中药材鹿心分子鉴定方法及检测试剂的研究

目的建立中药材鹿心DNA指纹鉴定方法,研发鹿心DNA检测试剂盒。方法以梅花鹿和马鹿mtDNA Cytb基因作为靶基因,设计小片段特异性引物,研制鹿心DNA提取及PCR检测试剂;应用分子克隆及测序技术,克隆鹿心对照药材;并对试剂的特异性、重现性、稳定性及灵敏度进行考察;对市售鹿心样品进行真伪鉴定。结果自主研发的试剂提取鹿心的DNA浓度纯度均达到PCR的要求;在退火温度为63℃时引物的特异性最强;克隆测序后的鹿心DNA序列与鹿心mtDNA Cytb特异指纹区段序列一致;自主研发的试剂重现性、稳定性良好,灵敏度达到0.5%;对市售30个鹿心样品进行检测,伪品率为40%。结论建立的鹿心DNA指纹鉴定方法特异、准确、可靠,研制的鹿心DNA检测试剂盒操作简便、结果稳定。

Study on molecular identification method and detection reagent of Chinese medicinal materials deer heart

Objective To establish a DNA fingerprint identification method for Chinese medicinal materials deer heart and develop DNA detection kit for deer heart.Methods Using the mtDNA Cytb gene of Cervus nippon and C.elaphus as the target gene,small fragment specific primers were designed and used to develop the deer heart DNA extraction reagent and PCR detection reagent.Using molecular cloning and the sequencing technology,standard materials to deer heart was cloned.And then the specificity,reproducibility,stability and sensitivity of the reagent was investigated.Finally the authenticity of the commercial deer heart samples was verified.Results The concentration and purity of DNA extracted by the self-developed reagent reached the requirement of PCR.The specificity of the primer was the strongest when the annealing temperature was 63℃.The DNA sequence of deer heart after cloning was consistent with the specific fingerprint section of sika deer heart mtDNA Cytb gene.Self-developed reagent had good reproducibility,stability and sensitivity up to 0.5%.Thirty baked deer heart samples for sale were tested and the rate of counterfeit was 40%.Conclusion The DNA fingerprint identification method of deer heart established in this study is specific,accurate and reliable.The DNA detection kit for deer heart which developed in this study is simple to use and the result is stable.