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1.
Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43+/-16 to 95+/-16%. The average amount of total mtDNA was 3800+/-1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging. 相似文献
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热启动PCR快速制备地高辛标记探针 总被引:7,自引:0,他引:7
介绍了一种在热启动PCR中,以Dig-11-dUTP部分代替dTTP,从少量基因组DNA中快速制备大量的地高辛标记的探针的方法,此探针灵敏度达0.03pg,并只和相关的DNA特异杂交. 相似文献
3.
目的评价基于磁珠法的荧光定量PCR检测HBV-DNA的临床应用。方法选用磁珠法定量检测试剂和煮沸法定量检测试剂,对一系列临床患者血清标本进行检测,比较两种试剂检出率的差异;通过浓度为1×108的样本的梯度稀释结果考察两者的灵敏度和线性范围。结果 68例临床样本中,磁珠法试剂检测的阳性率为69.12%,煮沸法试剂检测的阳性率为32.35%(P0.05);两种试剂对103IU/m L阳性样本检测结果:y=0.913x-0.261,r=0.919;磁珠法线性范围3.9×(101~108),灵敏度39 IU/m L,煮沸法线性范围2.4×(102~107),灵敏度240 IU/m L。结论磁珠法核酸提取试剂线性范围宽,灵敏度高,临床检出率明显高于煮沸法,对于高浓度和低浓度样本都能准确的定值,适合于乙肝治疗后监测与体检筛查。 相似文献
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The DNA-binding mode of antitumor and antiviral agents has been evaluated by electrochemiluminescence (ECL) of tris(1,10-phenanthroline)-ruthenium complex (Ru(phen)(3)(2+)) in the presence of oxalate ion in pH 7.3 Tris buffer solution. An emission of Ru(phen)(3)(2+) was observed repeatedly with a voltage above 1000mV subjected to a potential sweep from 0 to 1250mV. The addition of lambdaDNA into the solution containing 1 micro M of Ru(phen)(3)(2+) caused the decrease in the ECL intensity, which became half at a DNA concentration of 20 micro M. This is due to the binding of Delta-type of Ru(phen)(3)(2+) with DNA in the major groove of DNA. When the various concentrations of the drug were added to the solution containing 1& micro M Ru(phen)(3)(2+), the ECL intensity was not affected by the concentration of the drug in the absence of DNA. In the presence of DNA (10 micro M), however, two ECL emission patterns were observed when the concentration of the drug was varied. The pattern that the ECL intensity increased with increasing the drug concentration was observed for cisplatin, daunomycin, and DC92-B. This may have resulted from the DNA binding of the drug with a major groove site, where Ru(phen)(3)(2+) should bind. Ru(phen)(3)(2+) nonbinding to DNA might exist in the bulk solution and exhibits ECL emission. The drug exhibiting the drug-concentration-dependent ECL is classified as a drug with a major groove binding character. The addition of drugs, such as mitomycin C and duocarmycin SA, did not cause a change in the ECL intensity even in the presence of DNA. This result indicates that these drugs bind to DNA with minor groove binding. Since similar trends were observed for actinomycin D, distamycin A, doxorubicin, and chromomycin A3; these drugs are also considered as minor groove binding agents. All these results demonstrate that the DNA-binding mode of the drug can be evaluated easily by utilizing the ECL of Ru(phen)(3)(2+), which is used as the sensing probe. 相似文献
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为了优选快速、 灵敏、 特异的家蚕微孢子虫Nosema bombycis分子检测方法和DNA抽提方法, 本文通过对家蚕微孢子虫TaqMan探针荧光定量PCR检测方法和SYBR Green荧光定量PCR检测方法的建立以及反应体系优化, 并与普通PCR方法进行比较; 再采用4种不同DNA抽提方法分别对PCR和实时荧光定量PCR方法检测家蚕微孢子虫悬浮液的效果评价。结果显示: 不经过DNA抽提, 直接将家蚕微孢子虫发芽液进行PCR反应的效果优于其他方法, 检测灵敏度由高到低依次为直接法、 酚/氯仿抽提法、 动物组织DNA试剂盒抽提法和植物组织DNA试剂盒抽提法; TaqMan探针法检测家蚕微孢子虫发芽液的灵敏度和SYBR Green法相近, 达到微孢子102个/mL, 两者均优于普通PCR方法。实验表明, 直接采用发芽液结合荧光定量PCR方法检测家蚕微孢子虫最为简便、 快速、 灵敏。该研究结果将有助于提高家蚕微粒子病监控技术和检疫能力, 对家蚕微粒子病的检疫和防治具有积极意义。 相似文献
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Danièle Chevrier Michel Yvan Popoff Michel Pierre Dion DanielHermant Jean-Luc Guesdon 《FEMS immunology and medical microbiology》1995,10(3-4):245-252
