Allele-specific primers for diagnostic PCR authentication of Dendrobium officinale

Based on rDNA ITS sequences of D. officinale and the other 37 species of Dendrobium, a pair of allele-specific diagnostic primers, TP-JB01S and TP-JB01X, were designed to authenticate D. officinale from the other species. Before the diagnostic PCR, the primer pair, P1 and P2, for amplifying the whole ITS region was used to validate template DNA and to obtain the appropriate template DNA for the diagnostic PCR. Diagnostic PCRs were performed using the diagnostic primers with the total DNAs of the original plants as a template. When the annealing temperature was raised to 66 degrees C, only the template DNA of D. officinale could be amplified whereas the diagnostic PCRs of the other Dendrobium species were all negative. The diagnostic PCRs have been repeated many times and have played an important role in authenticating the stems of D. officinale in China. Compared with the authentication method by sequencing DNA fragments, the allele-specific diagnostic PCR is not only simpler and time-saving but also practical and effective.     

Molecular authentication of the traditional Chinese medicinal plant Euphorbia pekinensis

The ITS regions of Euphorbia pekinensis and six other Euphorbia species used as adulterants of E. pekinensis were sequenced to differentiate them. The sequences are identical among the individuals in the seven species studies. Diversity in DNA sequences among various species was found ranging from 8.3% to 43.8% in ITS1 and 7.6% to 36.6% in ITS2 region. Furthermore, based on the divergent ITS regions, species-specific primers, JDJp 1 and JDJp 2, were designed in the polymorphic regions of E. pekinensis to distinguish it from adulterants. These ITS-derived primers amplified a 281-bp-specific DNA fragment from E. pekinensis. No amplified product was observed using DNA of six adulterants.     

Authentication of Chinese crude drug, Gecko, by allele-specific diagnostic PCR.

Based on the sequences of the mitochondrial 12S rRNA gene fragment of 17 samples from Gekkonidae, Salamandridae, Agamidae and Hynobiidae, respectively, a pair of allele-specific primers was designed for differentiating the Chinese medicinal material Gecko from its adulterants by PCR. The results of amplification with the primers indicate that amplicons from the templates of Gekko gecko were clearly revealed by agarose gel electrophoresis, whereas no evident amplicons were found from other species. The primers were employed to identify crude drug samples from different sources. Among a total of 9 samples, 3 were diagnosed as genuine Gecko. This result is consistent with morphological identification and DNA sequence analyses.     

紫苏属药用植物的rDNA ITS区SNP分子标记与位点特异性PCR鉴别

目的分析单种属——紫苏属各变种间rDNA ITS区的序列以及存在的单核苷酸多态性(SNP)现象，设计出位点特异性PCR引物，用于紫苏属各变种间的分子标记鉴别。方法对紫苏属各变种多个体的rDNA ITS区全序列进行了准确测定，运用Clustral X 1.8，MEGA 3.0进行排序并进行SNP分析，从而设计出鉴别各变种的等位基因位点特异性PCR鉴别引物。结果紫苏属各变种(紫苏、白苏、鸡冠苏和耳齿紫苏等)的rDNA ITS区全序列共有615～618 bp的长度，ITS1为233～235 bp，5.8S为179 bp，ITS2为203～204 bp，GC含量为61.5%～61.9%。从rDNA ITS区碱基变异的整体情况来看，紫苏属各变种间不仅在非编码的转录间隔区ITS1和ITS2内存在非编码区单核苷酸多态性(ncSNP)，而且在保守的5.8S编码区内也存在3个位点的单核苷酸多态性，即编码区SNP(cSNP)，所有的SNP均只具2等位多态性。5.8S区cSNP的出现与产生该变异的变种出现的显著形态差异关联。本文还利用这些SNP位点设计出了鉴别紫苏属各变种的位点特异性PCR引物，无需测序即可对紫苏属的原植物及“苏子”、“苏叶”等药材进行有效准确的分子鉴别。结论紫苏属药用植物rDNA ITS区存在的SNP可用作紫苏属各变种鉴别的分子标记。     

球花石斛的位点特异性PCR鉴别研究

为建立球花石斛的DNA分子标记鉴别方法，本文根据测定及GenBank上登录的109种共计164个石斛样本的rDNA ITS序列，设计了特异性鉴别引物QH-JB1 和QH-JB2，并对球花石斛进行了位点特异性PCR鉴别研究。结果表明，当复性条件为63.5 ℃，1 min时，只有球花石斛的模板DNA能被扩增出约300 bp的阳性扩增带，而其他种石斛均为阴性。证明用本法鉴别球花石斛简便、省时，也适用于干燥球花石斛药材的鉴别，具有广泛的应用前景。     

