Analysis of the Authenticity of Stone Medicinal Plants by DNA Barcoding

The genus Pyrrosia belongs to the family Polypodiaceae and are medium-sized epiphytic ferns,where the dried leaves of Pyrrosia lingua,Pyrrosia sheareri,Pyrrosia lanceolata,and Pyrrosia calvata are commonly used in medicinal practice.In this study,the authenticity of the collected medicinal plant samples of Shiwei was identified with the help of DNA barcoding technology using the internal transcribed spacer 2(ITS2)as the identifying sequence.The experimental samples were analyzed using the basic local alignment search tool(BLAST)and the authenticity of the samples was further verified with the results of similarity comparison.The results proved that the sequences of the experimentally collected samples of Pyrrosia lingua,Pyrrosia sheareri,Pyrrosia lanceolata,and Pyrrosia calvata had a similarity of more than 97%when compared with the corresponding sequences that were uploaded on the Internet.

PCR Amplification System of DNA Barcoding Genes ITS, ITS2 and rbcL from Xanthium

[Objective] This study aimed to provide reference and reduce the workload for screening standard DNA barcoding genes of plants. [Method] Three DNA barcoding genes ITS, ITS2 and rbcL were amplified from seven Xanthium species under the same PCR condition： PCR amplification was started with initial denaturation at 95 ℃ for 4 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 52 ℃ for 45 s, and extension at 72 ℃ for 45 s; the amplification was completed by holding the reaction mixture at 72 ℃ for 10 min to allow complete extension of PCR, and the PCR products were stored at 4 ℃. [Result] Three DNA barcoding genes ITS, ITS2 and rbcL were all amplified successfully. [Conclusion] This study indicates that PCR amplification conditions for DNA barcoding genes ITS,ITS2 and rbcL in plants may be consistent.

A WATERMARKING ALGORITHM BASED ON PERMUTATION AND 2-D BARCODE

This letter presents a method for digital image watermarking for copyright protection. This technique produces a watermarked image that closely retains the quality of the original host image while concurrently surviving various image processing operations such as lowpass/highpass filtering, lossy JPEG compression, and cropping. This image watermarking algorithm takes full advantage of permutation and 2-D barcode (PDF417). The actual watermark embedding in spatial domain is followed using permutated image for improving the resistance to image cropping. Much higher watermark robustness is obtainable via a simple forward error correction technique, which is the main feature of PDF417 codes. Additional features of this technique include the easy determination of the existence of the watermark and that the watermark verification procedure does not need the original host image. The experimental results demonstrate its effectiveness.

Application of Evolutionary Encryption 2D Barcode Generation Technology in Agricultural Product Quality and Safety Traceability System

Two-dimensional(2D) barcode technology is an electronic tagging technology based on combination of computer and optical technology. It is an important way of information collection and input. 2D barcode technology has been widely used in various fields of logistics,production automation,and e-commerce,but it also has brought about a series of safety problems. Based on evolutionary encryption technology,this paper improved algorithm of traditional 2D barcode generation,to improve forgery- proof performance of 2D barcode. This algorithm is applied to agricultural products quality and safety traceability system and the results show that it is effective.

基于ITS 2序列的肉苁蓉种子DNA条形码鉴定研究

本研究基于ITS 2序列,对肉苁蓉和管花肉苁蓉的种子进行鉴定研究,旨在建立肉苁蓉种子的DNA条形码鉴定方法。通过提取种子的基因组DNA,经PCR扩增和双向测序,获得ITS 2序列。应用MEGA-X软件进行序列比对,计算K 2 P遗传距离,构建系统发育树。结果表明,所有种子样本均可以成功提取DNA和扩增ITS 2序列,种子的DNA分子量为10000 bp左右,扩增产物为500 bp左右。肉苁蓉种子的ITS 2序列长度为414~472 bp,GC含量为54.41%~55.07%;管花肉苁蓉种子的ITS 2序列长度为399~489 bp,GC含量为54.18%~55.14%。肉苁蓉种子和管花肉苁蓉种子的ITS 2序列共有61个差异位点,种内最大遗传距离小于种间最小遗传距离,二者在系统发育树中分别聚为一支。ITS 2序列可以作为肉苁蓉种子的DNA条形码鉴定序列。

