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运用基因芯片技术检测牛、山羊、猪和鸡源性成分 总被引:3,自引:0,他引:3
本研究通过对脊椎动物分子标记基因进行序列分析,最终选择线粒体DNA(mtDNA)16S rRNA基因为目标基因,利用一对通用引物,在该引物扩增区间设计了4条特异性基因芯片检测探针及2条质控探针用于对牛、山羊、猪、鸡等4种动物源性成分进行检测。通过对PCR扩增体系及杂交体系的优化,该检测方法能实现对上述4种动物源性成分同时进行快速、准确地检测,具有很好的特异性,灵敏度均达到1pg,最终建立了这4种动物源性基因芯片检测方法。该基因芯片检测技术将为我国进出口饲料中的动物源性成分的鉴别提供新的检测方法和技术支持。 相似文献
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运用基因导流杂交法在低密度基因芯片平台上检测乙肝病毒DNA(HBV DNA)。设计特异性引物,对HBV基因组DNA中的编码HBV多聚酶蛋白的一段序列进行PCR扩增;根据被扩增片段,设计保守的特异性探针,并将该探针固定在杂交膜上,制备低密度基因芯片:使用导流杂交法将上述扩增产物和低密度基因芯片进行杂交,根据显色反应判断被检测样本有无HBV DNA。基因导流杂交法在低密度基因芯片平台上可以方便、快速、准确地检测乙肝病毒。 相似文献
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目的:制备乙型脑炎病毒(JEV)可视化分型基因芯片。方法:根据JEV的基因组序列,应用生物学软件设计JEV分型引物及探针,制备其可视化分型基因芯片;用生物素标记的引物PCR扩增目的片段,并与固定于玻片上的探针杂交,加入链霉亲和素标记的纳米金,银增强实现可视化;进行特异性、灵敏性及重复性试验。结果:探针特异地与相应的标记目的基因片段杂交,并在芯片上呈现较强的阳性杂交信号;2号探针能特异性检出JEV,3、4号探针可分别对Ⅰ型和Ⅲ型JEV进行分型;芯片对JEV质粒检测的灵敏度达105拷贝/mL;以蓝耳病病毒等5种病毒为对照,芯片只对JEV响应,具有特异性;制备的基因芯片具有批间、批内重复性。结论:制备的基因芯片具有高特异性、灵敏性及重复性,可以快速、准确、高通量地对JEV进行可视化分型检测。 相似文献
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基因芯片技术是以基因序列为分析对象的生物芯片.是技术最成熟、最早进入应用和实现商业化的生物芯片。基因芯片是把大量已知序列探针集成在同一个基片上,经过标记的靶核苷酸序列与芯片特定位点上的探针杂交,通过检测杂交信号,对细胞或组织中大量的基因信息进行检测与分析。1991年Affymetfix公司的Fodor等人应用光刻技术研发了世界上第一张基因芯片。 相似文献
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对于基因表达芯片,特异性探针的选择是探针设计的重要环节,由于基因组序列数据量极大,不可能对每个候选探针都在全序列中进行特异性评价并进行取舍。对此问题,提出了一种采用马尔可夫链概率准则的探针特异性选择方法,即把基因组序列看作马尔可夫链,任何探针序列的互补序列作为它的一个子序列,都具有一定的出现概率,概率越小,越可能具有特异性。据此,选择其中概率最小的N个候选探针,能够大大减少进行特异性评价的探针数量,缩短探针设计的计算时间。对实际数据的测试结果表明,该方法选择的探针具有很高的特异性。 相似文献
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分子生物学及其技术的发展和变革开辟了生态学研究的新途径。分子生物学与生态学交叉的研究,越来越引起人们的重视,于是分子生态学便应运而生了。而分子微生物生态学则为其中的重要分支,它是运用分子的方法和技术,在基因水平上估计种的个体丰度,查明种的变异情况以及探究群落中微生物间相互关系的科学。生物技术的应用,使我们不必培养微生物,而直接通过对环境中的遗传物质的研究来达到目的,它为微生物生态学的研究开辟了新的途径。1用于分子微生物生态学的主要生物技术1.1核酸探针检测技术探针是能与特定核苷酸序列发生特异性互… 相似文献
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建立以Real-time PCR为基础的新型高致病性A(H5N8)亚型禽流感病毒NA基因检测方法。针对2016年6月起频繁暴发的H5N8禽流感疫情,从GenBank和Global Initiative on Sharing All Influenza Data(GISAID)下载2014年以来的H5N8亚型禽流感病毒的NA序列,通过序列比对,在相对保守区域设计适用于实时荧光逆转录聚合酶链式反应(rRT-PCR)的引物和探针。选用28株不同NA亚型的流感病毒进行特异性验证,结果显示本文设计的引物探针组合能够特异性检测高致病性H5N8亚型禽流感病毒的NA基因。灵敏度检测结果显示,本文设计的引物探针组合能检出最低23个拷贝的RNA。本文建立了高致病性H5N8亚型禽流感病毒NA基因特异性荧光定量检测方法,与世界卫生组织(WHO)推荐的A型流感病毒M基因、H5基因检测引物探针的最低检测限一致,可以组合用于H5N8亚型禽流感病毒的检测。 相似文献
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A fast and flexible approach to oligonucleotide probe design for genomes and gene families 总被引:1,自引:0,他引:1
MOTIVATION: With hundreds of completely sequenced microbial genomes available, and advancements in DNA microarray technology, the detection of genes in microbial communities consisting of hundreds of thousands of sequences may be possible. The existing strategies developed for DNA probe design, geared toward identifying specific sequences, are not suitable due to the lack of coverage, flexibility and efficiency necessary for applications in metagenomics. METHODS: ProDesign is a tool developed for the selection of oligonucleotide probes to detect members of gene families present in environmental samples. Gene family-specific probe sequences are generated based on specific and shared words, which are found with the spaced seed hashing algorithm. To detect more sequences, those sharing some common words are re-clustered into new families, then probes specific for the new families are generated. RESULTS: The program is very flexible in that it can be used for designing probes for detecting many genes families simultaneously and specifically in one or more genomes. Neither the length nor the melting temperature of the probes needs to be predefined. We have found that ProDesign provides more flexibility, coverage and speed than other software programs used in the selection of probes for genomic and gene family arrays. AVAILABILITY: ProDesign is licensed free of charge to academic users. ProDesign and Supplementary Material can be obtained by contacting the authors. A web server for ProDesign is available at http://www.uhnresearch.ca/labs/tillier/ProDesign/ProDesign.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. 相似文献
