Rapid visual detection of anisakid nematodes using recombinase polymerase amplification and SYBR Green I

Abstract

Anisakidosis is a food-borne parasitic disease (FBPD) caused by the third-stage larvae of the family Anisakidae. Therefore, it is important to develop a simple, rapid and equipment-free detection method for anisakids in fish samples or seafood since current methods are time-consuming and require complex instruments. In this study, a recombinase polymerase amplification (RPA)-based method was established for the first time to detect anisakids by targeting the internal transcribed spacer (ITS) regions. The detection results were visualized by including SYBR Green I (SG) in the method. The sensitivity of RPA-SG assay was 102 copies per reaction of recombinant plasmid (within 20 min at 37°C), similar to quantitative real-time PCR (qPCR). The assay had high specificity for detecting anisakids against other related parasites and host fish. In addition, the assay was further used to detect fresh marine fish contaminated with anisakids and it showed high precision. These results indicate that the novel RPA-SG assay suitable for visual detection of anisakids in the field and food safety control.

Recombinase polymerase amplification combined with lateral flow dipstick assay for rapid visual detection of A.simplex (s. s.) and A.pegreffii in sea foods

“…As a result, the combination of RPA with the LFD simplifies the test process and yields simpler visualization of results, making rapid on-site detection feasible. Compared to the combination of RPA with SYBR Green detection for Anisakis reported by Chen et al [ 47 ] for on-site detection, this study was simpler regarding the visualization results because of using the LFD. In addition, visualization results avoid the harm from UV light using SYBR Green and reduce the error possibility from naked-eye observation.…”

“…The RPA-LFD assay is a detection technique that combines recombinase polymerase amplification and visualization techniques, showing much strength in pathogen detection. The ITS sequences have high interspecies variability in Anisakis and are often used as target genes [ 47 ]. In this study, we focused on specific ITS sequences and used them as the detection target to develop an RPA-LFD assay.…”

Agarose Hydrogel-Boosted One-Tube RPA-CRISPR/Cas12a Assay for Robust Point-of-Care Detection of Zoonotic Nematode Anisakis

“…This method was compared with the other nucleic acid detection methods for Anisakis. As shown in Table 2, the sensitivity of the agarose-improved RPA-CRISPR/Cas12a assay was better than RPA (10 2 copies/μL), 40 qPCR (1.29 × 10 2 copies/μL), 41 and LAMP (10 2 copies/μL). 42,43 The detection limit was similar to that of the physical separation of the one-tube RPA-CRIAPR/Cas12a method (the CRISPR system was placed on the pipe cover; 22.4 copies/μL 8 ).…”

Re‐evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1–3

“…However, this LAMP assay is not able to identify the isolated parasites to the species level. Finally, recombinase polymerase amplification‐SYBR Green (RPA‐SG) uses ITS locus to differentiate between A. simplex , A. pegreffii , A. typica , but achieves sensitivity of 10 2 copies per reaction (qPCR) in 20 min at 37°C (Chen et al., 2022 ). A SYBR‐Green uses mtDNA cox 2 was developed to detect A. simplex (s. l) DNA in commercial fish‐derived food (Godinez‐Gonzales et al., 2017 ).…”