAH. This EHP RPA-LFS detection method established in this study has the advantages of shorter time, visible results, no dependence on expensive instruments, and lower detection costs, compared with qPCR and nested PCR detection methods. This method can achieve on-site rapid detection of EHP and is very suitable for small laboratories, providing a new assay for EHP detection.

Key words: Enterocytozoon hepatopenaei, on-site rapid detection, recombinase polymerase amplification, lateral flow strip, detection limit, specificity