Objective:To establish a quick

accurate and effective method for authenticity identification of Dioscoreae Rhizoma by using rapid and specific polymerase chain reaction (PCR) method. Method:Samples of Dioscoreae Rhizoma and its counterfeit products from different regions were collected. All samples were extracted for total DNA. Homologous comparative analysis of DNA barcoding rbcL fragments of Dioscoreae Rhizoma and its confused products was carried out. The differential primers for Dioscoreae Rhizoma were designed according to the identification site and the primer sequences were as follows Sy89:5'-AAGGACGATGCTACCACATCGACAC-3'

Sy90:5'-ATCCCAATAGGGGACGGCCA-3'. The 2-step method was used for PCR amplification

and the influencing factors of the PCR reaction system and reaction procedures were investigated. Result:A rapid and specific PCR reaction procedure for Dioscoreae Rhizoma was obtained by optimizing the factors affecting PCR annealing temperature

annealing time

denaturation time and number of cycle times

as well as by examining different Mix. The PCR amplification reaction solution was 50 μL

including Premix TaqTM 25 μL

1.5 μL each for upstream and downstream primers

1.5 μL DNA template

and 50 μL sterile double distilled water. The PCR amplification procedure was as follows:pre-denaturation at 98℃ for 1 min

denaturation at 98℃ for 10 s

renaturation at 68℃ for 15 s

30 cycles

and extension at 72℃ for 30 s. The results of 1% agarose gel electrophoresis on PCR products showed that there was a clear band near 250 bp

and there was no band in the counterfeit products. Conclusion:Rapid and specific PCR method can be used to easily and quickly identify Dioscoreae Rhizoma

providing technical support for the field application of molecular identification of Chinese medicinal materials.