Identification of uighur medicine Cichorium glandulosum and its adulterants by Allele-Specific PCR / 中草药

ABSTRACT

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.