铁皮石斛RPA-CRISPR/Cas12b一步法快速鉴定OA

Objective To develop a one-step rapid detection method based on recombinase polymerase amplification(RPA)and CRISPR/Cas12b technology for the rapid and visual on-site identification of Dendrobium officinale Kimura&Migo(D.officinale)and its medicinal materials.Methods The ITS2 sequences of D.officinale and its 13 common adulterants were aligned to design specific RPA primers and small guide RNA(sgRNA)of D.officinale.An RPA-CRISPR/Cas12b one-step detection system was constructed,and concentration of key reaction components and temperature were optimized.The specificity and sensitivity of the optimized method were systematically evaluated.Furthermore,commercially available medicinal materials were subjected to dual verification using both PCR sequencing and the established one-step method.Results The RPA-CRISPR/Cas12b one-step detection system for D.officinale was established and optimized with the following key parameters:The concentration ratio of Cas12b to sgRNA of 1:1,Cas12b protein concentration of 100 nmol/L,ssDNA fluorescent reporter concentration of 1.2 µmol/L,RPA primer concentration of 0.2 µmol/L,and an optimal reaction temperature of 40℃.Under these conditions,D.officinale could be identified by distinct green fluorescence within 35 minutes.The method exhibited excellent specificity,producing significant fluorescence signals only for D.officinale,and a sensitivity up to 0.005 ng/µL.Testing for 18 commercial D.officinale samples showed complete consistency between the one-step method and PCR sequencing results,accurately identifying 15 genuine and 3 adulterated samples,demonstrating the high accuracy of the method.Conclusion The established RPA-CRISPR/Cas12b one-step detection method in this study is accurate,rapid,easy-to-operate,and visually interpretable.It is suitable for the rapid on-site screening of D.officinale and its medicinal materials,providing a reliable solution to the challenge of on-site identification of traditional Chinese medicinal materials and offering a technical reference for the rapid screening of other medicinal materials.