Page 218 - 《水产学报》2025年第11期

葛辉，等 水产学报, 2025, 49(11): 119417

Establishment of RPA-CRISPR/Cas12a-based visual detection of
Vibrio chagasii

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GE Hui , WU Qisheng , NING Yue , FENG Junya , CHEN Haoxiang , LI Huiyao ,
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YE Jun , WU Liyun , WEN Ping , XU Chunyan , REN Lei 2*
1. Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province,
Fisheries Research Institute of Fujian, Xiamen 361013, China;
2. College of Materials, Xiamen University, Xiamen 361005, China
Abstract: Vibrio chagasii is an emerging pathogen in Crassostrea angulata aquaculture, causing significant economic losses
and posing risks to food safety. Traditional detection methods, such as PCR, are time-consuming and rely on complex equip-
ment, making them unsuitable for on-site rapid detection. Therefore, developing fast, sensitive, and simple detection methods is
of great importance. To meet the need for rapid detection of V. chagasii, this study integrated recombinase polymerase ampli-
fication (RPA) with the CRISPR/Cas12a system, aiming to establish real-time fluorescence detection method and test strip
detection based on RPA-CRISPR/Cas12a. Specific fragments targeting the toxR gene of V. chagasii were designed, and RPA
primers and crRNA sequences were optimized to ensure accurate gene amplification and identification. Upon recognition of the
RPA-amplified target gene, the CRISPR/Cas12a system activated its trans-cleavage activity, cleaving biotin-modified single-
stranded DNA fluorescent probes. Detection results were determined by the fluorescence brightness under blue light and the
color development of the test strip. The experiment used multiple Vibrio species to verify the specificity of the detection meth-
ods and evaluated the sensitivity of the two detection methods through different concentrations of V. chagasii bacterial solution.
The two methods were further compared with PCR using real samples. The established RPA-CRISPR/Cas12a-based fluores-
cence detection method and test strip detection demonstrated excellent specificity, accurately identifying V. chagasii, and the
whole detection process could be completed within 1 h. Both methods achieved a detection limit of 100 CFU/mL, showing high
consistency with PCR results (100% concordance). The RPA-CRISPR/Cas12a-based detection methods developed in this study
are fast, sensitive, and easy to operate, providing a reliable technical solution for the on-site detection of V. chagasii. This study
offers two novel, efficient, and convenient methods for the rapid detection of V. chagasii, contributing significantly to disease
control in aquaculture and ensuring food safety.
Key words: Vibrio chagasii; recombinase polymerase amplification (RPA); CRISPR/Cas12a; fluorescence detection; strip test
Corresponding author: REN Lei. E-mail: renlei@xmu.edu.cn
Funding projects: China Agriculture Research System (CARS-49); National Marine Aquatic Germplasm Resources Bank
(2024); Fujian Province Marine Service and High-quality Development of Fisheries Special Project (FJHY-YYKJ-2024-1-13);
Basic Research Special Project of Public Welfare Research Institutes of Fujian Province (2022R1013007); 2025 Marine and
Fisheries Comprehensive Service Special Fund Project (FYZF-KTYJ-2025-10)

中国水产学会主办 sponsored by China Society of Fisheries https://www.china-fishery.cn
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