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Use of quantitative real-time PCR (QPCR) with TaqMan probes is increasingly popular in various environmental works to detect and quantify a specific microorganism or a group of target microorganism. Although many aspects of conducting a QPCR assay have become very easy to perform, a proper design of oligonucleotide sequences comprising primers and a probe is still considered as one of the most important aspects of a QPCR application. This work was conducted to design group specific primer and probe sets for the detection of ammonia oxidizing bacteria (AOB) using a real-time PCR with a TaqMan system. The genera Nitrosomonas and Nitrosospira were grouped into five clusters based on similarity of their 16S rRNA gene sequences. Five group-specific AOB primer and probe sets were designed. These sets separately detect four subgroups of Nitrosomonas (Nitrosomonas europaea-, Nitrosococcus mobilis-, Nitrosomonas nitrosa-, and Nitrosomonas cryotolerans-clusters) along with the genus Nitrosospira. Target-group specificity of each primer and probe set was initially investigated by analyzing potential false results in silico, followed by a series of experimental tests for QPCR efficiency and detection limit. In general, each primer and probe set was very specific to the target group and sensitive to detect target DNA as low as two 16S rRNA gene copies per reaction mixture. QPCR efficiency, higher than 93.5%, could be achieved for all primer and probe sets. The primer and probe sets designed in this study can be used to detect and quantify the beta-proteobacterial AOB in biological nitrification processes and various environments. 相似文献
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A sensitive nonisotopic hybridization assay for HIV-1 DNA 总被引:8,自引:0,他引:8
We have developed a microtiter-based sandwich hybridization assay for the detection of low copy number HIV-1 sequences. The assay employs a capture DNA sequence covalently coupled to microtiter wells through linker arms. The detection probe is a biotin-labeled DNA fragment derived from sequences adjacent to the capture sequence. After hybridization in the presence of sample nucleic acid, the detection probe remains bound only if the sample contained complementary sequences spanning the junction between capture and detection probes. The amount of detection probe bound is quantified by incubation with a peroxidase-streptavidin conjugate and a colorimetric peroxidase substrate. This assay has been combined with enzymatic target amplification to achieve sensitive detection of HIV-1 in patient samples. Following amplification of HIV-1 DNA using the polymerase chain reaction technique, a 190-bp product is produced. This product is easily and specifically quantified using the sandwich hybridization assay. The resulting test can detect one HIV-1-infected cell in 10(5) cells or about 30 molecules of HIV-1 DNA. 相似文献
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Background
The detection of enriched DNA or RNA fragments by tiling microarrays has become more and more popular. These microarrays contain a high number of small probes covering genomic loci. However, to achieve high coverage the probe sequences cannot be selected for their hybridization properties. The affinity of the probes towards their targets varies in a sequence-dependent manner. In order to remove this bias a number of approaches have been developed and shown to increase the detection of enriched DNA or RNA fragments. However, these approaches also employ a peak detection algorithm that is different from the one used previously. Thus, it seems possible that the enhancement of detection is due to the peak detection algorithm rather than the sequence-dependent normalization. 相似文献15.
Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction 总被引:9,自引:0,他引:9
Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments. 相似文献
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Erik S. Wright L. Safak Yilmaz Andrew M. Corcoran Hatice E. ?kten Daniel R. Noguera 《Applied and environmental microbiology》2014,80(16):5124-5133
Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu). 相似文献
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Kousoulidou L Männik K Sismani C Zilina O Parkel S Puusepp H Tõnisson N Palta P Remm M Kurg A Patsalis PC 《Nature protocols》2008,3(5):849-865
High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400-600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4-5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype-phenotype correlations and the discovery of new genes. 相似文献