On-site Identification of Medicinal Bubali Cornu by Recombinase- mediated Amplification Technique / 中国实验方剂学杂志

ABSTRACT

Objective: To establish a rapid on-site method for identifying Chinese medical material recombinase-mediated amplification (RAA) technology for the use of identifying medicinal Bubali Cornu from yak horn. Method: Based on the differences of mitochondrial genome sequences between Bubali Cornu and adulterants,the specific RAA primer (SNJ-1.F,SNJ-1.R) and fluorescence probe SNJ-1.probe were designed by variation sites. Alkaline lysis method was used to extract DNA from milled samples,and optimize RAA reaction system. The incubation was made at 37℃ for 15-20 min, the reaction results were monitored through gel electrophoresis and a mobile fluorescence amplification instrument. The RAA identification result was compared with COI DNA sequencing. Result: After incubation at 37℃ for 20 min,about 140 bp of bright and simple bands was amplified from DNA templates of Bubalus bubalis,whereas Bos mutus were negative. By the Real-time fluorescent RAA identify method,all reactions in DNAs from Bubali Cornu samples were amplified from 6.21 to 8.37 min,whereas DNAs from yak samples were amplified after 10.08 min,COI sequencing results conformed to the Real-time fluorescent RAA identification. Conclusion: Specific RAA could rapidly identify Bubali Cornu in 20 minutes, and thus be applied in medical material and its products. This method is simple,rapid and sophisticated instrument-free,with promises in on-site identification of traditional Chinese medicine.