栀子栽培品种与近缘种的ITS2序列分析

【目的】栀子属植物是重要的观赏、药用、色素和油用资源植物,有着悠久的栽培历史。由于生长环境的不同,以及长期的人工栽培和选育,使其习性、叶的形状、花的形态、果实的形状及大小等发生诸多变异,使得栀子栽培群体的变异类型丰富,并形成了一些较为稳定的品种类型。开展栀子栽培品种和近缘种核糖体DNA内部转录间隔区2(nrDNA ITS2)序列变异程度、遗传距离和亲缘关系研究,为栀子属植物品种鉴定、遗传育种及多样性保护提供科学依据。【方法】以保存于江西省林业科学院中药资源圃内的栀子18个栽培品种和3个近缘种共38个样品为试验材料,进行聚合酶链式反应(PCR)直接测序法,对样品进行ITS2片段扩增和双向测序,所得序列用Codon Code Aligner v6.0.1软件拼接后,采用Lasergene Edit Seq 11.1软件进行平均碱基组成百分比统计,用MEGA7.0软件进行分析比对,获得序列长度、GC含量以及变异位点,基于K2P模型分析遗传距离,用UPGMA法构建系统聚类树。【结果】序列变异程度分析显示,狭叶栀子和栀子的ITS2序列长度均为347 bp,GC含量为66.86%,大黄栀子序列长度为355 bp,GC含量为67.61%,栀子栽培品种序列长度除‘燃叶’、‘金福水栀’为348 bp外,其他品种序列长度和栀子一致,均为347 bp,栀子栽培品种与近缘种间的平均GC含量相差不明显,GC含量为66.28%~67.15%,其中‘白蟾’GC含量最低,‘雀舌栀子’和‘银边雀舌’GC含量最高。所有样本ITS2序列比对共产生12个变异位点,10个碱基插入,其中8个碱基插入是大黄栀子所独有的,且存在5个特异的变异位点,一部分表型特征有共性的品种之间有共同的变异位点,如植株矮小、叶片小而窄长的品种‘雀舌栀子’和‘银边雀舌’在50 bp处,果实相对较大的品种‘分关1号’、‘金福水栀’和‘金元’在84 bp处等。一些品种具有特异的变异位点,如‘白蟾’(133 bp处)、‘小白蟾’(229 bp处)等。序列遗传距离分析显示,栀子属种间遗传距离普遍小于近缘种和品种间的遗传距离,大于栀子栽培品种之间的遗传距离,所有样本平均遗传距离为0.0055,大黄栀子和‘白蟾’间遗传距离最大,为0.0206。聚类分析结果显示,栀子属乔木树种大黄栀子单独成一支,与栀子属其它种和栀子栽培品种亲缘关系最远,狭叶栀子和野生栀子所有个体总体表现出较近的亲缘关系。【结论】分析结果表明,3种栀子属植物在ITS2序列长度和GC含量上表现出较高的保守性,研究发现的部分品种共同变异位点以及部分品种特异变异位点将对栀子品种鉴定有较大的应用价值。部分聚类结果和以表型数据为基础的数量分类结果类似,但并没有像数量分类结果那样以花重瓣、大果、小果等性状形成明显的系统分枝,研究结果对栀子品种起源、资源保护和种质创新等方面有重要的指导价值。

基于J2EE和XML的食品安全数据系统设计

为了解决大量的食品安全数据和复杂的信息来源严重制约食品安全信息系统发展的问题,以EJB为主要工具、采用XML构架采集复杂的多数据源,提出了一种基于J2EE技术的具有5层结构的食品安全数据平台设计方案.分别从数据结构、组织层次和标准格式三方面为食品信息组织提供支持,并分析了自动识别技术的应用.在保障数据的准确性与稳定性的前提下,该平台可以更好地实现与现有数据和设备资源的兼容,满足食品追踪与溯源的要求.

