Detection of Genetically Modified Crops by Combination of Multiplex PCR and Low-density DNA Microarray

Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified （GM） soybean. Seventeen capture probes （PCR products） and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean （GTS40-3-2）, maize （MonS10, Nk603, GA21）, canola （T45, MS1/RF1）, and rice （SCK） in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean （Roundup Ready GTS40-3-2） and canola （MS1/RF1）. Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS 1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

Prospects of DNA microarray application in management of chronic obstructive pulmonary disease:A systematic review

Chronic obstructive pulmonary disease(COPD)is incurable chronic disease which kills 3.3 million each year worldwide.Number of global cases of COPD is steadily rising alongside with life expectancy,disproportionally hitting middle-income countries like Russia and China,in such conditions,new approaches to the COPD management are desperately needed.DNA microarray technology is a powerful genomic tool that has the potential to uncover underlying COPD biological alteration and brings up revolutionized treatment option to clinicians.We executed systematic review studies of studies published in last 10 years regarding DNA microarray application in COPD management,with complacence to PRISMA criteria and using PubMed and Medline data bases as data source.Out of 920 identified papers,39 were included in the final analysis.We concluded that Genome-wide expression profiling using DNA microarray technology has great potential in enhancing COPD management.Current studied proofed this method is reliable and possesses many potential applications such as individual at risk of COPD development recognition,early diagnosis of disease,COPD phenotype identification,exacerbation prediction,personalized treatment optioning and prospect of oncogenesis evaluation in patients with COPD.Despite all the proofed benefits of this technology,researchers are still in the early stage of exploring it’s potential.Therefore,large clinical trials are still needed to set up standard for DNA microarray techniques usage implementation in COPD management guidelines,subsequently giving opportunity to clinicians for controlling or even eliminating COPD entirely.

DNA微阵列芯片法在非结核分枝杆菌菌种鉴定的应用探讨

目的为临床工作中非结核分枝杆菌(NTM)的感染和防治提供科学依据。方法收集疑似结核分枝杆菌感染患者标本(包括呼吸道标本痰液、灌洗液以及非呼吸道标本无菌体液)共1089例,采用DNA微阵列芯片法进行检测,分析分枝杆菌菌种的分布特点。结果1089份标本中检出结核分枝杆菌878份(80.62%),NTM211份(19.38%)。其中非结核分枝杆菌菌种分布有5种,胞内分枝杆菌153例(72.51%),鸟分枝杆菌28例(13.27%),龟脓分枝杆菌复合群19例(9%),堪萨斯分枝杆菌10例(4.74%),耻垢分枝杆菌1例(0.48%)。非结核分枝杆菌感染人员分布男性127例(60.19%),女性84例(39.81%),年龄分布以老年为主,60岁以上103例(48.82%),男性71例(68.93%),女性32例(31.07%)。结论疑似结核分枝杆菌患者检出NTM共有5种,排名前三的NTM种类是胞内分枝杆菌、鸟分枝杆菌和龟脓分枝杆菌复合群,感染人群以老年男性为主。

用DNA microarray快速检测淋球菌耐喹诺酮类药物基因突变

目的 研究 DNA m icroarray的制备及其检测淋球菌耐喹诺酮类药物基因突变的准确性。方法 根据淋球菌药敏及测序结果分别对淋球菌 gyr A和 par C基因的序列设计特异引物和探针并制作 DNA m icroarray。对淋球菌临床拭子进行 PCR扩增并荧光标记包含 gyr A和 par C基因的目的 DNA片段 ,与芯片杂交 ,同时以测序法进行双盲淋球菌耐喹诺酮类药物基因突变的检测。结果 87份泌尿生殖道试子全部可用 DNA m icroarray检测出来 ,芯片检测结果与药敏结果符合率为 10 0 % ,与测序结果符合率为 97.7%。结论 用 DNA microarray来检测淋球菌 gyr A和 par C基因突变快速、特异性高和灵敏度高 。

