目的 探讨利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)技术鉴别西洋参的方法。方法 针对西洋参的CYP450基因设计特异性引物,应用RT-LAMP(real time-LAMP)、琼脂糖凝胶电泳和可视化检测3种方法对LAMP检测方法进行优化,并对其灵敏性和特异性进行了评价。结果 LAMP反应体的最佳孵育时间为60 min,最佳反应温度为65 ℃,最适内外引物比为8∶1。反应体系的灵敏度达到0.5 ng·μL-1,特异性较好,能与近缘植物人参相区分开。将人参掺入西洋参模拟市场掺假行为,其检出率达到20%。对市售22份西洋参产品进行检测,其阳性率达到81%。结论 建立了一种快速、简便、特异、灵敏的西洋参LAMP检测方法,为人参和西洋参的快速检测和规范中药市场提供有力工具。
Abstract
OBJECTIVE To evaluate a loop-mediated isothermal amplification (LAMP) assay for detection of Panax quinuefolium. METHODS Based on the CYP450 gene of Panax quinuefolium, specific primers were designed. The LAMP assay was optimized by real time-LAMP (RT-LAMP), agarose gel electrophoresis and visual inspection, and its sensitivity and specificity were evaluated. RESULTS The optimal reaction time of LAMP reaction was 60 min, the optimal reaction temperature was 65 ℃, and the optimal ratio of internal and external primers was 8∶1. The sensitivity of the reaction system reached 0.5 ng·μL-1, and the specificity was good, which could be distinguished from the related plant Panax ginseng. The adulteration behavior in the market was simulated by adding Panax ginseng into Panax quinuefolium, and the detection rate reached 20%. The positive rate of 22 Panax quinuefolium products in the market was 81%.CONCLUSION These results demonstrate that we develop a rapid, simple, specific and sensitive LAMP assay for Panax quinuefolium, which provide a powerful tool for the rapid identification of Panax ginseng and Panax quinuefolium.
关键词
中药鉴定 /
环介导等温扩增技术 /
西洋参 /
人参
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Key words
authentication /
LAMP /
Panax quinquefolium /
Panax ginseng
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中图分类号：
R282
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参考文献
[1] CHEN C, CHIOU W, ZHANG J. Comparison of the pharmacological effects of Panax ginseng and Panax quinquefolium [J]. Acta Pharmacol Sin(中国药理学报),2008, 29(9): 1103-1108.
[2] JUNG J, KIM K H, YANG K, et al. Practical application of DNA markers for high-throughput authentication of Panax ginseng and Panax quinquefolius from commercial ginseng products [J]. J Ginseng Res, 2014, 38(2): 123-129.
[3] ZHENG Y L, ZHANG C X, LI X G, et al. Chemical composition of rhizome and root from Panax ginseng C. A. Mey. and Panax quinquefolius L. [J]. Chin Pharm J (中国药学杂志), 1989, 24(6): 332-334,379.
[4] LIU Y L, LEI R, WANG X L, et al. Researchon quality control methods of Panax ginseng, Panax quinquefolius, Panax notoginseng and related proprietary Chinese medicines based on Q-marker [J]. Chin Pharm J (中国药学杂志), 2019, 54(17): 1402-1410.
[5] LIM W, MUDGE K, WESTON L. Utilization of RAPD markers to assess genetic diversity of wild populations of north american ginseng (Panax quinquefolium) [J]. Planta Med, 2007, 73(1): 71-76.
[6] FUSHUMI H, KOMATSU K, ISOBE M, et al. Application of PCR-RFLP and MASA Analyses on 18S ribosomal RNA gene sequence for the identification of three Ginseng drugs [J]. Biol Pharm Bull, 1997, 20(7): 765-769.
[7] JEE J W, BANG K H, KIM Y C, et al. CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer) [J]. Mol Biolrep, 2012, 39(1): 729-739.
[8] KIM N K, CHOI H I, AHN I O, et al. EST-SSR marker sets for practical authentication of all nine registered ginseng cultivars in Korea [J]. J Ginseng Res, 2012, 36(3): 298-307.
[9] WANG H, LI G, KWON W S, et al. Development of EST intron-targeting SNP markers for Panax ginseng and their application to cultivar authentication [J]. Int J Mol Sci, 2016, 17(6): 884.
[10] SHARMA G, TEWARI R, DHATWALIA S K, et al. A loop-mediated isothermal amplification assay for the diagnosis of pulmonary tuberculosis [J]. Lett Appl Microbiol, 2019, 68(3): 219-225.
[11] KREITLOW A, BECKER A, SCHOTTE U, et al. Evaluation of different target genes for the detection of Salmonella spp. by loop-mediated isothermal amplification (LAMP) [J]. Lett Appl Microbiol, 2020, 72(4): 420-426.
[12] AHN S J, BAEK Y H, LLOREN K K S, et al. Correction to: Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loopmediated isothermal amplification (RT-LAMP) diagnostic platform [J]. BMC Infect Dis, 2020, 20(1): 965.
[13] CHAUDHARY A A, HEMANT, MOHSIN M, et al. Application of loop-mediated isothermal amplification (LAMP)-based technology for authentication of Catharanthus roseus (L.) G. Don [J]. Protoplasma, 2012, 249(2): 417-422.
[14] LI M, WONG Y L, JIANG L L, et al. Application of novel loop-mediated isothermal amplification (LAMP) for rapid authentication of the herbal tea ingredient hedyotisdiffusa willd [J]. Food Chem, 2013, 141(3): 2522-2525.
[15] SASAKI Y, NAGUMO S. Rapid identification of curcuma longa and C. aromatica by LAMP [J]. Biol Pharm Bull, 2007, 30(11): 2229-2230.
[16] WANG Y, LI X, LIN Y, et al. Structural variation, functional differentiation, and activity correlation of the cytochrome P450 gene superfamily revealed in ginseng [J]. Plant Genome, 2018, 11(3): 170106.
[17] HAN J Y, HWANG H S, CHOI S W, et al. Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in panax ginseng [J]. Plant Cell Physiol, 2012, 53(9): 1535-1545.
[18] YONGKIETTRAKUL S, KAMPEERA J, CHAREANCHIM W, et al. Simple detection of single nucleotide polymorphism in plasmodium falciparum by SNP-LAMP assay combined with lateral flow dipstick [J]. Parasitol Int, 2017, 66(1): 964-971.
[19] LI G, CUI Y, WANG H, et al. Molecular differentiation of russian wild ginseng using mitochondrial nad7 intron 3 region [J]. J Ginseng Res, 2017,41(3): 326-329.
[20] LI J, XIONG C, LIU Y, et al. Loop-mediated isothermal amplification (LAMP): Emergence as an alternative technology for herbal medicine identification [J]. Front Plant Sci, 2016,7: 1956.
[21] WANG R B, TIAN H L, WANG H T, et al. Development of indel markers for molecular authentication of Panax ginseng and P. quinquefolius [J]. China J Chin Mater Med(中国中药杂志), 2018, 43(7): 1441-1445.
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脚注
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基金
吉林省科技发展计划项目资助(20190304108YY);大学生创新项目资助(S202110201058)
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