Identification of Aucklandia Costus, Dolomiaea Souliei and Inula Racemosa by PCR-RFLP Based on ITS Sequence
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摘要:
目的 利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)技术, 建立快速鉴别木香、川木香和藏木香的方法。 方法 通过比对木香、川木香和藏木香3个物种ITS基因序列, 筛选限制性内切酶并设计鉴别引物。优化PCR扩增和酶切反应条件, 并进行方法学考察和统计学分析。 结果 退火温度为60 ℃、循环数为30时, 能检出500~750 bp单一DNA条带; 底物为10 μL、酶切时间为15 min时, BsaHⅠ酶可将木香、川木香及藏木香在一定范围内切割成大小不一的DNA条带, 方法学和统计学结果较好。 结论 建立的PCR-RFLP方法能够准确地鉴别木香、川木香及藏木香3种药材, 避免此3类药材的混用, 保证临床用药安全。 Abstract:OBJECTIVE To establish a rapid method for distinguishing Aucklandia costus, Dolomiaea Souliei and Inula racemosa by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). METHODS The ITS gene sequences of Aucklandia costus, Dolomiaea souliei and Inula racemosa were compared, restriction enzymes were screened and identification primers were designed. The PCR amplification and digestion reaction conditions were optimized, and the methodological examinations and statistical analysis were carried out. RESULTS When the annealing temperature was 60 ℃ and the cycle number was 30, a single DNA band of 500~750 bp could be detected. When the substrate was 10 μL and the digestion time was 15 min, BsaHI enzyme could cut Aucklandia costus, Dolomiaea souliei and Inula racemosa into DNA bands of different sizes in a certain range. The results of methodology and statistics were good. CONCLUSION The PCR-RFLP method established in this study can accurately identify the three medicinal materials of Aucklandia costus, Dolomiaea souliei and Inula racemosa, avoid the mixed use of these three medicinal materials, and ensure the safety of the clinical medication. -
Key words:
- Aucklandia costus /
- Dolomiaea souliei /
- Inula racemosa /
- PCR-RFLP /
- ITS sequence
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图 1 川木香、木香和藏木香ITS序列图
Figure 1. ITS sequence diagram of Dolomiaea souliei, Aucklandia costus and Inula racemosa samples
图 2 川木香、木香和藏木香NJ进化树聚类图
Figure 2. NJ evolutionary tree clustering of Dolomiaea souliei, Aucklandia costus and Inula racemosa
图 3 不同条件PCR扩增电泳图
注: M.D2000 maker; K.空白对照; 1, 4.川木香; 8, 15.木香; 20.藏木香; A.退火温度; B.循环次数; C.DNA聚合酶类型
Figure 3. Electrophoresis of PCR amplification under different conditions
图 4 不同条件酶切电泳图
注: MI.DNA Marker I；K.空白对照；A.酶切时间; B.底物量
Figure 4. Enzyme digestion electrophoresis under different conditions
图 5 PCR扩增和酶切反应稳定性考察电泳图
注: M.D2000 maker; MI.DNA Marker I; K.空白对照; CMX.川木香对照药材; 1~7, 22~26.川木香; MX.木香对照药材; 8~15, 27~31.木香; ZMX.藏木香对照药材; 16~21, 32~36.藏木香; A~B.PCR扩增稳定性考察; C~D.酶切反应稳定性考察
Figure 5. Electrophoretic diagram of stability investigation of PCR amplification and enzyme digestion reaction
图 6 适用性考察电泳结果图
注: MI.DNA Marker I; K.空白对照; CMX.川木香对照药材; ZMX.藏木香对照药材; MX.木香对照药材; 1~4.川木香掺藏木香; 5~8.木香掺藏木香
Figure 6. Electrophoretic results of applicability test
图 7 Agilent Surecycler型PCR仪扩增结果适用性图
注: M.D2000 maker; K.空白对照; CMX.川木香对照药材; ZMX.藏木香对照药材; MX.木香对照药材; 1~7.川木香; 8~15.木香; 16~21.藏木香
Figure 7. Applicability of amplification results of Agilent Surecycler PCR instrument
图 8 酶切结果适用性图
注: MI.DNA Marker I; K.空白对照; CMX.川木香对照药材; ZMX.藏木香对照药材; MX.木香对照药材; 1~7.川木香; 8~15.木香; 16~21.藏木香
Figure 8. Applicability of enzyme digestion results
图 9 川木香、木香和藏木香的AOD值比较
注：CMX.川木香; ZMX.藏木香; MX.木香；与CMX组比较，***P < 0.001, ****P < 0.000 1;与MX组比较，#P < 0.05。
Figure 9. Optical density analysis of Dolomiaea souliei, Aucklandia costus and Inula racemosa
表 1 木香、川木香和藏木香样品来源信息表
Table 1. Source information of Dolomiaea souliei, Aucklandia costus and Inula racemosa samples
序号 名称 拉丁学名 产地/来源 采样日期 1~3 川木香 Dolomiaea souliei (Franch.) 四川阿坝州 2020.12 4~7 川木香 Dolomiaea souliei (Franch.) 四川凉山州 2020.12 8~11 木香 Aucklandia costus Falc. 陕西商洛市 2020.10 12~13 木香 Aucklandia costus Falc. 云南迪庆州 2020.10 14~15 木香 Aucklandia costus Falc. 甘肃华亭市 2021.01 16 藏木香 Inula racemosa Hook.f. 西藏林芝市 2021.02 17~19 藏木香 Inula racemosa Hook.f. 甘肃卓尼县 2020.10 20 藏木香 Inula racemosa Hook.f. 甘肃合作市 2020.10 21 藏木香 Inula racemosa Hook.f. 宁夏隆德县 2020.11 22~26 川木香 Dolomiaea souliei (Franch.) 成都荷花池药材市场 2021.11 27~31 木香 Aucklandia costus Falc. 兰州黄河药材市场 2021.11 32~36 藏木香 Inula racemosa Hook.f. 兰州黄河药材市场 2021.11 
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