基于LPC2138的超市收银机系统设计

给出一种基于Philips公司的ARM7 LPC2138微控制器的超市收银管理系统的设计方法。该系统不仅实现商品价目表(PLU),销售日志保存、记录和打印,中英文字符和数字输入等基本功能,还实现了对超市环境参数监测报警、语音播报及语音识别等功能。

白前及其混伪品的ITS2分子鉴定

目的:采用ITS2序列鉴定白前及其易混伪品,为准确鉴别白前和保证临床用药的安全性提供参考。方法:白前与其混伪品的ITS2序列从Gen Bank数据库中获取,然后使用MEGA 6.06软件进行种内、种间序列变异分析、构建邻接系统树(NJ树)并计算遗传距离,运用ITS2数据库相似性搜索引擎预测ITS2二级结构。结果:白前ITS2序列长度在246~270 bp范围内,种内变异少且种间存在明显差异,与所有混伪品种间遗传距离为0.026~0.309。NJ树显示白前独聚一支,另外白前有明显ITS2二级结构特征,可与其混伪品明显区分。结论:ITS2序列能够有效地区分白前与其混伪品,为白前的用药安全提供技术保障。

RNA barcode segments for SARS-CoV-2 identification from HCoVs and SARSr-CoV-2 lineages

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the pathogen responsible for coronavirus disease 2019(COVID-19),continues to evolve,giving rise to more variants and global reinfections.Previous research has demonstrated that barcode segments can effectively and cost-efficiently identify specific species within closely related populations.In this study,we designed and tested RNA barcode segments based on genetic evolutionary relationships to facilitate the efficient and accurate identification of SARS-CoV-2 from extensive virus samples,including human coronaviruses(HCoVs)and SARSr-CoV-2 lineages.Nucleotide sequences sourced from NCBI and GISAID were meticulously selected and curated to construct training sets,encompassing 1733 complete genome sequences of HCoVs and SARSr-CoV-2 lineages.Through genetic-level species testing,we validated the accuracy and reliability of the barcode segments for identifying SARS-CoV-2.Subsequently,75 main and subordinate species-specific barcode segments for SARS-CoV-2,located in ORF1ab,S,E,ORF7a,and N coding sequences,were intercepted and screened based on single-nucleotide polymorphism sites and weighted scores.Post-testing,these segments exhibited high recall rates(nearly 100%),specificity(almost 30%at the nucleotide level),and precision(100%)performance on identification.They were eventually visualized using one and two-dimensional combined barcodes and deposited in an online database(http://virusbarcodedatabase.top/).The successful integration of barcoding technology in SARS-CoV-2 identification provides valuable insights for future studies involving complete genome sequence polymorphism analysis.Moreover,this cost-effective and efficient identification approach also provides valuable reference for future research endeavors related to virus surveillance.

基于DNA条形码(ITS2)的中药材防风与其混伪品的鉴定

目的通过ITS2序列分析,对防风及其易混伪品进行鉴定,确保用药安全。方法从Gen Bank数据库下载中药材防风与其混伪品的DNA条形码ITS2序列,利用MEGA 6.06软件进行比对分析,计算GC含量及变异位点、种内、种间遗传距离,并构建NJ系统发育树,应用ITS2数据库网站预测其ITS2二级结构。结果序列分析与遗传距离结果显示,防风与其混伪品存在明显差异,基于ITS2序列的NJ树及其ITS2二级结构均可以将防风与其混伪品进行区分。结论 ITS2能够准确鉴定防风及其易混伪品,可用于中药材的快速鉴别。

基于ITS 2序列的15种淫羊藿属药用植物分类鉴定研究

为探讨内转录间隔区2(ITS2)序列及其二级结构在15种淫羊藿属(Epimedium)中的鉴定效率。利用试剂盒提取叶片总DNA,对ITS2序列进行PCR扩展与测序,计算K2P遗传距离,构建系统发育树,预测其二级结构。结果表明,ITS2序列在15种淫羊藿属植物中的通用性好,扩增成功率与测序成功率均为100%。从K2P种间和种内遗传距离看,ITS2在淫羊藿属中的种间变异较小,部分种类种内遗传距离大于种间遗传距离,鉴定成功率约为60%。采用比对法(Blast)分析,ITS2鉴定效率为75%。由ITS2系统进化树可知,淫羊藿属与鬼臼属各为一支,易于分开。除薄叶淫羊藿、竹山淫羊藿、巫山淫羊藿外,其余种类均能较好的进行聚类;除粗毛淫羊藿、保靖淫羊藿外其余均不能与NCBI下载到的序列进行较好聚类。研究表明,15种淫羊藿属植物ITS2序列在ML系统进化树中具较好的鉴别能力,但淫羊藿属各样本种内与种间遗传较为混乱,遗传距离复杂,ITS2二级结构形态结构亦较为相似,将各种间进行区分存在一定难度,不适合单独作为该属条形码使用。

