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| Identification of Cirsium japonicum，Carduus nutans and Cirsium leo by specific PCR-RFLP |
| Hits 689 Download times 508 Received:August 07, 2023 |
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| DOI
10.11656/j.issn.1673-9043.2023.06.15 |
| Key Words
Cirsium japonicum;Carduus nutans;Cirsium leo;PCR-RFLP;ITS sequence |
| Author Name | Affiliation | E-mail | | ZHANG Shihua | Gansu University of Chinese Medicine, Lanzhou 730030, China | | | LAI Jing | Gansu Institute for Drug Control, Lanzhou 730070, China | | | NI Lin | Gansu Institute for Drug Control, Lanzhou 730070, China | | | SONG Pingshun | Gansu Institute for Drug Control, Lanzhou 730070, China | | | LI Chenquan | Gansu University of Chinese Medicine, Lanzhou 730030, China | | | GUO Zhaohui | Gansu Institute for Drug Control, Lanzhou 730070, China | 1282664933@qq.com |
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| Abstract
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| [Objective] To establish a rapid method to identify the adulterants of Cirsium japonicum:Carduus nutans and Cirsium leo by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP).[Methods] By comparing the ITS gene sequences,the restriction endonucleases of Carduus nutans and Cirsium leo were screened and the primers were designed. The annealing temperature,cycle number and applicability of different enzymes of PCR reaction were investigated,and the digestion time and substrate amount were optimized. At the same time,the adaptability,specificity and stability of the method were investigated.[Results] When the annealing temperature is 58-60℃ and the number of cycles is 30,a 379 bp DNA band can be amplified from the sample. Substrate is 8 μL. When the digestion temperature was 37℃ and the digestion reaction was 120 min,ZraI enzyme cut two DNA bands of 120 bp and 259 bp from Carduus nutans;DNA substrate is 8 μL. When the digestion temperature was 37℃ and the digestion reaction was 60 min,ApaLI enzyme cut two DNA bands of 126 bp and 253 bp from Cirsium leo.[Conclusion] The PCR-RFLP method established in this study can accurately identify the adulterants of Cirsium japonicum:Carduus nutans and Cirsium leo,avoid the mixing of these herbs,and ensure the safety of clinical medication. |
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