基于RAA-CRISPR/Cas12a技术的桃成分单管一体化快速检测方法

Rapid Single-Tube Detection of Peach(Prunus persica)-Derived Ingredients by Recombinase-Aided Amplification Combined with Clustered Regularly Interspaced Short Palindromic Repeat(CRISPR)/CRISPR-Associated Proteins(Cas)12a

通过设计特异性的桃重组酶介导的等温扩增(recombinase-aided amplification,RAA)和CRISPR RNA(crRNA)引物,建立RAA技术结合CRISPR/Cas12a系统的单管一体化桃成分快速检测方法,并分析该方法的特异性、灵敏度和检出限,同时对市售样品进行检测。结果显示,该方法不依赖大型仪器设备,在30 min内即可完成桃成分的快速检测;与其他常见水果物种之间无交叉污染,表现出良好的特异性;该方法的灵敏度为1.0×10-1 ng/μL;检出限为1%;通过对20份市售样品检测,结果显示本方法与实时聚合酶链式反应法检测结果一致。因此,本研究建立的RAA-CRISPR/Cas12a单管一体化桃成分快速检测方法具有特异性好、灵敏度高的优点,为桃成分快速检测提供了一种新的技术。

A rapid single-tube method for the detection of peach-derived ingredients was established using recombinaseaided amplification(RAA)combined with clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPRassociated proteins(Cas)12a.Specific primers for RAA and CRISPR RNA(crRNA)were designed.The specificity,sensitivity and detection limit of the developed method were analyzed,and it was applied to commercial samples.The results showed that the method enabled rapid detection of peach-derived ingredients without the need for large-scale instruments in less than 30 minutes.No cross-contamination with other common fruit species was observed,indicating good specificity.The sensitivity of this method was 1.0×10-1 ng/μL,and the limit of detection was 1%.The results of detection of 20 commercial fruit juice samples by this method were consistent with those of real-time polymerase chain reaction(PCR).Therefore,the single-tube integrated RAA-CRISPR/Cas12a method has the advantages of high specificity and sensitivity,providing a new technology for rapid detection of peach-derived ingredients.