甲基化芯片技术检测粪便DNA甲基化在海南地区少数民族人群大肠癌筛查中的应用

目的 分析甲基化芯片技术检测粪便DNA甲基化在海南地区少数民族人群大肠癌筛查中的应用价值。方法 选取2020年6月—2022年6月海南省人民医院就诊的大肠癌高危少数民族人群102例。另选取同期该院招募的30例健康志愿者作为对照组。留取所有患者在结肠镜检查前自然排出或服用泻药后的第一段粪便,通过甲基化芯片技术检测粪便DNA甲基化。根据肠镜病理检查结果,将102例患者分为大肠癌组、腺瘤组、增生性息肉组。对比4组粪便DNA中VAV3、IKZF1、RIMS1基因甲基化状态。将肠镜病理检查结果作为金标准,评估粪便DNA中VAV3、IKZF1、RIMS1基因联合检测大肠癌、腺瘤的诊断效能。结果 大肠癌组VAV3、IKZF1、RIMS1基因单独及联合检测甲基化率高于腺瘤组、增生性息肉组、对照组(P <0.05)。腺瘤组与增生性息肉组VAV3、IKZF1、RIMS1基因单独及联合检测甲基化率比较,差异无统计学意义(P>0.05)。VAV3、IKZF1、RIMS1基因联合检测大肠癌的特异性、敏感性、阴性预测值、阳性预测值、准确率最高,分别为100.00%、92.31%、92.59%、100.00%和96.08%。粪便DNA中VAV3、IKZF1、RIMS1基因联合检测腺瘤的特异性、敏感性、阴性预测值、阳性预测值、准确率最高,分别为97.30%、71.43%、90.00%、90.91%和90.20%。结论 海南地区少数民族人群大肠癌患者粪便DNA中VAV3、IKZF1、RIMS1基因表现出较高的甲基化水平,且3个基因联合检测可提升大肠癌、腺瘤的诊断效能。

Subtype Identification of Avian Influenza Virus on DNA Microarray

We have developed a rapid microarray-based assay for the reliable detection of H5, H7 and H9 subtypes of avian influenza virus （AIV）. The strains used in the experiment were A/Goose/Guangdong/1/96 （H5N1）, A/African starling/983/79 （H7N1） and A/Turkey/Wiscosin/1/66 （H9N2）. The capture DNAs clones which encoding approximate 500-bp avian influenza virus gene fragments obtained by RT-PCR, were spotted on a slide-bound microarray. Cy5-labeled fluorescent cDNAs, which generated from virus RNA during reverse transcription were hybridized to these capture DNAs. These capture DNAs contained multiple fragments of the hemagglutinin and matrix protein genes of AIV respectively, for subtyping and typing AIV. The arrays were scanned to determine the probe binding sites. The hybridization pattern agreed approximately with the known grid location of each target. The results show that DNA microarray technology provides a useful diagnostic method for AIV.

Experimental genomics:The application of DNA microarrays in cellular and molecular biology studies

The genome sequence information in combination with DNA microarrays promises to revolutionize the way of cellu-lar and molecular biological research by allowing complex mixtures of RNA and DNA to interrogated in a parallel and quantita-tive fashion. DNA microarrays can be used to measure levels of gene expression for tens of thousands of gene simultane-ously and take advantage of all available sequence information for experimental design and data interpretation in pursuit of biological understanding. Recent progress in experimental genomics allows DNA microarrays not simply to provide a cata-logue of all the genes and information about their function, but to understand how the components work together to comprise functioning cells and organisms. This brief review gives a survey of DNA microarrays technology and its applications in ge-nome and gene function analysis, gene expression studies, biological signal and defense system, cell cycle regulation, mechanism of transcriptional regulation, proteomics, and the functionality of food component.

Polyurethane Molecular Stamps for the in situ Synthesis of DNA Microarray

Fabrication of polyurethane molecular stamps (PU stamps) based on polypropylene glycol (PPG) and toluene diisocyanate (TDI), using 3, 3-dichloro-4, 4-methylenedianiline (MOCA) as the crosslinker, is reported. It was shown from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, guaranteeing the well distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays after hybridization confirmed polyurethane is an excellent material for molecular stamps when transferring polar chemicals and conducting reactions on interfaces by stamping.

Development and evaluation of a DNA microarray assay for the simultaneous detection of nine harmful algal species in ship ballast and seaport waters

Rapid,high-throughput and reliable methods are urgently required to accurately detect and monitor harmful algae,which are responsible for algal blooms,such as red and green tides. In this study,we successfully developed a multiplex PCR-based DNA microarray method capable of detecting nine harmful algal species simultaneously,namely A lexandrium tamarense,Gyrodinium instriatum,Heterosigma akashiwo,Karenia mikimotoi,Prorocentrum donghaiense,Prorocentrum minimum,Ulva compressa,Ulva ohnoi and Ulva prolifera. This method achieved a limit of detection(LOD) of 0.5 ng of genomic DNA(orders of magnitude of the deci-nanogram range) in the tested algae cultures. Altogether,230 field samples from ship ballast waters and seaport waters were used to evaluate the DNA microarray. The clinical sensitivity and specificity of the DNA microarray assay in detecting field samples were 96.4% and 90.9%,respectively,relative to conventional morphological methods. This indicated that this high-throughput,automatic,and specific method is well suited for the detection of algae in water samples.

Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

The double-stranded DNA (dsDNA) probe contains two different protein binding sites. One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme. The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

应用DNA微阵列芯片检测青年肺结核患者结核菌耐药性的研究

目的:以结核菌比例法药敏试验为标准,分析DNA微阵列芯片(以下简称DNA微阵列)在聊城市青年肺结核患者痰标本中进行结核分枝杆菌异烟肼和利福平耐药性检测的效能。方法:选取聊城市2019年10月-2021年9月确诊的18~45岁的青年肺结核患者,对其中痰涂片抗酸杆菌阳性的215例患者,通过DNA微阵列和比例法药敏对痰标本进行异烟肼和利福平的耐药性检测,分析DNA微阵列对异烟肼、利福平、耐多药结核诊断的敏感性、特异性、阳性预测值和阴性预测值,并与比例法进行一致性比较。结果:DNA微阵列检测耐多药结核的敏感度和特异度分别为71.4%和99.0%,阳性预测值和阴性预测值分别为83.3%和98.0%,Kappa=0.75,说明两种方法在耐多药结核检测中有高度一致性。DNA微阵列检测异烟肼耐药性的敏感度和特异度分别为76.5%和97.2%,阳性预测值和阴性预测值分别为83.9%和95.7%,Kappa=0.76,说明两种方法在异烟肼耐药性检测中有高度一致性。DNA微阵列检测利福平耐药性的敏感度和特异度为82.1%和98.9%,阳性预测值和阴性预测值分别为92.0%和97.4%,Kappa=0.85,说明两种方法在利福平耐药性检测中基本一致。结论:DNA微阵列适用于青年结核患者快速筛查和耐药性检测,为聊城青年结核病的防控能提供有力支持。

用氨基修饰的载玻片制作cDNA微阵列

cDNA微阵列已在基因差异表达、寻找新基因等研究方面获得广泛应用 ,但有关cDNA微阵列的制作 ,目前多采用多聚赖氨酸修饰的载玻片为探针固定载体 ,固定效果较差 .用氨基硅烷处理的载玻片为载体制作cDNA微阵列 ,然后考察其固定效率、检测灵敏度、稳定性、实用性等指标 .结果表明 ,用氨基硅烷处理的载玻片具有比多聚赖氨酸更令人满意的核酸固定效率、检测灵敏度 ,且稳定实用 .因此 ,用氨基硅烷修饰的载玻片为探针固定载体制作cDNA微阵列较为理想 .

16SrDNA基因芯片检测临床常见感染性细菌

目的 提高细菌和临床微生物检测的速度和准确性 ,建立含有 2 0种细菌探针在内的感染性细菌检测用基因芯片模型。方法 使用 16SrDNA克隆探针和合成的寡核苷酸探针两种 ,利用点样仪制成基因芯片。细菌DNA经过 16SrDNA通用引物扩增后与芯片上的探针杂交 ,然后用荧光扫描仪检测信号。结果 基因芯片能够用于细菌检测 ,克隆探针具有广泛和灵敏的特点 ,但是交叉反应明显 ;寡核苷酸探针具有较高的特异性 ,但是灵敏度稍差。结论 cDNA探针和寡核苷酸探针结合或设计几个不同的探针来指向同一株细菌很可能是将来基因芯片的检测方向。

鼠疫耶尔森菌DNA标识序列的鉴定及其应用研究

目的 确定鼠疫耶尔森菌DNA标识序列 ,以用于鼠疫耶尔森菌的快速检测和鉴定。方法 通过芯片比较基因组杂交和PCR验证 ,鉴定鼠疫耶尔森菌DNA标识序列 ,针对标识序列设计特定的PCR引物。结果 鼠疫耶尔森菌基因组中存在 3个区段 ,共 2 8条基因 ,均是鼠疫耶尔森菌DNA标识序列。针对其中 3条基因设计PCR引物 ,能特异性扩增出鼠疫耶尔森菌 ,与来自其他非鼠疫耶尔森菌的DNA无交叉反应。结论 鼠疫耶尔森菌存在DNA标识序列 ,并能用于鼠疫耶尔森菌的快速检测与鉴定。

鼠疫耶尔森菌全基因组DNA芯片的研制及用于比较基因组学分析

目的 研制鼠疫耶尔森菌全基因组DNA芯片 ,并将其用于比较基因组分析。方法 挑选出 4 0 0 5条鼠疫耶尔森菌基因 ,PCR扩增各基因 ,以纯化的PCR产物点样制备芯片 ,采用双色荧光杂交策略 ,进行芯片比较基因组杂交。结果 设计了若干质控杂交组合 ,芯片杂交结果与全基因组测序结果完全一致。

分子印章法DNA芯片的合成(Ⅰ)——PDMS印章的固定与收缩研究

用MicronXYZScope考察了用不同方法处理的玻璃片对膜厚为 0 .6～ 1 .0mm ,阵点高度为 1 5μm的聚二甲基硅氧烷 (PDMS)印章的粘附固定情况以及对印章收缩的影响 .经硅烷化的玻璃片与PDMS印章结合牢固 ,虽然印章表面阵点的面积收缩为 0 2 86% ,但由于分子间的强相互作用力以及分子之间的铆合作用 ,使其印章的线收缩为 0 0 4 0 3% .这一结论保证了寡核苷酸合成过程中一套印章在合成载片位点上的多次准确定位以及印章多次压印偶联反应不发生错位 ,亦即为分子印章法DNA芯片的正确合成提供了依据 .

