Research On Rapid Detection Method For Aeromonas Veronii Based On CRISPR/Cas System

Aeromonas veronii(A.veronii)belongs to the family Aeromonas(Aeromonadaceae),Aeromonas(Aeromonas),also known as Aeromonas veronii,Aeromonas veronii,Aeromonas versonii,is a gram-negative,rod-shaped bacterium that does not produce spores and is anaerobic in nature.The bacterium is commonly found in various environments,including fresh water,sewage,soil,and even seawater.It can also be present in milk,tap water,and food.The bacterium has a blunt and round shape at both ends,with a size range of approximately 0.3-0.7μm×1.2-2.5μm.It has flagella,and can move.The bacteria exhibited robust growth on standard nutrient agar,and after 24 h of cultivation,the colonies were circular with the diameter of approximately1.3 mm.They displayed neat edges,smooth surface,gray-white,opaque,and slightly raised in the center of the colonies.A.veronii can infect not only aquatic animals such as fish,but also mammals including humans.It can lead to various health issues such as gastroenteritis,peritonitis,meningitis,sepsis and traumatic infections,resulting in significant economic losses for the aquaculture industry and posing a severe threat to human health.Currently,several countries have classified A.veronii and related species as quarantine organisms for water quality and food safety.Moreover,A.veronii has emerged as a significant pathogen affecting both humans and fish.In this study,A.veronii was selected as the experimental subject to develop rapid,efficient and sensitive detection techniques,which was of great significance for the detection of food pathogens in daily life and the improvement of food quality and safety.Firstly,the target strain was isolated,purified,cultured,and then identified by bacterial morphology and universal primer amplification sequencing.The specific genes for the experiment were identified through ordinary PCR amplification.Based on this,the relevant specific primers and the cr RNA required for the CRISPR/Cas12a system were designed.Its activity and working conditions were explored,and the specificity was evaluated through PCR amplification,including ordinary PCR and gel electrophoresis,and real-time PCR.The activity was achieved by assembling CRISPR/Cas12a with the cr RNA complex to cleave the target DNA,and fluorescence is generated through fluorescence reporter modification,which produces a fluorescence map.Based on this map,the fluorescence intensity resulting from the interaction of different dosages of the system on the target gene is observed to determine the optimal dosage ratio.The optimization results of CRISPR/Cas system demonstrated that optimal components for the 25μL system were CRISPR/Cas12a(0.5μL),CRISPR/Cas12a reaction buffer(2.5μL),cr RNA(2.5μL),ds DNA(2.5μL),and fluorescent reporter FQ(2.5μL).The sensitivity of the experiment was measured using various methods to derive the detection limit.The minimum detection limit of the target strain DNA was 1.09×10~2 copies/μL by ordinary PCR,1.09×10~1 copies/μL by real-time PCR,while the CRISPR/Cas system could reach 1.09×10~0 copies/μL or even lower.