Study On Molecular Identification Of Two Genuine Traditional Chinese Medicine In AnHui

Agkistrodon acutus (Guenther) and Chaenomeles speciosa (Sweet) Nakai, the two typical Chinese crude drug in AnHui were selected to carry out molecular identification:(1) Sequencing of Cyt b gene of genuine Chinese crude drug Agkistrodon acutus (Guenther) and its adulterants collected from the markets was performed. The results of compare of these sequences indicated that there were many other snakes sold in the name of Agkistrodon acutus (Guenther) on the markets; The difference of Cyt b gene sequence between Agkistrodon acutus (Guenther) and its adulterants was obvious: the difference rate in Agkistrodon acutus (Guenther) was 0% to 0.91%, the difference rate between Agkistrodon acutus (Guenther) and its adulterants was 18.57%～23.78%.(2) At the same time, based on the analysis of DNA sequence data of mitochondrial Cyt b gene from Agkistrodon acutus (Guenther) and its adulterants, a pair of specialized primers named as qsJBll and qsJBhl was designed for the PCR identification of Agkistrodon acutus (Guenther). The results showed that only Agkistrodon acutus (Guenther) samples could be distinguished when the anneal temperatures was 64℃, however no any significant positive amplification was showed from its adulterants at this temperature. The diagnostic primers designed in the present study were highly specific for Agkistrodon acutus (Guenther). This PCR identification could be used efficiently and reliably in identification of traditional Chinese medicine Agkistrodon acutus (Guenther).(3) The AFLP analysis system on Chaenomeles speciosa (Sweet) Nakai was established by studying and optimizing the AFLP main factors such as method of DNA extraction, enzymes restriction, adapter-ligation, pre-amplification and selective amplification. A brief AFLP analysis on fourteen samples of Chaenomeles speciosa (Sweet) Nakai from different regions was performed by using selected a pair of primers, EcoRI-ACA and MseI-CAA. Clear, distinctive and various bands were obtained after selective amplification, which suggested that the reaction system was exercisable and reliable for the AFLP analysis on genuine Chaenomeles speciosa (Sweet) Nakai, and provided a foundation for studying the genuineness and genetic diversity on Chaenomeles speciosa (Sweet) Nakai resources.(4) The ITS gene of Chaenomeles speciosa (Sweet) Nakai in XuanCheng was sequenced, The sequences length was 594 bp, and the homology of nucletide with Chaenomeles speciosa (AF186530) was 95%.