齿瓣石斛的位点特异性PCR鉴别

目的设计出齿瓣石斛的位点特异性鉴别引物,仅通过PCR就能完成对齿瓣石斛真伪进行准确鉴别。方法根据齿瓣石斛及其他枫斗类、黄草类石斛的rDNA ITS序列数据库,设计了齿瓣石斛的位点特异性PCR鉴别引物JB-Chiban-01S和JB-Chiban-01X。然后,对38种石斛属植物模板DNA进行了PCR扩增,阳性者即为齿瓣石斛正品。结果当退火温度设定为58℃时,只有齿瓣石斛的模板DNA能被扩增出来,而其他的37种石斛属植物均为阴性。该鉴别反应重复性好,已在鉴别齿瓣石斛时发挥重要作用。结论运用位点特异性鉴别引物能成功地对齿瓣石斛进行PCR鉴别,与DNA测序鉴别方法相比,位点特异性PCR具有高效、准确、简便、省时等优点。     

Authentication of stems of Dendrobium officinale by rDNA ITS region sequences

The rDNA ITS regions of five Dendrobium species were sequenced. Each Dendrobium species was found to have a unique sequence in the ITS region, so that they could be easily distinguished at the DNA level. The aligned 644 bp of the ITS region includes 235 bp ITS1, 163 bp 5.8S, and 246 bp ITS2. One hundred and eighty-nine sites are variable. The sequences of D. officinale could be easily distinguished from the other four adulterant species according to the sequence variation at 11 sites, 7 in ITS1, 1 in 5.8S, and 3 in ITS2. These could be used as molecular characters to distinguish the stems of D. officinale from the adulterants.     

Differentiation of medicinal Codonopsis species from adulterants by polymerase chain reaction-restriction fragment length polymorphism

DNA sequence analysis of rDNA internal transcribed spacer (ITS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were exploited for their applications in differentiating medicinal species Codonopsis pilosula, C. tangshen, C. modesta, and C. nervosa var. macrantha, from two related adulterants Campanumoea javania and Platycodon grandiflorus. The data demonstrated that the rDNA ITSI and ITSII sequences of the four Codonopsis are highly homologous but not identical, and are significantly different from those of the two adulterants. The sequence difference allows effective and reliable differentiation of Codonopsis from the adulterants by PCR-RFLP.     

花椒及其混淆品的rDNA ITS区序列分析与鉴别

目的研究不同居群的花椒及其混淆品的rDNA ITS区碱基序列的特征及其差异，为花椒的鉴别提供可靠的分子标记。方法运用PCR产物直接测序和克隆测序法对甘肃、陕西、四川、河北等7个花椒居群及3个混淆种的rDNA ITS区(包括ITS1，5.8S，ITS2)碱基序列进行序列测定。结果首次报道花椒ITS区的碱基序列，序列总长度为619-620 bp，长度变异较少，与混淆种长度仅相差4 bp。花椒各居群中，rDNA ITS区碱基序列有15个变异位点、12个信息位点、3个特异性识别位点。与混淆品间的碱基差异则较为显著，多达71个变异位点，有4个花椒特异性识别位点。结论依据花椒ITS区的序列特征可准确鉴别各居群的花椒及其混淆品；亲缘关系密切的花椒居群在地理位置上也非常靠近；rDNA ITS序列特征可作为花椒种内和种间鉴别的有效分子标记。     

Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA

Herba Dendrobii (Shihu) is a commonly used Chinese medicine derived from the stem of several orchid species belonging to the genus Dendrobium. It is rather expensive and adulteration is frequent. Proper authentication of the medicinal species is necessary to protect consumers and support conservation measures. DNA sequences of the internal transcribed spacer 2 (ITS 2) of 16 Dendrobium species were shown to be significantly different from one another by an average of 12.4% and from non-orchids and Pholidota (an adulterant of Shihu) by 29.8% and 18.8%, respectively. The intra-specific variation among the Dendrobium species studied was only about 1%. Therefore, ITS 2 regions could be adopted as a molecular marker for differentiating medicinal Dendrobium species from one another and also from non-orchids and adulterants.     

Molecular authentication of the animal crude drug Sailonggu (bone of Myospalax baileyi)

Two pairs of allele-specific diagnostic primers (SL1L/SL1H and SL2L/SL2H) for distinguishing the Chinese crude drug Sailonggu (bone of plateau zokor, Myospalax baileyi) from its substitutes were designed based on complete sequences of mitochondrial 12S rRNA and cytochrome b genes of the original animals of Myospalacinae, bamboo rat Rhizomys sinensis and black lipped pika Ochotona curzoniae. Total DNA was extracted from crude drug samples and original animals. Allele-specific diagnostic PCRs were performed using these primers with the total DNA as a template annealing at 65 degrees C. Positive amplifications were obtained from all DNA templates of Sailonggu and M. baileyi, whereas negative amplifications resulted from those of other zokors, the bamboo rat and black lipped pika. These results indicate that Sailonggu samples can be definitely distinguished from their substitutes by diagnostic PCR, and no incorrect discrimination was found under the same reaction conditions. Each of the two diagnostic primer pairs can be used to distinguish crude drug Sailonggu from its substitutes or adulterants. The three Sailonggu samples studied were diagnosed as genuine Sailonggu. In addition, the results of sequence alignment and phylogenetic analysis are congruent with that of the allele-specific diagnostic PCR.     

