CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

Abstract

Vibrio parahaemolyticus is one of the major pathogenic Vibrio species that contaminate seafood. Rapid and accurate detection is crucial for avoiding foodborne diseases caused by pathogens and is important for food safety management and mariculture. In this study, we established a system that combines chemically enhanced clustered regularly interspaced short palindromic repeats (CRISPR) and recombinase-aided amplification (RAA) (CE–RAA–CRISPR) for detecting V. parahaemolyticus in seafood. The method combines RAA with CRISPR-associated protein 12a (Cas12a) for rapid detection in a one-pot reaction, effectively reducing the risk of aerosol contamination during DNA amplifier transfer. We optimized the primers for V. parahaemolyticus, determined the optimal crRNA/Cas12a ratio, and demonstrated that chemical additives (bovine serum albumin and L-proline) could enhance the detection capacity of Cas12a. The limit of detection (at optimal conditions) was as low as 6.7 × 101 CFU/mL in pure cultures and 7.3 × 101 CFU/g in shrimp. Moreover, this method exhibited no cross-reactivity with other microbial pathogens. The CE–RAA–CRISPR assay was compared with the quantitative polymerase chain reaction assay using actual food samples, and it showed 100% diagnostic agreement.

Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a

“…Based on fluorescence change from no template control to no DNase added, 0.1 U, 0.5 U, and 1 U DNase/L treatment reaction removed 81%, 84%, and 85% of the plasmid, respectively. The DNase step is commonly used as a cleanup step for any free DNA not already digested by Benzonase, commonly added to remove genomic DNA even in upstream processing [28]. Thus, it would be unlikely such a high amount of free transgene DNA would be present in samples for genome titer testing.…”

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

“…23 Some researchers optimized the reaction buffer by adding L-proline and bovine serum albumin to enhance the detection capability of the CRISPR/Cas12a system. 24 Lin et al completed the physical separation of the RPA and CRISPR systems by adding glycerol, obviously improving the sensitivity of the one-tube RPA-CRISPR system. 25 Yin et al utilized the density difference of sucrose concentration to spatially separate the RPA and CRISPR systems.…”

“…Some physical separation methods were adopted to isolate the RPA and CRISPR systems, like adding Cas12a reagents to the cover or wall of the PCR tube and adding the RPA system into the bottom of the tube . Some researchers optimized the reaction buffer by adding l -proline and bovine serum albumin to enhance the detection capability of the CRISPR/Cas12a system . Lin et al completed the physical separation of the RPA and CRISPR systems by adding glycerol, obviously improving the sensitivity of the one-tube RPA-CRISPR system .…”

Establishment and application of a rapid visualization method for detecting Vibrio parahaemolyticus nucleic acid

“…Vibrio parahaemolyticus , which is a commonly encountered Gram-negative bacterium, is a halophilic Vibrio species that can be detected not only in various marine products, such as fish, shrimp, and shellfish [1] , but also in ready-to-eat food items [2] . Infection with V. parahaemolyticus not only gives rise to gastrointestinal diseases characterized by symptoms such as watery diarrhoea, nausea, vomiting, and abdominal cramps, but can also lead to wound infections.…”

“…Currently, there are several commonly used methods for detecting V. parahaemolyticus , including pathogen-based diagnosis, immunological diagnosis, and molecular diagnosis [9] . Among these methods, isolation and culture identification are considered the ‘gold standard’ for V. parahaemolyticus detection [3] ; however, this approach is time-consuming and labor-intensive and does not meet the requirements for rapid testing [1] . Immunological diagnosis techniques, such as enzyme-linked immunosorbent assays (ELISAs) and colloidal gold detection, require the preparation of high-quality antigens and specific antibodies, resulting in high costs and lengthy procedures.…”