鲉形目鱼类DNA条形码分析及鲉科DNA条形码电子芯片建立

为建立鲉形目内各物种的快速鉴别方法，本研究扩增了我国47个铀形目物种并下载了GenBank上收录的鲉形目共计23科85属233种873条细胞色素C氧化酶I基因（COI）序列，分析该基因结构特性。结果表明，鲉形目DNA条形码基因的碱基变异中，不变位点508个，占总位点数的82．1％；转换位点70个，颠换位点41个，分别占总位点数的11．3％和6．6％；种内遗传距离为0-0．019，平均遗传距离为0．003±0．0034；属内种间遗传距离为0．003-0．258，平均值为0．086±0．038；基于233个物种的COI序列构建的系统进化树显示，227个物种（97．4％）能聚为独立分支，且具有较高支持度。在此基础上以鲇科11属39种鱼类的DNA条形码为例，筛选出25个种的37个特异性探针，以此探针对鲉科鱼类进行物种快速鉴定成功率为64．1％，研究结果表明DNA条形码芯片用于鲉科的鉴定具有一定的可行性。

DNA芯片技术研究进展

DNA芯片技术是近年来发展迅速的生物高技术 .其基本过程是采用寡核苷酸原位合成或显微打印手段 ,将大量探针片段有序地固化于支持物如硅芯片的表面 ,然后与扩增、标记的生物样品杂交 ,通过对杂交信号的检测分析 ,即可得出样品的遗传信息 .该技术不仅可以对遗传信息进行定性、定量分析 ,而且扩展到基因组研究和基因诊断等方面的应用 .尽管目前在硬件和软件上还面临一些困难 ,但其发展和应用的前景广阔 .

奶牛乳房炎主要致病菌16S rDNA基因芯片检测方法的建立

本研究建立了快速检测奶牛乳房炎主要致病菌的基因芯片方法,试验以奶牛乳房炎4种主要致病菌的16S rDNA基因作为基因芯片检测的靶片段,设计和筛选通用性引物和特异性探针,对通用引物进行荧光标记、PCR扩增、芯片杂交和信号扫描分析,根据杂交信号强度和聚类分析结果,判定奶牛乳房炎感染的致病菌种类。实验结果显示:以16S rDNA为靶基因检测奶牛乳房炎主要致病菌(无乳链球菌、肺炎克雷伯氏菌、奇异变形杆菌、金黄色葡萄球菌)的基因芯片方法,可以快速、特异、准确地对这4种试验菌株进行检测和鉴定。该检测方法的建立将为流行病学调查、食品卫生监督、乳房炎的防控等提供快速、有效的检测方法,具有良好的市场应用前景。

用酿酒酵母全基因组DNA芯片研究盐酸小檗碱的药理作用机制

目的 :利用自己构建的酿酒酵母全基因组DNA芯片 ,分析盐酸小檗碱处理酵母细胞后对其整个基因表达谱的影响 ,在基因水平上分析盐酸小檗碱的药理机制。方法 :用盐酸小檗碱 (10 0 μg/ml)处理酵母细胞 90min后 ,抽提细胞的RNA进行反转录荧光标记cDNA ,与DNA芯片进行杂交。用激光扫描仪检测杂交信号。结果 :共有 5 45个基因〔占全部基因 (6 183个 )的 9%〕受到诱导 ,38个基因 (占全部基因的 0 6 % )受到抑制。被诱导的基因包括与细胞生长、蛋白质合成及应激反应相关的基因。受抑制的基因包括与氧化磷酸化过程相关的基因和好氧基因。与金属离子平衡 ,特别是与铁离子和铜离子平衡有关的基因表达有较大变化。结论 :处理 90min后 ,酵母细胞在转录水平上表现出对盐酸小檗碱处理的适应性。盐酸小檗碱的药理作用可能和引起细胞内的氧浓度变化及氧化还原反应产生的自由基有关。细胞容易对盐酸小檗碱产生抗药性的原因是由于与细胞抗药性相关的基因受到了明显的诱导。