Rapid Visual Nucleic Acid Detection Of Vibrio Alginolyticus By RPA Combined With CRISPR/Cas13a

Vibrio alginolyticus（V.alginolyticus）is an important zoonotic pathogen.Infection of aquaculture animals can cause huge economic losses,while infection of humans can induce serious bloodstream or skin infections,food-borne diarrhoea and related complications.Traditional testing methods are complicated and require professional instruments,which limits the early diagnosis of V.alginolyticus.Therefore,the establishment of a rapid on-site detection method for V.alginolyticus is significance for saving lives and economic losses in mariculture.The gyrB gene encodes the B subunit of DNA gyrase,which is relatively conserved and valuable for the identification of Vibrio spp.It is commonly used as a target for nucleic acid detection of Vibrio spp.In this study,the phylogenetic evolutionary tree of gyrB gene of Vibrio spp.was performed using MEGA software to verify the effectiveness of the gene for interspecific discrimination of similar Vibrio spp.The gyrB gene of V.parahaemolyticus standard strain,V.fluvialis standard strain,V.harveyi standard strain and V.vulnificus standard strain were compared with the gyrB gene of V.alginolyticus standard strain,and specific primers of recombinase polymerase amplification（RPA）and CRISPR RNA（cr RNA）sequences was designed for the conserved region of the gyrB gene of V.alginolyticus.The nucleic acid template was rapidly amplified by RPA technology at thermostatic conditions,and the detection sensitivity and specificity of this system was improved by combining with CRISPR/Cas13a reaction system.The result was visualized by fluorescent or Lateral flow dipstick.Plasmids containing the gyrB gene were constructed and used as template for sensitivity validation by serial dilution of the plasmids.The specificity of the method was evaluated using the genomic DNA of closely related marine Vibrio species（V.parahaemolyticus,V.fluvialis,V.metschnikovii）and common clinical pathogens（Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa）.The feasibility of the above two assays in clinical applications were validated by thirty-five wild V.alginolyticus strains isolated and identified in our laboratory.The experimental results showed that both the RPA-CRISPR/Cas13a fluorescence method and the RPA-CRISPR/Cas13a-LFD method exhibited high sensitivity.The limit of detection for RPA-CRISPR/Cas13a fluorescence method and RPA-CRISPR/Cas13a-LFD method were1copy·μL^-1 and 1×10¹copies·μL^-1,respectively.Both RPA-CRISPR/Cas13a fluorescence method and RPA-CRISPR/Cas13a-LFD method have good specificity and no cross-reactivity with closely related marine Vibrio species and common clinical pathogens.Positive detection rate of 35 wild V.alginolyticus strains by RPA-CRISPR/Cas13a fluorescence method and RPA-CRISPR/Cas13a-LFD method was 100%（35/35）,which was consistent with the results of MALDI-TOF MS mass spectrometry identification and Taqman-qPCR detection.The specificity and sensitivity of the above two methods were 100%.This confirms that the above two methods can be used for clinical nucleic acid detection of wild Vibrio alginolyticus.In summary,the RPA-CRISPR/Cas13a-based Vibrio alginolyticus nucleic acid detection method established in this study are fast,highly sensitive,highly specific,visualize results and not dependent on complex equipment,compared with the common V.alginolyticus detection methods such as bacterial culture identification method and Taqman-qPCR method.It is suitable for V.alginolyticus nucleic acid detection when lacking professional instruments and personnel,and can shorten the clinical testing time to improve detection efficiency.