Abstract A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella . One pair of oligonucleotide primers was designed to amplify a 93-bp fragment of a gene required for the invasion of HeLa cells by Salmonella ser Typhi strain Ty2. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. The described combination of microplate sandwich hybridisation and PCR seems to be a suitable method for rapid detection of Salmonella subspecies I. It only requires a thermal cycler and a conventional microtiter reader, and can be readily done on a large scale. 相似文献
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A simple method of DNA extraction from soil for detection of composite transgenic plants by PCR 总被引:2,自引:1,他引:2
Dieter Ernst Evi Kiefer Alain Drouet Heinrich Sandermann 《Plant Molecular Biology Reporter》1996,14(2):143-148
This new and simple method of DNA extraction from composite soil allows the isolation of plant DNA with high efficiency, quality
and reproductivity. The method is based on a simple CaCl2-precipitation step and requires no additional purification steps to eliminate humic acids. The extracted DNA was obtained
in sufficient purity and quantity to allow direct detection of transgenes by PCR. Furthermore, the simple procedure allows
the assay of many samples at the same time. 相似文献
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目的 通过对PCR引物、反应条件及样本处理条件的摸索,建立一种不经过分离培养和DNA提取纯化步骤,直接用乳酸杆菌特异性引物快速定性、半定量妇女阴道分泌物标本中乳酸杆菌的方法.方法 设计乳酸杆菌属特异性引物;对样本处理方法和PCR反应条件进行摸索;比较用PCR法定量阴道分泌物中乳酸杆菌的结果和稀释滴种法定量结果的相关性.结果 (1)用浓度为2%的Tritonx-100处理阴道标本后可在220 bp的位置得到最佳的特异性扩增区带;(2)将MgCl2在PCR反应体系中的终浓度调节至2.5 mmol/L时,可以产生最强的特异性反应扩增带;(3)用PCR法分析阴道分泌物所得菌数与培养所得菌数之间明显相关(P<0.05);用PCR法分析阴道分泌物所得灰度值与培养所得菌数之间明显相关(P<0.05).结论 将阴道分泌物标本经过简单的离心、洗涤处理后,直接应用PCR法,就可以快速地定性和半定量妇女阴道分泌物标本中的乳酸杆菌. 相似文献
10.
Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction 总被引:10,自引:0,他引:10
López-Andreo M Lugo L Garrido-Pertierra A Prieto MI Puyet A 《Analytical biochemistry》2005,339(1):73-82
Six TaqMan real-time polymerase chain reaction (PCR) systems using minor groove binding (MGB) probes have been developed for the detection quantitation of bovine, porcine, lamb, chicken, turkey, and ostrich DNA in complex samples. Species-specific amplification was achieved by combining only two fluorogenic probes and 10 oligonucleotide primers targeting mitochondrial sequences, decreasing the cost of the assay significantly. The limits of detection ranged from 0.03 to 0.80 pg of template DNA. Analysis of experimental mixtures containing two to four different species showed the suitability of the assay for detection of more than 1% of pork, chicken, or turkey and of more than 5% of cattle or lamb. The quantitation accuracy in samples containing 10-100% of beef or pork DNA was close to 90%. The system is complemented with one additional TaqMan MGB detector based on consensus sequence segments of the nuclear 18S ribosomal RNA gene. A method to evaluate the presence of unknown eukaryotic DNA in a mixture, where data derived from the species-specific detection are compared with the experimental values obtained from the general 18S detector, is presented. This method allows the validation of the quantitative measurements, providing an internal control of the total content of PCR-amplifiable DNA in the sample. The system was tested on DNA mixtures containing different shares of up to four different species and on DNA extracted from processed commercial food samples. 相似文献
11.
