目的: 建立针对真菌类中药材茯苓的聚合酶链式反应(polymerase chain reaction, PCR)鉴定技术,从分子生物学角度出发,对茯苓的真伪进行鉴定。方法: 对经典十六烷基三甲基溴化铵(CTAB)法进行改良,提取样品基因组DNA,以茯苓内转录间隔区(internal transcribed space,ITS)为目的序列,设计特异性引物,优化PCR反应体系,建立茯苓PCR鉴定方法,同时克隆特异性扩增的目的条带,测序鉴定其准确性,最后进行方法学验证。结果: 提取的基因组DNA浓度较高、纯度较好、完整度良好。琼脂糖凝胶电泳结果表明正品茯苓可以在199 bp处出现单一明亮的特异性扩增条带,而伪品不出现扩增条带,引物的特异性为100%,克隆后的目的片段电泳图谱清晰明亮,克隆片段与Genbank数据库中已登记的茯苓物种序列相似性高达100%。在操作人员不知样品真伪的情况对样本检测,目标条带出现位置与预期一致;灵敏度检测结果表明,DNA检测下限可达1 ng·μL-1;挑选3位实验室人员进行茯苓真伪检测,结果相同且和预期一致,建立的茯苓PCR鉴定方法特异性良好,准确性高,重复性好。结论: PCR鉴定技术为茯苓的真伪鉴别提供了简便可靠、特异性良好,并且准确性高的鉴别方法。采用分子克隆技术可以成功保存茯苓标准对照品,测序结果准确可靠。
Objective: To establish PCR identification technology of Poria cocos (Schw.) Wolf, a Chinese medicinal material of the fungus, and identify the authenticity of Poria cocos (Schw.) Wolf from the perspective of molecular biology. Methods: The classic CTAB method was improved and used to extract the genomic DNA from the samples, specific primers were designed with the internal transcription spacer of Poria cocos (Schw.) Wolf as the target sequence, and PCR reaction system was optimized. PCR identification method of Poria cocos (Schw.) Wolf was established. At the same time, the target band specifically amplified was cloned and sequenced to identify its accuracy. Finally, the method was verification. Results: The extracted genomic DNA was of high concentration, good purity, and good integrity. The results of agarose gel electrophoresis showed that the genuine Poria cocos (Schw.) Wolf could appear a single bright specific amplified band at 199 bp, but the counterfeit could not appear amplified band. The specificity of the primer was 100%. It was cloned and the target fragment was clear and bright after the electrophoresis pattern. And the cloned fragment had 100% similarity with sequence of Poria cocos (Schw.) Wolf registered in Genbank database. When the operator didn’t know the authenticity of the samples, the samples were tested, and the position of the target bands were consistent with the expectation. The sensitivity test results showed that the lower limit of DNA detection was up to 1 ng·μL-1. Three laboratory staff were selected for testing the authenticity of Poria cocos (Schw.) Wolf, and the results were the same and consistent with expectations. The established PCR identification method showed good specificity, high accuracy and repeatability. Conclusion: PCR identification technology provides a simple, reliable, specific and highly accurate identification method for the authenticity of Poria cocos (Schw.) Wolf. Its standard reference materials can be successfully stored by using molecular cloning technology and the sequencing results are accurate and reliable.
