RPA-CRISPR/Cas12a-based visual and rapid detection of Anisakis
To achieve rapid,simple,and efficient detection of Anisakis in seafood to control its prevalence,recombinase polymerase amplification(RPA)primers and CRISPR RNA(crRNA)for the conserved sequence of internal transcribed space in Anisakis were designed,the RPA-CRISPR/Cas12a fluorescence detection system was established and reaction conditions such as crRNA concentration were optimized.The detection limit,specificity,and repeatability of this method were evaluated,and its actual detec-tion ability was verified through artificially contaminated samples.The results showed that the RPA-CRISPR/Cas12a fluorescence detection system could complete the reaction within 40 min,and with the sensitivity of 1 copy/μL.This method could only detect Anisakis and not react with Hysterothy-lacium aduncum,Ligula intestinalis,Trichiurus lepturus tissue and Larimichthys polyactis tissue.The RPA-CRISPR/Cas12a fluorescence visualization detection system was fast,sensitive,and specific,and could be applied to the detection of Anisakis in marine products.
国家科技期刊平台
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