Shepherd Mervyn Henry Robert 《Molecular breeding : new strategies in plant improvement》1998,4(6):509-517
DNA melting curves of genotype-specific PCR fragments were used to differentiate between species and amongst varieties of cereals. Melting curves were generated by ramping the temperature of PCR fragments through their dissociation temperature in the presence of a double-stranded DNA binding dye. Genotypes were discriminated by differences in the position and shape of the melting curve which is a function of the fragment's sequence, length and GC content. Amplification of 5S ribosomal RNA genes generated species-specific fragments for six of the major cereal crops. Of the 15 possible pairwise comparisons, 13 distinctions could be reliably made using melting curve position data. Wheat varieties were identified by the melting profiles of PCR products generated using microsatellite primers. DNA melting curve analysis was conveniently coupled with capillary-PCR using a LightCycler instrument to provide a rapid method of genotyping in cereals. 相似文献
12.
目的探讨双重荧光定量PCR技术的优化条件,建立基于TaqMan探针技术荧光定量法检测同时检测解脲支原体和巨细胞病毒的新方法。方法分别采用普通定性PCR扩增母婴垂直传播常见的病原体(解脲支原体和巨细胞病毒)测序鉴定,然后分别采用TaqMan探针的单重和双重定量PCR技术对解脲支原体和巨细胞病毒同时定性定量检测。结果解脲支原体和巨细胞病毒单种定性PCR检测均为阳性,TaqMan探针单重和双重定量PCR检测解脲支原体和巨细胞病毒阳性率和特异性均为100%,相同样品TaqMan探针单重、双重定量PCR分别检测的结果符合率100%。结论TaqMan探针双重荧光定量PCR技术可同时检测两种靶分子,结果可靠,应用前景广阔。 相似文献
13.
Lucia Ma?arová Katarína Trnková Soňa Feiková Margita Obernauerová 《Experimental parasitology》2010,126(1):37-41
Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence.Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri. 相似文献
14.
Jizhong Zhou Liyou Wu Ye Deng Xiaoyang Zhi Yi-Huei Jiang Qichao Tu Jianping Xie Joy D Van Nostrand Zhili He Yunfeng Yang 《The ISME journal》2011,5(8):1303-1313
To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating β-diversity but less on α-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative analysis of the β-diversity of microbial communities. 相似文献
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Ken Aoshima Kentaro Takahashi Masayuki Ikawa Takayuki Kimura Mitsuru Fukuda Satoshi Tanaka Howell E Parry Yuichiro Fujita Akiyasu C Yoshizawa Shin-ichi Utsunomiya Shigeki Kajihara Koichi Tanaka Yoshiya Oda 《BMC bioinformatics》2014,15(1)
Background
Label-free quantitation of mass spectrometric data is one of the simplest and least expensive methods for differential expression profiling of proteins and metabolites. The need for high accuracy and performance computational label-free quantitation methods is still high in the biomarker and drug discovery research field. However, recent most advanced types of LC-MS generate huge amounts of analytical data with high scan speed, high accuracy and resolution, which is often impossible to interpret manually. Moreover, there are still issues to be improved for recent label-free methods, such as how to reduce false positive/negatives of the candidate peaks, how to expand scalability and how to enhance and automate data processing. AB3D (A simple label-free quantitation algorithm for Biomarker Discovery in Diagnostics and Drug discovery using LC-MS) has addressed these issues and has the capability to perform label-free quantitation using MS1 for proteomics study.Results
We developed an algorithm called AB3D, a label free peak detection and quantitative algorithm using MS1 spectral data. To test our algorithm, practical applications of AB3D for LC-MS data sets were evaluated using 3 datasets. Comparisons were then carried out between widely used software tools such as MZmine 2, MSight, SuperHirn, OpenMS and our algorithm AB3D, using the same LC-MS datasets. All quantitative results were confirmed manually, and we found that AB3D could properly identify and quantify known peptides with fewer false positives and false negatives compared to four other existing software tools using either the standard peptide mixture or the real complex biological samples of Bartonella quintana (strain JK31). Moreover, AB3D showed the best reliability by comparing the variability between two technical replicates using a complex peptide mixture of HeLa and BSA samples. For performance, the AB3D algorithm is about 1.2 - 15 times faster than the four other existing software tools.Conclusions
AB3D is a simple and fast algorithm for label-free quantitation using MS1 mass spectrometry data for large scale LC-MS data analysis with higher true positive and reasonable false positive rates. Furthermore, AB3D demonstrated the best reproducibility and is about 1.2- 15 times faster than those of existing 4 software tools.Electronic supplementary material
The online version of this article (doi:10.1186/s12859-014-0376-0) contains supplementary material, which is available to authorized users. 相似文献16.