[1] 田双双, 赵晓梅, 刘勇, 等. 茯苓药材和饮片质量标准研究[J].中国中药杂志, 2020, 45(8):1734
TIAN SS, ZHAO XM, LIU Y, et al. Studies on quality standards of Poria[J].China J Chin Mater Med, 2020, 45(8):1734
[2] WU P, TAN HY, ZHAN JF, et al. Optimization of bioprocess extraction of Poria cocos polysaccharide (PCP) with Aspergillus niger β-glucanase and the evaluation of PCP antioxidant property[J].Molecules, 2020, 25(24):5930
[3] AI JX, WANG XS, GAO LJ, et al. PCR-fingerprint profiles of mitochondrial and genomic DNA extracted from Fetus cervi using different extraction methods[J].Mitochondrial DNA A DNA Mapp Seq Anal, 2017, 28(6):781
[4] 徐岩, 段思琪, 翟英南, 等. 基于Cyt b基因的PCR技术对牛鞭药材的鉴定方法研究[J].中国药房, 2019, 30(24):3408
XU Y, DUAN SQ, ZHAI YN, et al. Study on identification methods of Bullwhip by PCR technology based on Cyt b gene[J].China Pharm, 2019, 30(24):3408
[5] 梁恒兴, 王巍, 段庆梓, 等. 基于PCR的冬虫夏草检测方法研究[J].中药材, 2018, 41(3):546
LIANG HX, WANG W, DUAN QZ, et al. A new methods based on PCR for identification of Cordyceps sinensis[J].Chin Med Mater, 2018, 41(3):546
[6] 杨健, 李靖, 薛维娜, 等. 实时荧光定量PCR法鉴别川贝母掺伪[J].中成药, 2020, 42(5):1262
YANG J, LI J, XUE WN, et al. Identification adulteration in Fritillariae Cirrhosae by real-time fluorescence quantitative PCR method[J].Chin Tradit Pat Med, 2020, 42(5):1262
[7] 赵云冬, 王天添, 陈思秀, 等. 阿胶真伪鉴定方法的建立与应用[J].食品与发酵工业, 2021, 47(12):140
ZHAO YD, WANG TT, CHEN SX, et al. Establishment and application of Corii asini identification method[J].Food Ferment Ind, 2021, 47(12):140
[8] 刘丽, 肖炳燚, 罗晖明, 等. 多重PCR方法同时鉴别人参、西洋参、三七[J].药物分析杂志, 2016, 36(4):668
LIU L, XIAO BY, LUO HM, et al. Identification of Panax ginseng, Panax quinquefolium and Panax notoginseng by multiplex PCR assay[J].Chin J Pharm Anal, 2016, 36(4):668
[9] 刘富艳, 袁媛, 金艳, 等. 多重PCR同时鉴别5种药用海马[J].中国中药杂志, 2018, 43(23):4562
LIU FY, YUAN Y, JIN Y, et al. Multiplex PCR to simultaneous identification of five traditional Chinese medicinal seahorses[J].Chin J Chin Master Med, 2018, 43(23):4562
[10] KACHIPRATH B, JAYANATH G, SOLOMON S, et al. CTAB influenced differential elution of metagenomic DNA from saltpan and marine sediments[J].3 Biotech, 2018, 8(1):44
[11] GUNNELS T, CREWELL M, MCFERRIN J, et al. The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements[J].PloS One, 2020, 15(11):e0236774
[12] HOWARD C, LOCKIE-WILLIAMS C, SLATER A. Applied barcoding: the practicalities of DNA testing for herbals[J].Plants(Basel), 2020, 9(9):1150
[13] 陈思秀, 张馨方, 刘玟妍, 等. 阿胶中动物源性DNA提取方法的改进及驴源性成分鉴定[J].中国药学杂志, 2019, 54(22):1840
CHEN SX, ZHANG XF, LIU WY, et al. Improvement of extraction method of animal-derived DNA from colla corii asini and identification of donkey-derived components[J].Chin Pharm J, 2019, 54(22):1840
[14] 吴文如, 安鑫, 来慧丽, 等. 基于ITS2序列位点特异性PCR鉴别广藿香及其混伪品[J].时珍国医国药, 2019, 30(4):897
WU WR, AN X, LAI HL, et al. Identification of Pogostemon cablin (Blanco) Benth. with the counterfeit products by allele-specific PCR based on ITS2 sequence[J].Lishizhen Med Mater Med Res, 2019, 30(4):897
[15] WANG YS, YUAN GX, ZHANG LH, et al. Establishment of mink heart identification method based on mitochondrial cytochrome b gene and development of its detection kit[J].Mitochondrial DNA A DNA Map Seq Anal, 2019, 30(2):325
[16] 李萧涵, 李明成, 周亭亭, 等. 中药材川贝母标准品特异DNA指纹片段的克隆及鉴定[J].中国药学杂志, 2016, 51(8):616
LI XH, LI MC, ZHOU TT, et al. Cloning and identification of a specific standard DNA fingerprint fragment from Chinese herbal medicine Fritillaria cirrhosa D. Don[J].Chin Pharm J, 2016, 51(8):616
[17] 王巍, 尚柯, 段庆梓, 等. 市售三七粉中三七的DNA检测方法研究[J].时珍国医国药, 2019, 30(6):1399
WANG W, SHANG K, DUAN QZ, et al. Research on DNA detection method of Panax notoginseng in commercial Panax notoginseng powder[J].Lishizhen Med Mater Med Res, 2019, 30(6):1399
[18] 周亭亭, 李明成. 基于DNA指纹鉴定技术的市售川贝母质量考察分析[J].北华大学学报(自然科学版), 2017, 18(6):743
ZHOU TT, LI MC. Quality of commercially available Unibract fritillary Bulb based on DNA fingerprint identification technology[J].J Beihua Univ (Nat Sci), 2017, 18(6):743
