An improved recombinase polymerase amplification assay for visual detection of Vibrio parahaemolyticus with lateral flow strips

Abstract

Vibrio parahaemolyticus is an important pathogenic bacterium in both food safety management and mariculture. Rapid and accurate detection technologies are critical for effective control of its outbreak and spreading. Conventional technologies and polymerase chain reaction (PCR)‐based approaches have limited usage because of the requirement of laboratory instruments and trained personnel. Using the isothermal recombinase polymerase amplification (RPA) technology, several detection assays have been developed with added convenience. Combining the lateral flow strip (LFS) test with RPA can further simplify the detection. In this study, an improved RPA assay using LFS for visual detection of V. parahaemolyticus was developed. Primers were designed targeting the virulence genes and screened for amplification efficiency, nonspecific amplification, and primer–dimer formation. Probes were designed for the best primer pairs, and the weakness of LFS tests, being easily affected by primer‐dependent artifacts, was overcome by sequence modifications on primers and probe. The RPA–LFS assay took 25 min at 35 to 45 °C, and showed excellent specificity. It detected as low as one colony forming unit (CFU) of V. parahaemolyticus per reaction without DNA purification, or 10 CFU/10 g spiked food samples with 2 hr of enrichment. The detection limit was better than the currently available RPA‐based detection methods. Application of the RPA–LFS assay for simulated samples or real clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. The RPA–LFS assay provided a rapid, accurate, and convenient V. parahaemolyticus detection method suitable for on‐site detection in resource‐limited conditions. Practical Application This research developed a rapid and visual detection technology for Vibrio parahaemolyticus that is not dependent on complicated equipment. The detection process takes 25 min and the result is read with the naked eye. A detection kit can be developed based on this technology for on‐site detection of V. parahaemolyticus in resource‐limited regions for food safety management and mariculture.

A Real-Time Recombinase Polymerase Amplification Method for Rapid Detection of Vibrio vulnificus in Seafood

“…In other reports, the limit of detection of V. vulnificus was 0.1-1 CFU/reaction with qPCR-based methods or 10-100 CFU/reaction with LAMP (Panicker and Bej, 2005;Wang et al, 2016;D'Souza et al, 2019). The limit of detection of the real-time RPA method was also comparable with that of other RPA-based methods for detection of other pathogens, such as ∼10 CFU per reaction (Vibrio harveyi and Listeria monocytogenes) or 50 copies per reaction (Vibrio parahaemolyticus) (Garrido-Maestu et al, 2019;Pang et al, 2019;Yang et al, 2020).…”

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

“…The amplification works between 25 and 42℃ that has greatly reduced equipment dependence (Piepenburg et al., 2006). 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).…”

Rapid visual detection of Micropterus salmoides rhabdovirus using recombinase polymerase amplification combined with lateral flow dipsticks

“…When using LFD for the visualization of RPA detection, it is necessary to avoid false‐positive signals from primer dimers. In several studies, base substitutions were made for the probe and reverse primer to eliminate possible matches between the two (Wang et al., 2021; Yang et al., 2020), which requires thorough analyses of the primer and probe sequences.…”