A sensitive method for the determination of levamisole in serum by electrochemiluminescence 下载免费PDF全文
A novel method was developed for the determination of levamisole by electrochemiluminescence. The method was based on electrochemiluminescence signal enhancement produced by Ru(bpy)32+, which reacted with the tertiary amine group of levamisole on a platinum electrode in 12 mmol/L borate buffer (pH 9). A linear relationship between the luminous intensity and concentration of levamisole in the range 0–1 × 10–7 mol/L was obtained and the detection limit was 1.76 × 10–11 mol/L. The method is sensitive, selective, simple and convenient. The method has been successfully applied to the analysis of levamisole in serum. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
17.
Traunšek U Toplak N Jeršek B Lapanje A Majstorović T Kovač M 《Journal of microbiological methods》2011,85(1):40-46
Listeriosis is a serious food-borne infection with mortality rates approaching 30%. Therefore, the rapid, cost-effective, and automated detection of Listeria monocytogenes throughout the food chain continues to be a major concern. Here we describe three novel quantitative real-time PCR assays for L. monocytogenes based on amplification of a target hlyA gene with SYBR Green I chemistry and hydrolysis probe (TaqMan MGB probe). In order to offer sensitive, rapid and robust tool of additional economical value the real-time PCR assays were designed and optimized to only 5 μl-reactions. All assays were evaluated by using different non-reference Listeria strains isolated from various food matrices. Results demonstrated specificity to L. monocytogenes with accurate quantification over a dynamic range of 5-6 log units with R2 higher than 0.98 and amplification efficiencies reaching above 92%. The detection and quantification limits were as low as 165 genome equivalents. Comparison of novel assays to commercially available TaqMan® Listeria monocytogenes Detection Kit and previously published studies revealed similar specificity, sensitivity and efficiency, but greater robustness and especially cost-efficiency in the view of smaller reaction volumes and continuous increase in sample throughput. 相似文献
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实时荧光PCR检测水产品中副溶血性弧菌 总被引:1,自引:0,他引:1
目的探索副溶血性弧菌快速检测法,应用于日常监测及食物中毒的快速查源。方法用副溶血性弧菌实时荧光试剂盒对水产品样本进行检验,以副溶血性弧菌toxR基因为靶序列,设计1对引物和探针,采用热裂解法提取DNA。结果实时荧光PCR从42份水产品样品的增菌液中检出13份样品副溶血性弧菌阳性,与传统培养法相比一致性极好(K=0.943,K〉0.75)。结论实时荧光PCR方法在副溶血性弧菌的检验方面较传统方法具有快速、灵敏、特异性强等优势,具有广阔的应用前景。 相似文献
20.
Peeters S Stakenborg T Colle F Liu C Lagae L Van Ranst M 《Biotechnology progress》2010,26(6):1678-1684
The performance of various molecular techniques using complex biological samples greatly depends on the efficient separation and purification of DNA targets. In recent years, magnetic separation technology making use of small magnetic beads, has gained immense popularity. Most of these methods rely on the non-specific adsorption of DNA/RNA. However, as presented here, when functionalizing the beads with complementary DNA probes, the target of interest can selectively be isolated. Such sequence specific purification was evaluated for short DNA targets by means of simple fluorescent measurements, resulting in purification efficiencies around 80%. Besides standard fluorescent techniques, a real-time PCR (qPCR) method was applied for monitoring the purification of longer DNA targets. This qPCR method was specifically optimized for directly quantifying the purification efficiency of low concentrated DNA targets bound to magnetic beads. Additionally, parameters possibly affecting the magnetic isolation, including the length of the used capture probe or the hybridization location, were investigated. Using optimized conditions in combination with qPCR, purification efficiencies between 60% and 80% were observed and this over a large concentration window. These data also show the power of a direct qPCR approach to monitor the magnetic isolation of DNA at very low concentrations. 相似文献