章秀梅;李丹;刘丛彬;俞年军;李永杰;谢冬梅;
ZHANG Xiu-mei;LI Dan;LIU Cong-bin;YU Nian-jun;LI Yong-jie;XIE Dong-mei;Anhui University of Chinese Medicine;Institute of Traditional Chinese Medicine Resources Protection and Development, Anhui Academy of Chinese Medicine;Anhui Province Key Laboratory of Research & Development of Chinese Medicine;the First Affiliated Hospital of Dalian Medical University;

• 1:安徽中医药大学
• 2:安徽省中医药研究院中药资源保护与开发研究所
• 3:中药研究与开发安徽省重点实验室
• 4:大连医科大学附属第一医院

摘要(Abstract)：

建立一种能同时鉴别苍耳子及其混淆品蒙古苍耳子和意大利苍耳子的方法。对植物苍耳、蒙古苍耳和意大利苍耳的基因组序列进行测序，分别获得约2.21、2.24、2.54 Gb大小的基因序列；结合BLASTN法和Bowtie 2软件筛选出76条特异性contigs并设计对应引物；通过PCR扩增，筛选出3对扩增稳定、重复性好的特异性鉴别引物并建立多重PCR反应体系，并对其退火温度、DNA模板用量、循环数、DNA聚合酶种类和不同PCR仪等扩增条件进行优化。结果表明，在退火温度为52℃,DNA模板用量为30 ng,循环次数为35时，苍耳子能同时扩增出262、458 bp的片段，蒙古苍耳子扩增出260、454、927 bp的片段，意大利苍耳子扩增出260、926 bp的片段。应用该多重PCR方法，对18批苍耳样品、17批蒙古苍耳样品和12批意大利苍耳样品进行验证，结果表明所建立的多重PCR鉴别方法可以准确、快速开展苍耳子及其混淆品的鉴别，为中药苍耳子的分子鉴定提供了新的方法。
The purpose of this study is to establish a molecular method to identify Xanthii Fructus and two adulterants, the fruits of Xanthium mongolicum and X. italicum. Xanthii Fructus is the fruit of X. sibiricum, which is a Chinese herbal medicine used clinically to treat allergic rhinitis. The fruits of X. mongolicum and X. italicum have strong morphological similarities with Xanthii Fructus, while their safety of medication cannot be guaranteed. The genomes of X. sibiricum, X. mongolicum, and X. italicum were sequenced, which generated sequences of 2.21, 2.24, and 2.54 Gb, respectively. Based on the 76 specific contigs screened out by BLASTN and Bowtie 2, the corresponding primers were designed by Primer 5.0. Three pairs of primers with stable amplification efficiency and good reproducibility were screened out to establish a multiplex PCR method based on the PCR amplification results. Further, the annealing temperature, the amount of DNA template, the number of cycles, different DNA polymerases, and different PCR thermal cyclers were optimized. Fragments of 262 bp and 458 bp from X. sibiricum, 260, 454, and 927 bp from X. mongolicum, and 260 bp and 926 bp from X. italicum were amplified under the following conditions: the annealing temperature of 52 ℃, 35 cycles, 30 ng template DNA. Then, the established method was used to detect 18 samples of X. sibiricum, 17 samples of X. mongolicum, and 12 samples of X. italicum. The results showed that all the samples had positive results, which were consistent with the morphological identification results, thus proving the stability and reliability of the established method. Combining genome sequencing technology and multiplex PCR method to identify Xanthii Fructus and its adulterants can not only obtain the difference in genetic background but also facilitate the design of reliable primers. The multiplex PCR have high specificity and repeatability, providing a new method for the molecular identification of Xanthii Fructus.

关键词(KeyWords)： 苍耳子;混淆品;基因组;多重PCR;分子鉴定
Xanthii Fructus;adulterant;genome;multiplex PCR;molecular identification

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81503190);; 安徽中医药大学2021年度校级探索性科研项目(2021hxts22)

作者(Authors): 章秀梅;李丹;刘丛彬;俞年军;李永杰;谢冬梅;
ZHANG Xiu-mei;LI Dan;LIU Cong-bin;YU Nian-jun;LI Yong-jie;XIE Dong-mei;Anhui University of Chinese Medicine;Institute of Traditional Chinese Medicine Resources Protection and Development, Anhui Academy of Chinese Medicine;Anhui Province Key Laboratory of Research & Development of Chinese Medicine;the First Affiliated Hospital of Dalian Medical University;

DOI: 10.19540/j.cnki.cjcmm.20220115.103

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