利用ITS2序列识别盾蚧科昆虫

[目的]盾蚧科隶属于半翅目蚧总科,大多数种类是危害很严重的经济害虫。由于其外部形态特征简单,导致识别该类群物种相对困难。利用DNA条形码为解决这个问题提供有效的方法。[方法]共采集了5个省47个样本,属于7属11个种,并从GeneBank下载了4属5个物种的序列。采用distance-based方法和tree-based方法对ITS2基因片段是否可以作为盾蚧科DNA条形码的补充做了分析。[结果]虽然ITS2没有Barcode gap存在,然而所有的分支都表现出了明显的单系群,表现出较高的物种识别能力。同时,Best Match(BM)和Best closed match(BCM)也表现出了超过90%的识别能力。[结论]这些结果证实ITS2在盾蚧科物种的识别有效性方面具有较高的应用价值。

ITS2序列作为青风藤DNA条形码的研究

采用DNA试剂盒法提取采自不同地区的青风藤及其类似植物的基因组DNA,设计通用ITS2引物进行PCR扩增和测序,运用生物信息学软件对序列进行分析,从而对青风藤及其类似植物进行分子鉴定。结果表明：产地为陕西、贵州、湖北的青风藤样品序列长度分别为380~437、429~436、434~438 bp,GC含量在54.2%~55.7%,平均GC含量约为55%。青风藤及其类似植物的ITS2序列存在较大差异,且平均K2P遗传距离明显大于青风藤的种内平均K2P遗传距离。这说明内转录间隔区2（ITS2）作为DNA条形码可准确地鉴别青风藤与其他类似植物,为中药材的鉴别提供了新途径。

Identification of Lablab Semen Album by DNA Barcode Technology

[Objectives] To identify ITS2 barcode of Lablab Semen Album and its adulterants,and provide a new method for the identification of Lablab Semen Album. [Methods] The ITS2 sequence was amplified by PCR and sequenced bi-directionally. After splicing by Codon Code Aligner,the data were processed with the aid MEGA software to construct the cluster dendrogram( neighbor-joining,NJ tree). [Results]The ITS2 sequence of Lablab Semen Album had length of 218 bp; the constructed cluster dendrogram indicated that all species were monophyletic and could be distinguished from other species. [Conclusions] The ITS2 barcode can be used for rapid identification of Lablab Semen Album and its adulterants and this experiment further verified that DNA barcode technology is effective in identification of traditional Chinese medicines.

基于DNA条形码和PCR-RFLP技术的进口紫草ITS2序列特征研究

目的:基于DNA条形码和PCR-RFLP技术研究进口紫草ITS2序列的特征,为市场紫草药材和饮片的质量控制与真伪鉴别提供参考依据。方法:选用ITS2区域作为对进口紫草和紫草对照药材进行比较、鉴定的DNA条形码序列,并基于DNA条形码和PCR-RFLP技术比较不同来源进口紫草的ITS2序列与紫草对照药材的异同。结果:39份进口紫草样品经限制性内切酶AluI酶切后,其产物的琼脂糖凝胶电泳检测结果显示,仅DH3在500 bp左右有条带,而在100~300 bp无条带,其余样品均在100~300 bp有2条或3条明显条带;进口紫草样品与紫草对照药材的ITS2序列进行比对,其中样品DH3与紫草对照药材的碱基差异最多,有15个碱基差异,样品F2与紫草对照药材的ITS2序列一致,其他进口紫草样品与紫草对照药材的碱基差异为1~9个碱基;从聚类结果中可以看出进口紫草样品DH3与其他进口紫草样品和紫草对照药材均明显区分,独自为一枝,而与紫草对照药材共同聚为一枝且支持率≥50%的样品共有14个。结论:选用ITS2区域,基于DNA条形码和PCR-RFLP技术,比较了进口紫草与紫草对照药材ITS2序列的异同,为紫草药材及饮片的有效鉴定提供参考依据,为紫草药材的市场监管提供有力保障。

