摘要
目的:为了快速准确地鉴定中毒事件中毒源植物的种类,对有毒药用植物开展基于DNA条形码技术的分子鉴定研究。方法:选取11种有毒药用植物,对植物叶片组织材料进行硅胶干燥、沸水加热及胃液消化等21种不同条件的处理,采用直接聚合酶链式反应(PCR)法,选用6种引物扩增常用的5个植物DNA条形码序列,检测不同处理方法下扩增与测序的成功率。对于通过测序获得的DNA条形码序列,使用Blast法开展鉴定分析工作。结果:硅胶干燥材料的PCR成功率普遍低于未处理或短时间沸水处理的新鲜材料。短时间沸水加热处理并不会影响PCR的成功率,而沸水处理超过15 min时PCR扩增成功率急剧下降。在胃液结合短时间沸水处理条件下,PCR成功率仍然可以维持在较高的水平,而PCR扩增成功率同样在沸水处理材料15 min后显著下降。通过测序获得的DNA条码序列使用Blast方法进行鉴定,其科、属水平的准确率较高,而种水平的准确率普遍较低。内转录间隔区(ITS)片段种级鉴定准确率显著高于叶绿体区段,而叶绿体区段中psb A-trn H的种级鉴定准确率最高。结论:直接PCR扩增获取DNA条形码的方法在实际中毒案例植物的鉴定中具有一定的应用前景。
Objective:In order to quickly identify the poisonous plants on an incident of poisoning,we conducted molecular identification experiments based on DNA barcoding technique using different kinds of poisonous plant materials under various treatments.Methods:We conducted molecular identification experiments based on DNA barcoding technique to test direct PCR amplification using 11 poisonous plant materials under 21 different treatments,including silica-gel drying,boiling,digesting by gastric juice,etc.An economical and fast direct PCR amplification method was proposed and applied.Six primers for five commonly used plant DNA barcodes were used for amplification and sequencing.The success rate of PCR amplification and sequencing were calculated for each sample treatment.Blast method was utilized to assess applicability of each DNA barcode.Results:The results showed that the PCR success rates were lower for silica-gel dried samples than those of the fresh or short-time boiled samples.Short term boiling and gastric acid after short boiling did not significantly lower PCR success.However,if the boiling time exceeded 15 minutes,the PCR success was dropping significantly.Blast identification results showed that at the family and generic level,the five barcodes obtained high accuracy rates,whereas at the species level the accuracy of identification was decreased.ITS region obtained the highest identification accuracy at species level,and psb A-trn H was tested to be the most accurate barcode among the tested plastid regions.Conclusion:The methods used in this study showed potential usefulness for poisonous plant identification on scene of poisoning case.
作者
赵喆
李明宇
何仟
程瑾
谢磊
ZHAO Zhe;LI Ming-yu;HE Qian;CHENG Jin;XIE Lei(Beijing Forestry University,Beijing 100083,China;National Institute of Occupational Health and Poison Control,Chinese Center for Disease Control and Prevention,Beijing 100050,China)
出处
《中国现代中药》
CAS
2020年第10期1600-1606,共7页
Modern Chinese Medicine
基金
职业病与中毒控制检测鉴定及技术储备项目。
关键词
DNA条形码
沸水处理
分子鉴定
胃液处理
直接PCR
药用植物
有毒植物
DNA barcode
boiling water treatment
molecular identification
gastric juice treatment
direct PCR
medicinal plant
poisonous plant
