霍山石斛的分子特异性鉴别

Specific PCR Identification between Dendrobium huoshanense and Its Adulterants

目的基于ITS序列上变异位点设计特异性引物对霍山石斛及其常见混伪品(铁皮石斛和河南石斛)进行有效鉴别。方法利用最大简约法(MP)和贝叶斯法(BI)构建霍山石斛及常见混伪品的系统发育树,并基于ITS序列上变异位点以霍山石斛为正品设计特异性PCR引物SH-CP9s/SH-CP9a、SH-CP25s/SH-CP25a、SH-CP29s/SH-CP29a,优选扩增条件,用于霍山石斛及其混伪品鉴别。结果在系统发育树中霍山石斛聚类为一个单系。通过对PCR反应的退火温度、DNA模板用量进行优化,并对不同型号的PCR仪和Taq酶进行考察,分别获得霍山石斛的特异性PCR反应程序。在PCR产物中,正品出现目的条带,而混淆品不出现条带。结论 ITS序列能够作为霍山石斛的DNA鉴别条形码,且设计的特异性引物能够有效地区别霍山石斛与其混伪品。

OBJECTIVE To design specific identification primers based on ribosomal DNA ITS sequences and accurate identification of Dendrobium huoshanensis and its adultments.METHODS Phylogenetic tree of D. huoshanense and D. officinale and D. henanense by maximum parsimony （MP） and Bayesian inference （BI） were constructed. Specific primers SH-CP9s/SH-CP9a, SH-CP25s/SH-CP25a, SH-CP29s/SH-CP29a for PCR identification of D. huoshanensis based on mutation sites on ITS sequences were designed.RESULTS In the phylogenetic tree, D. huoshanense and D. officinale and D. henanense were clustered into monophyletic group. The specific PCR reaction procedure of D. huoshanense was obtained by investigating the annealing temperature and the amount of DNA template, and different types of PCR and Taq enzymes. In the PCR product, D. huoshanense appearance of the band, and adultments with no band.CONCLUSION The ITS sequences can be used as DNA Barcoding identification of D. huoshanensis, and identify D. huoshanense and its adultments by the designed primers.