基于RNA条形码技术对新型冠状病毒的鉴定

旨在设计一种通过RNA条形码片段对SARS-CoV-2进行更快、更准确鉴定的技术。该技术基于NCBI数据库对所有Beta-CoV属以及7种HCoVs的序列进行筛选并构建序列库。通过生物信息学方法对序列库进行遗传多样性分析、遗传距离分析以及系统发育树的构建,从而测试条形码片段在不同情况下对鉴定SARS-CoV-2的准确性与稳定性。基于SNP位点对序列进行剪裁并利用NCBI的Blast功能将剪裁的片段进行打分,得到鉴别效果最优的条形码片段并可视化。结果表明,RNA条形码片段可以准确地将SARS-CoV-2从序列库内包含的所有毒株中鉴定出来,而通过Blast打分以及P值检验最终得到的2条条形码片段(分别位于ORF1ab和S基因编码区)对鉴定SARS-CoV-2具有很好的稳定性。RNA条形码技术不仅有利于探索SARS-CoV-2全基因组序列多态性与物种特异性遗传标记的关系,还有利于为SARS-CoV-2的鉴定技术提供新思路。

Identification of medicinal plants within the Apocynaceae family using ITS2 and psbA-trnH barcodes

To ensure the safety of medications,it is vital to accurately authenticate species of the Apocynaceae family,which is rich in poisonous medicinal plants.We identified Apocynaceae species by using nuclear internal transcribed spacer 2(ITS2)and psb Atrn H based on experimental data.The identification ability of ITS2 and psb A-trn H was assessed using specific genetic divergence,BLAST1,and neighbor-joining trees.For DNA barcoding,ITS2 and psb A-trn H regions of 122 plant samples of 31 species from 19 genera in the Apocynaceae family were amplified.The PCR amplification for ITS2 and psb A-trn H sequences was 100%.The sequencing success rates for ITS2 and psb A-trn H sequences were 81%and 61%,respectively.Additional data involved 53 sequences of the ITS2 region and 38 sequences of the psb A-trn H region were downloaded from Gen Bank.Moreover,the analysis showed that the interspecific divergence of Apocynaceae species was greater than its intra-specific variations.The results indicated that,using the BLAST1 method,ITS2 showed a high identification efficiency of 97%and 100%of the samples at the species and genus levels,respectively,via BLAST1,and psb A-trn H successfully identified 95%and 100%of the samples at the species and genus levels,respectively.The barcode combination of ITS2/psb A-trn H successfully identified 98%and 100%of samples at the species and genus levels,respectively.Subsequently,the neighbor joining tree method also showed that barcode ITS2 and psb A-trn H could distinguish among the species within the Apocynaceae family.ITS2 is a core barcode and psb A-trn H is a supplementary barcode for identifying species in the Apocynaceae family.These results will help to improve DNA barcoding reference databases for herbal drugs and other herbal raw materials.

基于杜娟属植物的DNA条形码序列筛选

我国的杜鹃属有毒植物约在60种以上,如羊踯躅等药用植物具有强毒付作用,而目前杜娟属的分类十分复杂。DNA barcoding技术是一种新的物种鉴定技术,COI序列已成功应用于动物的鉴定,但是目前国际上还没有找到一个通用序列能鉴定所有植物物种。本文对杜娟属257个物种ITS、ITS2、matK、PsbA-trnH序列比较,运用Wilcoxon秩和检验比较不同序列的变异性,然后用Taxon DNA软件评估这四个序列的barcoding gap。筛选适合于杜娟属的DNA条形码序列。分析结果表明:matK和psbA-trnH并不适合于杜娟属的DNAbarcoding鉴定。ITS2和ITS序列都可以有效的对杜娟属植物进行DNA barcoding鉴定,但ITS在物种中的扩增效率较低,因此推荐将ITS2序列作为一个潜在的杜娟属植物DNA barcoding鉴定候选序列。

基于二维条码PDF417的数字图像水印算法

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