Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay

Abstract

Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3–7 min at 39°C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms.

Simultaneous visual diagnosis of acute hepatopancreatic necrosis disease and Enterocytozoon hepatopenaei infection in shrimp with duplex recombinase polymerase amplification

“…In the industrial standard nested PCR assays, pirA and pirB genes and swp gene were selected as the amplification targets for the two diseases, respectively (Dangtip et al., 2015; Jaroenlak et al., 2016). Our previous studies that developed fluorescent RPA assays for the two diseases also used pirB gene and swp gene as the diagnostic markers (Ma et al., 2021; Yu et al., 2021). This study further confirmed that the two diagnostic markers were highly specific to the respective diseases.…”

“…Primer and probe sequences for the RPA‐LFD assay in this study were derived from our previous studies reporting RPA assays combined with fluorescent signal reading for the diagnostic of AHPND and EHP infection (Ma et al., 2021; Yu et al., 2021) The target genes for AHPND and EHP were the binary toxic photorhabdus insect‐related gene ( pirB on the toxin plasmid pVPA3‐1, GenBank accession number NC_025152.1) and the spore wall protein gene ( swp , GenBank accession number KX258197.1), respectively. The sequences were analysed with the Primer Premier 5 software, and the finalized primer and probe sequences are shown in Table 2.…”

“…The RPA technology has been applied to POCT diagnostic assays for a number of infectious diseases in aquaculture (Dong et al., 2020; Jaroenram & Owens, 2014; Soliman et al., 2018; Yang et al., 2020). In particular, RPA assays combined with fluorescent signal reading have been used for the diagnostic of AHPND and EHP infection (Ma et al, 2021; Yu et al., 2021).…”

Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

“…Therefore, it is impossible to obtain accurate information regarding E. artemiae infection via microscopic examination. Based on this, highly sensitive molecular methods, such as PCR (Tang et al, 2015), nested PCR (Jaroenlak et al, 2016), qPCR (Liu et al, 2018;Piamsomboon et al, 2019), loopmediated isothermal amplification (Suebsing et al, 2013;Cai et al, 2018), and recombinase polymerase amplification (Ma et al, 2021), are used instead of microscopic methods. Compared with conventional PCR, qPCR has the advantages of high sensitivity, good repeatability and quantification.…”

Combining Direct PCR Technology and Capillary Electrophoresis for an Easy-to-Operate and Highly Sensitive Infectious Disease Detection System for Shrimp

“…To make molecular diagnostics more convenient, new detection methods have been developed in recent years. Examples include recombinase polymerase amplification, loopmediated isothermal amplification, and isothermal PCR technology, combined with the use of portable equipment to shorten the PCR detection time [15][16][17][18][19][20][21][22]. Another example is capillary electrophoresis (CE), which reduces post-PCR analysis time [23].…”