Establishment of a Cas13a-RPA Detection Method for the Causative Pathogen Vibrio splendidus of Sea Cucumber Apostichopus japonicus Skin Ulcerative Syndrome
LIU Ji, CHEN Yanru, HOU Zhumei, XU Dongxue, GU Yuanxue, SONG Wenqi, SONG Yize, XIA Bin
School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China
Abstract：In order to prevent and control the skin ulcerative syndrome (SUS) of sea cucumber Apostichopusjaponicus caused by Vibriosplendidus and maintain the healthy and sustainable development of the sea cucumber aquaculture industry, this study used the characteristic of LwCas13a protein when specific crRNA in LwCas13a binds to the target RNA, its collateral cleavage activity can be activated to degrade non-targeted exogenous RNA. Combined this characteristic with the recombinase polymerase amplification (RPA) technique, a rapid, sensitive and specific quantitative detection method for gyrB gene of V. splendidus—RPA-Cas13a was established. This method can realize rapid real-time detection of V. splendidus at constant 37 ℃ within 60 mins. Sensitivity test showed that the detection limit of this method is 550 copies/μL(gyrB), which is consistent with the sensitivity level of fluorescence quantitative PCR (qPCR); Specificity test demonstrated a relatively high specificity with the fact that it had no cross-reactions with other Vibrio species, e.g. V. vulnificus, V. harveyi and V. anguillarum, and other common aquatic animal bacterial pathogens, including Pseudoalteromonas nigrifaciens, Aeromonashydrophlia and Edwardsiellatarda. The detection rate of 57 environmental samples including skin mucus of A. japonicus (32 samples), culture pond seawater (15 samples) and surface sediments (10 samples) was 8.77%, highly consistent with that of traditional fluorescence qPCR method (Kappa=1.00) with no statistical difference (P>0.05). In conclusion, this study established a novel rapid, simple, sensitive and specific RPA-Cas13a quantitative detection method for V. splendidus, which provides a powerful molecular tool for preventing and controlling the occurrence of sea cucumber SUS
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2023, Vol. 42 
