基于重组酶介导等温扩增-CRISPR/Cas12a的青鱼物种快速鉴定研究

Rapid identification study of Mylopharyngodon piceus species based on recombinase-mediated isothermal amplification-CRISPR/Cas12a

目的 建立一种快速、特异、灵敏地鉴定青鱼物种的方法。方法 以青鱼线粒体16SrRNA基因的保守序列设计特异性crRNA,并根据crRNA设计重组酶介导等温扩增(recombinase-mediatedisothermal amplification,RAA)引物;建立青鱼的RAA-CRISPR/Cas12a快速鉴定方法,并对该方法的检测时间进行优化;同时,评估该方法的特异性和灵敏度,并对青鱼及其近缘物种的市售样品进行检测。结果 该方法仅需25 min完成检测;对青鱼物种的鉴定表现出显著的特异性,并对其近缘物种如草鱼、赤眼鳟、鳡鱼、倒刺鲃的检测结果均呈阴性;方法灵敏度为1.0×10-3ng/μL(以基因组DNA计);与DNA条形码技术相比,该方法在市售样品的检测结果上表现一致。结论 建立了青鱼物种的RAA-CRISPR/Cas12a现场快速检测方法,该方法具备快速、特异、灵敏的特点,为水产市场或野外环境中快速检测青鱼物种提供了技术手段。

Objective To establish a method for rapid,specific and sensitive identification of Mylopharyngodon piceusspecies.Methodss A specific crRNA was designed with the conserved sequence of Mylopharyngodon piceus mitochondrial 16S rRNA gene.Recombinase-mediated isothermal amplification(RAA)primers were designed based on the crRNA.A rapid identification method,RAA-CRISPR/Cas12a was developed for Mylopharyngodon piceus,and the detection time of this method was optimized.The specificity and sensitivity of the method were assessed,and commercially available samples of Mylopharyngodon piceus and its closely related species were tested.Results The method achieved rapid detection in just 25 minutes.It exhibited remarkable specificity in identifying Mylopharyngodon piceus,yielding negative results for Ctenopharyngodon idellus,Squaliobarbus curriculus,Elopichthys bambusa and Spinibarbus caldwelli.The sensitivity of the method was 1.0×10^(-3)ng/μL(calculated based on genomic DNA).The method showed consistent results in testing commercially available samples compared to the DNA barcoding technique.Conclusion A rapid RAA-CRISPR/Cas12a for on-site detection of Mylopharyngodon piceus species has been established.The method is rapid,specific and sensitive.This method can provide technical support for the rapid identification of Mylopharyngodon piceus species in the aquatic market or field environment.