A strategy for single nucleotide polymorphism analysis of chimerism for somatic cell therapy

Background aimsChimerism is an important outcome measure in hematopoietic cell transplantation as well as somatic cell therapy. Commonly used methods to estimate chimerism are restricted by either gender or inefficient sensitivity. In principle, real-time polymerase chain reaction (PCR)-based assays can be used to assess single nucleotide polymorphisms (SNP), which are a vast resource of molecular markers, and such assays demonstrate a substantially higher sensitivity (0.001%), but the specificity is unclear because of a low-level signal from mismatched sequences.MethodsIn this study, we cloned 14 pairs of SNP selected from the SNP HapMap database and examined the specificity and sensitivity of their detection by real-time PCR using two primer/fluorescent probe pairs to allow genotyping of the two possible variant alleles. Clinical donor–recipient pairs from 18 families were used to explore the efficacy of using SNP assays to measure chimerism.ResultsWe found that the polymorphic nucleotide influences the ability to distinguish the signal generated by the target and mismatched sequences. Moreover, the specific fluorescent reporter probe can affect the difference in signal intensity between the target and mismatched sequences. Real-time PCR SNP assays can attain a sensitivity of 0.1–0.5% with 100% specificity. When comparing possible clinical donor–recipient pairs, we found an average 3.3 out of 14 SNP were informative.ConclusionsBy optimal selection of the polymorphic sequences and fluorescent reporter, the real-time PCR SNP assay is superior to the short-tandem repeat chimerism assay and broadly applicable. This strategy may be applied in future clinical trials of bone marrow cell therapy.     

Optimized Quantification of Fragmented,Free Circulating DNA in Human Blood Plasma Using a Calibrated Duplex Real-Time PCR

Background

Duplex real-time PCR assays have been widely used to determine amounts and concentrations of free circulating DNA in human blood plasma samples. Circulatory plasma DNA is highly fragmented and hence a PCR-based determination of DNA concentration may be affected by the limited availability of full-length targets in the DNA sample. This leads to inaccuracies when counting PCR target copy numbers as whole genome equivalents.

Methodology/Principal Findings

A model system was designed allowing for assessment of bias in a duplex real-time PCR research assay. We collected blood plasma samples from male donors in pools of 6 to 8 individuals. Circulatory plasma DNA was extracted and separated by agarose gel electrophoresis. Separated DNA was recovered from the gel in discrete size fractions and analyzed with different duplex real-time PCR Taqman assays detecting a Y chromosome-specific target and an autosomal target. The real-time PCR research assays used differed significantly in their ability to determine the correct copy number ratio of 0.5 between Y chromosome and autosome targets in DNA of male origin. Longer PCR targets did not amplify quantitatively in circulatory DNA, due to limited presence of full-length target sequence in the sample.

Conclusions

PCR targets of the same small size are preferred over longer targets when comparing fractional circulatory DNA concentrations by real-time PCR. As an example, a DYS14/18S duplex real-time PCR research assay is presented that correctly measures the fractional concentration of male DNA in a male/female mixture of circulatory, fragmented DNA.     

Development and utility of an internal threshold control (ITC) real-time PCR assay for exogenous DNA detection

Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct) for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct) or negative (i.e. Ct greater than the ITC Ct). The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.     

A Quick Method for Species Identification of Japanese Eel (<Emphasis Type="Italic">Anguilla japonica</Emphasis>) Using Real-Time PCR: An Onboard Application for Use During Sampling Surveys

To compensate for the limited number of morphological characteristics of fish eggs and larvae, we established a convenient and robust method of species identification for eggs of the Japanese eel (Anguilla japonica) using a real-time polymerase chain reaction (PCR) that can be performed onboard research ships at sea. A total of about 1.2 kbp of the mitochondrial 16S ribosomal RNA gene sequences from all species of Anguilla and 3 other anguilliform species were compared to design specific primer pairs and a probe for A. japonica. This real-time PCR amplification was conducted for a total of 44 specimens including A. japonica, A. marmorata, A. bicolor pacifica, and 6 other anguilliform species. Immediate PCR amplification was only observed in A. japonica. We then tested this method under onboard conditions and obtained the same result as had been produced in the laboratory. These results suggest that real-time PCR can be a powerful tool for detecting Japanese eel eggs and newly hatched larvae immediately after onboard sampling during research cruises and will allow targeted sampling efforts to occur rapidly in response to any positive onboard identification of the eggs and larvae of this species.     

Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR

Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.     

Interactive Fluorophore and Quencher Pairs for Labeling Fluorescent Nucleic Acid Hybridization Probes

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.     

Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations

Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. The ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).     

Aptamer-Based Sensitive Detection of Target Molecules via RT-PCR Signal Amplification

In the efforts to explore an aptamer-based approach for target sensing and detection with higher sensitivity and specificity, instead of directly labeling aptamer with fluorophores, we proposed a new strategy by attaching a polymerase chain reaction (PCR) template to an oligonucleotide aptamer selected by systematic evolution of ligands by exponential enrichment (SELEX), so that after aptamer target binding, the template moiety serves as the PCR template in real-time quantitative PCR (RT-PCR), and therefore, the binding event can be reported by the following RT-PCR signals. Using the subtractive SELEX method, the oligonucleotide aptamers specific for the Fc fragment of mouse IgG were selected and subjected to coupling with the PCR dsDNA template by using overlap and the asymmetric extension PCR method. The target binding affinity of the PCR template tethered aptamer has been proven by electrophoretic mobility shift assay (EMSA), and further template tethered aptamer mediated real-time quantitative PCR (A-PCR) was conducted to validate the application for such a template tethered aptamer to be a sensitive probe for IgG detection. The results show that the protocols of A-PCR can detect 10-fold serial dilutions of the target, demonstrating a new mechanism to convert aptamer target binding events to amplified RT-PCR signal, and the feasibility of the PCR template tethered aptamer as a facile, specific, and sensitive target probing and detection is established. This new approach also has potential applications in multiple parallel target detection and analysis in a wide range of research fields.     

Identification of hepatotoxin-producing cyanobacteria by DNA-chip

We developed a new tool to detect and identify hepatotoxin-producing cyanobacteria of the genera Anabaena , Microcystis , Planktothrix , Nostoc and Nodularia . Genus-specific probe pairs were designed for the detection of the microcystin ( mcyE ) and nodularin synthetase genes ( ndaF ) of these five genera to be used with a DNA-chip. The method couples a ligation detection reaction, in which the polymerase chain reaction (PCR)-amplified mcyE / ndaF genes are recognized by the probe pairs, with a hybridization on a universal microarray. All the probe pairs specifically detected the corresponding mcyE / ndaF gene sequences when DNA from the microcystin- or nodularin-producing cyanobacterial strains were used as template in the PCR. Furthermore, the strict specificity of detection enabled identification of the potential hepatotoxin producers. Detection of the genes was very sensitive; only 1–5 fmol of the PCR product were needed to produce signal intensities that exceeded the set background threshold level. The genus-specific probe pairs also reliably detected potential microcystin producers in DNA extracted from six lake and four brackish water samples. In lake samples, the same microcystin producers were identified with quantitative real-time PCR analysis. The specificity, sensitivity and ability of the DNA-chip in simultaneously detecting all the main hepatotoxin producers make this method suitable for high-throughput analysis and monitoring of environmental samples.     

Exhaustive computational identification of pathogen sequences far-distant from background genomes: Identification and experimental verification of human-blind dengue PCR primers

We recently developed novel algorithms for exhaustive identification of all nucleotide subsequences present in a pathogen genome which differ by at least a chosen number of mismatches from the sequences of host/background organisms. This type of exhaustive computational analysis will be useful in reducing false positives and cross-reactivity in PCR and hybridization assays. We present the first experimental test of the method by showing that the subsequences identified when used as 18-mer PCR primers can detect the presence of dengue virus (DENV) even in the presence of a large excess of complex human genomic DNA. From our computations, 715 serotype-specific primer pairs were identified for three different DENV serotypes in which each primer sequence lies at least two mismatches from the nearest human sequence. DNA clones of representative strains of DENV-1, DENV-2, and DENV-4 viruses were subjected to real-time PCR testing using eight primer pairs each. Efficiencies were uniformly very high (mean+/-S.D.=99.6+/-3%), and amplification of human DNA was never observed within 35 cycles, even at a 5.5-fold molar excess of human DNA. Exhaustive primer/probe screening can potentially produce more selective and sensitive diagnostic assays for pathogens, especially in the presence of complex backgrounds.     