基于matK和ITS2及二级结构对药材香薷及其混伪品的鉴别研究

目的 快速、准确鉴别药材香薷及其混伪品，保障香薷的药材质量和用药安全。方法 收集石香薷、江香薷和香薷植物材料分别进行matK和ITS2序列的扩增与测序，测序结果经Codon Code Aligner软件校对，同时从GenBank下载石香薷、江香斋及其易混品种海州香薷、香薷、密花香薷、牛至等物种的matK和ITS序列。其中，ITS序列经隐马尔可夫模型去除两端的5.8S和28S序列，共得到16个物种的ITS2序列50条；经Clustal软件校对共获得9个物种的matK序列28条。通过Mega7.0软件分析matK和ITS2序列，计算所有物种种内和种间遗传距离，构建邻接法(neighbor joining，NJ)聚类树，通过ITS2 Database预测ITS2二级结构，采用4Sale软件比对二级结构，通过ProfDistS软件构建基于联合ITS2一级序列及其二级结构的剖面邻接(profile neighbor-joining，PNJ)系统发育树。结果 基于matK和ITS2序列的遗传距离均表明香薷正品与其各种混伪品之间存在明显barcoding gap。NJ和PNJ进化树的拓扑关系一致，可以区分药材香薷及其混伪品。香薷的ITS2二级结构与其各混伪品具有显著差异。结论 建议matK和ITS2序列均可以作为鉴别香薷与其混伪品的DNA条形码，ITS2二级结构信息的加入可丰富鉴定结果，为香薷药材的准确鉴别、香薷属与石荠苎属植物的科学分类提供参考。     

Use of the potential DNA barcode ITS2 to identify herbal materials

A potential DNA barcode, ITS2, was studied to discriminate herbal materials to confirm their identities and ensure their safe application in pharmaceuticals. Here, a total of 4385 samples of 2431 species were collected, and these samples are from 61 commonly used herbs and their closely related species or adulterants. Based on assessments of the extent of genetic divergence, the DNA barcoding gap and the ability for species discrimination, our results suggest that ITS2 is a powerful tool for distinguishing herbs. For the first dataset including 61 herbs, ITS2 correctly identified 100 % of them. For the second dataset containing 51 herbs and their 2382 closely related species, ITS2 could discriminate correctly 48 herbs from their closely related species. For the third dataset comprising 34 herbs and their 111 adulterants, ITS2 could distinguish successfully all the herbs from their adulterants. In conclusion, the ITS2 region is an efficient marker for the authentication of herbal materials, and our study will accelerate the process of the application of the DNA barcoding technique in differentiating herbs.     

PCR-based sensitive detection of medicinal fungi Hericium species from ribosomal internal transcribed spacer (ITS) sequences

Based on phylogenetic analysis of rDNA internal transcribed spacer (ITS) sequences, a pair of specific primers were designed for differentiating the Chinese traditional medicine Hericium species from other mushrooms by PCR. PCR was performed, with total DNAs as a template at an annealing temperature of 52-57 degrees C. Positive amplification was obtained from H. erinaceus with all DNA templates from different resources, but not from other related species. The result indicated that H. erinaceus could be clearly distinguished from other fungi by detection PCR, and no incorrect discrimination was found under the same reaction conditions. The primers were also successfully employed to identify H. erinaceus with different tissue types.     

Molecular authentication of the ethnomedicinal plant Sabia parviflora and its adulterants by DNA barcoding technique

Sabia parviflora Wall. ex Roxb. is a traditional herb widely used by Chinese people, especially by the Buyi ethnic group which resides in Guizhou and Yunnan provinces. According to the Chinese Ethnic Pharmacopeia, the species is commonly used for soothing the liver and for the treatment of icteric hepatitis, hemostasis, and inflammation. However, due to the similar morphological characters of Sabia species and higher market demands, there are many substitutes and adulterants of S. parviflora. In this study, the differential identification of 6 Sabia species and 7 adulterants were investigated through DNA sequence analysis of three candidate DNA barcodes (trnH-psbA, rbcL-α, matK). Based on sequence alignments, we concluded that not only the trnH-psbA spacer sequence can distinguish S. parviflora from other Sabia species, but the matK + rbcL-α sequences also can differentiate it from the substitutes and adulterants. The classification tree of all samples based on rbcL-α sequences indicated that the rbcL region can identify samples into a family/genus level. Our results suggest that the three candidate barcodes can be used for the identification of S. parviflora and to distinguish it from common substitutes or adulterants.     

ITS2+psbA-trnH复合序列鉴定徐长卿、白薇和白前

目的：建立以ITS2+psbA-trnH复合序列鉴定徐长卿、白薇和白前及其同属近缘混伪品的DNA条形码鉴定方法。方法：搜集徐长卿、白薇、白前及其近源混伪品,采用改良的CTAB法提取DNA,通过实验分别获得ITS2和psbA-trnH序列,将同一样本的ITS2序列与psbA-trnH序列整合得到43条ITS2+psbA-trnH复合序列,从GenBank数据库中下载来源于同一样本的ITS2序列与psbA-trnH序列整合得到14条ITS2+psbA-trnH 网上复合序列,用MEGA 6.05软件分析徐长卿、白薇、白前及其近源混伪品的复合序列变异位点,计算种内、种间的K2P距离,并构建NJ系统聚类树。结果：徐长卿、白薇和白前药材ITS2+psbA-trnH复合序列比对后产生17个变异位点;徐长卿、白薇和白前基源物种种内最大K2P距离均小于其与混伪品的种间最小K2P距离;系统NJ树能准确将徐长卿白薇和白前及其近缘混伪品。结论：该研究建立了应用ITS2+psbA-trnH复合序列鉴定徐长卿、白薇、白前及其近缘混伪品的DNA条形码鉴定方法。     

Molecular authentication of Dendrobium loddigesii Rolfe by Amplification Refractory Mutation System (ARMS)

As a widely used medicinal plant, Dendrobium loddigesii Rolfe is always a possible target for fraudulent labeling. The identification of D. loddigesii is generally difficult from its morphological and chemical appearance only. In order to develop a convenient and efficient identification method for D. loddigesii, six pairs of diagnostic ARMS primers were designed based on nrDNA ITS sequences of D. loddigesii and eleven adulterants. The results showed that one diagnostic primer pair (FJB-04-forward, FJB-04-reverse) could be used to authenticate D. loddigesii by generating a fragment of 353 bp at annealing temperatures from 48 degrees C to 62 degrees C while the other diagnostic primer pair (FJB-03-forward, FJB-03-reverse) took on the same effect at annealing temperatures from 49 degrees C to 55 degrees C. This points out the potential of ARMS analysis for authentication of D. loddigesii.     

安罗替尼联合AP方案治疗晚期非小细胞肺癌的临床研究

目的 基于线粒体cytochrome coxidase I（CO I）和16 S rRNA基因开发DNA双重条形码鉴定方法,由此验证并补充形态鉴定,以期建立一种准确、有效地鉴定地龙及混淆品原动物的方法。方法 对收集到的66份样品依据形态特征进行初步鉴定,使用优化后的引物同时扩增CO I和16 S rRNA序列。优化一步法双重PCR实验条件。用MEGA 5.1计算地龙及其混淆品的种内、种间遗传距离,基于K2P模型构建NJ树。结果 CO I和16 S rRNA双重DNA条形码鉴定与形态鉴定结合,可准确鉴别地龙及混淆品原动物。结论 形态鉴定是分子鉴定的基础,分子鉴定是形态鉴定的有力补充。二者结合可最大程度地实现地龙及其混淆品原动物的准确鉴定。DNA双重条形码鉴定方法也可为地龙药材鉴定以及其他动物药材的分子鉴定提供参考。     

冬虫夏草的分子鉴定方法

目的 建立专属性强的冬虫夏草分子鉴定方法.方法 通过在ITS1区设计冬虫夏草的特异性引物,并进行不同条件的优化试验,建立冬虫夏草的酶联免疫吸附法(PCR)鉴别体系.结果 建立的酶联免疫吸附法(PCR)体系对冬虫夏草有164bp条带的扩增,而伪品无扩增现象.结论 所建立的PCR体系能够准确、快速的鉴别冬虫夏草及其伪品.     

通关藤及其常见混伪品的ITS2条形码鉴别研究

目的:采用ITS2序列分析,对通关藤及其混淆品进行鉴别,确保用药安全。方法:从GenBank下载通关藤序列,使用MEGA5.0软件对测序序列及下载序列进行对比分析,统计变异位点、计算遗传距离、构建样品NJ树,运用NCBI Blast进行物种鉴定,并与性状鉴别结果进行核对。结果:ITS2序列分析与遗传距离计算结果显示各通关藤样品间有明显差异,NJ树聚类结果能明显区分通关藤正品与伪品。结论:ITS2可准确鉴别通关藤及其伪品,可用于中药材真伪的快速鉴别,应用前景广阔。