目的 建立维吾尔药毛菊苣及其易混品菊苣的位点特异性PCR鉴别方法。方法 采集不同地理区域的毛菊苣及其易混品，提取样品基因组总DNA。通过对其叶绿体rbcL基因片段进行扩增、测序，进行多重序列比对后根据其变异位点设计位点特异性鉴别引物，对其反应条件进行优化，确定检出限，建立位点特异性PCR鉴别方法。结果 建立的位点特异性PCR鉴别体系优化结果为30 μL反应体系包含Taq酶0.25 U、10×buffer 2.5 μL、dNTP 2.0 μL、正、反向引物各0.5 μL、总DNA 2 μL、ddH₂O 22.25 μL。适宜的扩增条件为94℃预变性3 min；94℃变性30 s，55℃退火30 s，72℃延伸1 min，32个循坏；最后72℃延伸7 min。对20份毛菊苣与菊苣药材进行扩增，其中毛菊苣能够扩增出约230 bp目的片段条带，而菊苣样品则不能扩增出明显的目的条带。结论 建立并优化了毛菊苣及其易混品菊苣位点特异性PCR鉴别技术，能够准确而可靠的达到2种药材的有效鉴别。

Objective To establish molecular identification method of Cichorium glandulosum and its adulterants Cichorium intybus by Allele-Specific PCR. Methods The samples of C. glandulosum and C. intybus were collected in different geographical areas. The DNA was extracted, and rbcL gene segments were amplified and sequenced directionally. The multiple sequences were aligned by using Clustal W. Specific primers were designed and amplified according to its variable sites, and PCR reaction system was optimized to determine detection limits and establish Allele-Specific PCR identification method. Results According to Allele-Specific PCR system established in this study for C. glandulosum, the optimization results was a total of 30 μL reaction system containing TaqDNA polymerase 0.25 μL, 10×buffer 2.5 μL, dNTP 2.0 μL, primer 0.5 μL, template DNA 2 μL, and ddH₂O 22.25 μL. The most suitable PCR amplification procedure is one cycle of predegeneration at 94℃ for 3 min; 32 cycles of denaturing at 94℃ for 30 s, annealing at the primer temperature 55℃ for 30 s and extending at 72℃ for 1 min, and extending at 72℃ for 7min. Through the detection of 20 medicinal materials of C. glandulosum and C. intybus, the result showed that 230 bp amplified band of target fragment was identified for C. glandulosum but no amplified band was observed for its adulterants. Conclusion In this study, we established and optimized the Allele-Specific PCR identification technology of C. glandulosum and its adulterants C. intybus, which can accurately, reliably, and effectively identify these two medicinal materials.

新疆维吾尔自治区卫生计生委青年医学科技人才项目（WJWY-201842）