肉苁蓉饮片基原植物PCR-RFLP鉴别方法研究OA

[Objective]To establish a polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP)identification method for Cistanche deserticola and Cistanche tubulosa,the original plants of Cistanche herbs slices.[Methods]The ITS sequences of C.deserticola and C.tubulosa were compared and analyzed with SanpGene software to screen for heterogeneous restriction endonuclease sites,and PCR amplification products of their ITS sequences were indentified using restriction endonucleases.The DNA extraction method for C.deserticola and C.tubulosa slices,annealing temperature,cycle number,and amplification efficiency of different enzymes for PCR amplification,as well as the enzyme digestion reaction time and substrate amount were optimized,and the PCR-RFLP identification method was established.On this basis,the stability and accuracy of this method were investigated.[Results]The suitable extraction method for DNA from herbs slices was the reagent kit method,and the suitable annealing temperature and cycle number for PCR amplification were 47℃and 35,respectively.The suitable substrate amount for the NcoI restriction enzyme digestion reaction was 7.5 μL,and the suitable temperature and reaction time was 37℃and 60 min,respectively.On the above conditions,the PCR amplification product of C.deserticola was digested into two bands,which were located between 100 bp-250 bp and 500 bp-750 bp,respectively.However,the PCR amplification product of C.tubulosa was not digested and displayed one band,and only a distinct band at 750 bp was observed.[Conclusions]The study established a PCR-RFLP identification method for the original plants of the medicinal herb C.deserticola and C.tubulosa.This method was faster and more accurate than microscopic and component identification,and could provide technological reference for the identification of Cistanche herbs slices in the market.