Visual Identification and Serotyping of Toxigenic Vibrio cholerae Serogroups O1 and O139 With CARID

Abstract

There is a growing demand for rapid, sensitive, field-deployable nucleic acid tests for cholera, which usually occurs in rural areas. In this study, we developed a Cas12a-assisted rapid isothermal detection (CARID) system for the detection of toxigenic V. cholerae serogroups O1 and O139 by combining recombinase-aided amplification and CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). The results can be determined by fluorescence signal and visualized by lateral flow dipstick. We identified 154 V. cholerae strains and 129 strains of other intestinal diarrheagenic bacteria with a 100% coincidence rate. The limit of detection of CARID was 20 copies/reaction of V. cholerae genomic DNA, which is comparable to that of polymerase chain reaction (PCR) and qPCR. Multiple-CARID was also established for efficiency and economic considerations with an acceptable decrease in sensitivity. Simulated sample tests showed that CARID is suitable for complex samples. In conclusion, CARID is a rapid, sensitive, economically efficient, and portable method for the detection of V. cholerae, which makes it suitable for field responses to cholera.

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

“…However, RPA assay associated with aerosol contamination due to production of amplicons. Therefore, the addition of templates and transfer of amplification products should be conducted in different rooms (Lu et al, 2022). Designing portable and contamination-prevention cartridges can also prevent contamination.…”

Advances in cholera research: from molecular biology to public health initiatives

“…In endemic settings, the use of rapid, affordable, and accurate serotyping techniques is critical for identifying pandemic cholera in the field and implementing swift public health response measures. A lateral flow dipstick method, Cas12a-assisted rapid isothermal detection (CARID), was recently developed to identify O1 and O139 serogroups in complex samples using recombinase-aided amplification and CRISPR-Cas ( Lu et al, 2022 ). Of note, the World Health Organization assembled the Global Task Force on Cholera Control (GTFCC) which has established a roadmap to ending cholera by 2030 and is referenced several times in this text.…”