细叶石斛的位点特异性PCR鉴别

Allele-specific Diagnostic PCR Authentication of D.hancockii from the Other Dendrobium Species

根据细叶石斛及其它 37种枫斗类和黄草类石斛的rDNAITS序列,我们设计了位点特异性PCR鉴别引物XY JB0 1S和XY JB0 1X,对细叶石斛进行了成功的DNA分子鉴别.在进行位点特异性鉴别PCR之前,首先运用扩增ITS区的通用引物P1、P2对模板DNA进行扩增,以验证模板的可靠性和扩增的合适浓度.当退火温度上升为 64℃,只有细叶石斛的模板DNA能被扩增出来,而其它的 37种石斛属植物均为阴性.该鉴别反应重复性好,已在鉴别细叶石斛中发挥重要作用.与DNA测序鉴别方法相比,位点特异性PCR具有简单、省时、高效、准确等优点.

Based on rDNA ITS sequences of D. hancockii and the other 37 species of Dendrobium, which are all used for the materials of various 'Fengdou' and 'Huangcao', the new allele specific diagnostic primers XYJB01S and XYJB01X have been designed to authenticate D. hancockii from the other species. Before the diagnostic PCR, the primer pair P1 and P2 for amplifying the whole ITS region should be used to validate template DNA at first so as to obtain the appropriate template DNA concentration for the diagnostic PCR. When the annealing temperature was raised to 64℃, only the template DNA of D. hancockii could be amplified whereas the diagnostic PCR of the other 37 species were all negative. The diagnostic PCR have been repeated for many times and have played an important role in identifying D. hancockii in China. Thus, the allele specific diagnostic primers have been designed to authenticate D. hancockii efficiently. Compared with the authentication method by sequencing DNA fragments, the allele specific diagnostic PCR was not only simple and timesaving but also practical and effective.