双链探针实时荧光PCR核酸检测新技术研究*

目的:采用一种“双链探针”实时荧光PCR技术,提高HBV核酸检测灵敏度,并在同一反应管中实现代谢酶CYP2C19^*2基因型检测。方法:采用双链探针与TaqMan探针同时检测不同浓度HBV血清样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和结果统计分析;采用双链探针检测代谢酶CYP2C19^*2不同基因型样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和基因型确定。结果:不同浓度HBV血清样本检测,双链探针荧光本底低,检测灵敏度更高,与TaqMan探针检测结果相比,两者核酸检测Ct值存在显著性差异(P<0.05);双链探针检测36份样本的代谢酶CYP2C19^*2基因型,检测结果与Sanger测序结果完全一致。结论:双链探针实时荧光PCR检测技术可完成目的基因的高灵敏核酸检测,也可实现基因型分析。     

Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes

Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.     

Validation of an algorithm for automatic quantification of nucleic acid copy numbers by real-time polymerase chain reaction

Real-time quantitative polymerase chain reaction (PCR) with on-line fluorescence detection has become an important technique not only for determination of the absolute or relative copy number of nucleic acids but also for mutation detection, which is usually done by measuring melting curves. Optimum assay conditions have been established for a variety of targets and experimental setups, but only limited attention has been directed to data evaluation and validation of the results. In this work, algorithms for the processing of real-time PCR data are evaluated for several target sequences (p53, IGF-1, PAI-1, Factor VIIc) and compared to the results obtained by standard procedures. The algorithms are implemented in software called SoFAR, which allows fully automatic analysis of real-time PCR data obtained with a Roche LightCycler instrument. The software yields results with considerably increased precision and accuracy of quantifications. This is achieved mainly by the correction of amplification-independent signal trends and a robust fit of the exponential phase of the signal curves. The melting curve data are corrected for signal changes not due to the melting process and are smoothed by fitting cubic splines. Therefore, sensitivity, resolution, and accuracy of melting curve analyses are improved.     

新型MGB探针在沙眼衣原体实时PCR检测中的应用

为建立基于TaqMan-MGB探针的沙眼衣原体DNA荧光定量PCR检测方法,探讨其临床应用价值,用 PCR法扩增沙眼衣原体隐蔽质粒pLVG440 2 464～2 980 nt段,并克隆入pMD18-T载体用作参比模板,设计一对引物和一个TaqMan-MGB探针,优化反应条件,建立沙眼衣原体DNA荧光定量PCR检测系统,并运用该系统同时应用连接酶链式反应(LCR)法对临床标本进行检测.结果显示所建立的沙眼衣原体DNA荧光定量PCR检测系统,最低检测限度为1 DNA拷贝每反应;在10⁰～10⁹ DNA拷贝每反应范围内,C_t值(每个反应管内的荧光信号达到设定的域值时所经历的循环数）和DNA拷贝数呈线性关系（r＞0.990）;对临床标本检测结果同LCR分析结果吻合率为100%.以上结果表明,所建立的基于TaqMan-MGB探针的沙眼衣原体DNA荧光定量PCR检测系统具有敏感性高、特异性强和线性检测范围广等特点,适用于对沙眼衣原体进行大规模筛选.     

PCR bias in ecological analysis: a case study for quantitative Taq nuclease assays in analyses of microbial communities

Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69-75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 10(4)-fold excess of phylogenetically similar DNA and of an up to 10(7)-fold excess of dissimilar DNA.     

A set of external reference controls/probes that enable quality assurance between different microarray platforms

RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration–response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT–PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT–PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.     

Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes

The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage lambda-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.     

Rapid and inexpensive species differentiation using a multiplex real-time polymerase chain reaction high-resolution melt assay

We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers.