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目的研究能够特异性鉴别鼓槌石斛的PCR引物,并优化PCR检测体系,确立一种快速、准确鉴别鼓槌石斛的方法。方法从GenBank数据库中下载石斛属rDNAITS序列170余条,运用MEGA5.0对所有序列进行同源性比较,找出各变异位点,在非保守区设计1对特异性引物。对35份石斛属植物DNA模板进行特异性引物PCR扩增,显示阳性者为鼓槌石斛。结果将退火温度升至58℃时,鼓槌石斛能被该引物特异性地扩增出来,而其他石斛属植物均为阴性,且该引物的灵敏度可达到0.69 ng/μL。结论建立一种特异性鉴别鼓槌石斛的方法,运用该特异性引物可实现从同源物种中快速、准确地鉴定出鼓槌石斛,具有特异性好,操作简便、高效、准确等优点。
Objective To study PCR primers that can specifically identify Dendrobium chrysotoxum, optimize PCR detection system and establish a rapid and accurate method for the identification of Dendrobium chrysotoxum. Methods A total of 170 rDNAITS sequences from Dendrobium were downloaded from the GenBank database. The homologies of all the sequences were compared using MEGA5.0 to find out the variation sites. One pair of specific primers was designed in the non-conservative region. Specific primers were amplified by PCR from 35 Dendrobium DNA templates, indicating that the positive ones were Dendrobium chrysotoxum. Results When the annealing temperature was raised to 58 ℃, Dendrobium chrysotoxum could be amplified specifically by this primer while other Dendrobium plants were negative, and the sensitivity of this primer was 0.69 ng / μL. Conclusion The method of specific identification of Dendrobium chrysotoxum is established. By using this specific primer, Dendrobium chrysotoxum can be quickly and accurately identified from the same species, which has the advantages of good specificity, simple operation, high efficiency and accuracy.