Competitive reporter monitored amplification (CMA)--quantification of molecular targets by real time monitoring of competitive reporter hybridization

Background

State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests.

Methodology and Principal Findings

The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique.

Conclusions and Significance

The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.     

Nucleic acid hybridization assays employing dA-tailed capture probes. I. Multiple capture methods

A quantitative hybridization assay termed "reversible target capture" is described. The technique is designed to extensively purify the target nucleic acid from crude cell lysates in about 1 h without phenol extraction. Simple, rapid methods are described that explain how each process in the assay is optimized. The procedure involves hybridizing the target nucleic acid in solution with a dA-tailed capture probe and a labeled probe. The capture probe-target-labeled probe "ternary complex" is then captured on magnetic beads containing oligo(dT). After the excess unhybridized labeled probe, cell debris, and other sample impurities are washed away, the intact ternary complex is further purified by chemical elution from the beads and recapture on fresh beads. The ternary complex is then eluted thermally and recaptured on a third set of beads or on poly(dT) filters. This triple capture method results in a detection limit of approximately 0.2 amol (100 fg) of target with 32P-labeled riboprobes. This is approximately 1000 times more sensitive than sandwich assays employing only a single capture step. The method is illustrated by detecting Listeria cells in the presence of heterologous bacteria. With three rounds of target capture, as few as six Listeria cells have been detected in the presence of 1.25 x 10(7) control cells.     

Electric chips for rapid detection and quantification of nucleic acids

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.     

Electrochemical sandwich assay for attomole analysis of DNA and RNA from beer spoilage bacteria Lactobacillus brevis

Attomole (10(-18)mol) levels of RNA and DNA isolated from beer spoilage bacterial cells Lactobacillus brevis have been detected by the electrochemical sandwich DNA hybridization assay exploiting enzymatic activity of lipase. DNA sequences specific exclusively to L. brevis DNA and RNA were selected and used for probe and target DNA design. The assay employs magnetic beads (MB) modified with a capture DNA sequence and a reporter DNA probe labeled with the enzyme, both made to be highly specific for L. brevis DNA. Lipase-labeled DNAs captured on MBs in the sandwich assay were collected on gold electrodes modified with a ferrocene (Fc)-terminated SAM formed by aliphatic esters. Lipase hydrolysis of the ester bond released a fraction of the Fc redox active groups from the electrode surface, decreasing the electrochemical signal from the surface-confined Fc. The assay, shown to be efficient for analysis of short synthetic DNA sequences, was ineffective with genomic double stranded bacterial DNA, but it allowed down to 16 amole detection of 1563 nts long RNA, isolated from bacterial ribosomes without the need for PCR amplification, and single DNA strands produced from ribosomal RNA. No interference from E. coli RNA was registered. The assay allowed analysis of 400 L. brevis cells isolated from 1L of beer, which fits the "alarm signal" range (from 1 to 100 cells per 100mL).     

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.     

Application of a unique server-based oligonucleotide probe selection tool toward a novel biosensor for the detection of Streptococcus pyogenes

We developed a software program for the rapid selection of detection probes to be used in nucleic acid-based assays. In comparison to commercially available software packages, our program allows the addition of oligotags as required by nucleic acid sequence-based amplification (NASBA) as well as automatic BLAST searches for all probe/primer pairs. We then demonstrated the usefulness of the program by designing a novel lateral flow biosensor for Streptococcus pyogenes that does not rely on amplification methods such as the polymerase chain reaction (PCR) or NASBA to obtain low limits of detection, but instead uses multiple reporter and capture probes per target sequence and an instantaneous amplification via dye-encapsulating liposomes. These assays will decrease the detection time to just a 20 min hybridization reaction and avoid costly enzymatic gene amplification reactions. The lateral flow assay was developed quantifying the 16S rRNA from S. pyogenes by designing reporter and capture probes that specifically hybridize with the RNA and form a sandwich. DNA reporter probes were tagged with dye-encapsulating liposomes, biotinylated DNA oligonucleotides were used as capture probes. From the initial number of capture and reporter probes chosen, a combination of two capture and three reporter probes were found to provide optimal signal generation and significant enhancement over single capture/reporter probe combinations. The selectivity of the biosensor was proven by analyzing organisms closely related to S. pyogenes, such as other Streptococcus and Enterococcus species. All probes had been selected by the software program within minutes and no iterative optimization and re-design of the oligonucleotides was required which enabled a very rapid biosensor prototyping. While the sensitivity obtained with the biosensor was only 135 ng, future experiments will decrease this significantly by the addition of more reporter and capture probes for either the same rRNA or a different nucleic acid target molecule. This will lead to the possibility of detecting S. pyogenes with a rugged assay that does not require a cell culturing or gene amplification step and will therefore enable rapid, specific and sensitive onsite testing.     

Telomerase catalyzed fluorescent probes for sensitive protein profiling based on one-dimensional microfluidic beads array

A nucleic acid-based signal amplified method for multiple proteins detection based on one-dimensional beads array using telomerase catalyzed fluorescent probes has been developed in this paper. The biotin labeled fluorescent probes were synthesized by telomerase in homogeneous solution. Approximately 360-480 fluorescein molecules were inserted in each probe. The limit of detection for p53 protein is 1.1 pM (S/N=3) and a 3 orders of linear dynamic range is obtained. The sensitivity is nearly two orders higher than the two-site "sandwich" immunoassay using the same platform. Using this method, cellular p53 protein contents of as few as 85 CNE2 cells per mul sample can be determined specifically. The expression changes of p53, c-myc and beta-actin in CNE2 cells were further examined as a function of anti-cancer drug treatment, and the results are consistent with our previous reports. Compared with immuno-polymerase chain reaction and immuno-rolling circle amplification, this method is simple, fast, cheap and suitable for multi-protein analysis. The multiplexed proteins profiling of cellular samples should facilitate the new opportunities to the fundamental research of tumor development and progression, especially to the low abundant tumor-associated proteins analysis.     

Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Molecular beacons represent a new family of fluorescent probes for nucleic acids, and have found broad applications in recent years due to their unique advantages over traditional probes. Detection of nucleic acids using molecular beacons has been based on hybridization between target molecules and molecular beacons in a 1:1 stoichiometric ratio. The stoichiometric hybridization, however, puts an intrinsic limitation on detection sensitivity, because one target molecule converts only one beacon molecule to its fluorescent form. To increase the detection sensitivity, a conventional strategy has been target amplification through polymerase chain reaction. Instead of target amplification, here we introduce a scheme of signal amplification, nicking enzyme signal amplification, to increase the detection sensitivity of molecular beacons. The mechanism of the signal amplification lies in target-dependent cleavage of molecular beacons by a DNA nicking enzyme, through which one target DNA can open many beacon molecules, giving rise to amplification of fluorescent signal. Our results indicate that one target DNA leads to cleavage of hundreds of beacon molecules, increasing detection sensitivity by nearly three orders of magnitude. We designed two versions of signal amplification. The basic version, though simple, requires that nicking enzyme recognition sequence be present in the target DNA. The extended version allows detection of target of any sequence by incorporating rolling circle amplification. Moreover, the extended version provides one additional level of signal amplification, bringing the detection limit down to tens of femtomolar, nearly five orders of magnitude lower than that of conventional hybridization assay.     

A sensitive nonisotopic hybridization assay for HIV-1 DNA

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.     

Highly sensitive and selective colorimetric genotyping of single-nucleotide polymorphisms based on enzyme-amplified ligation on magnetic beads

Herein we report a new strategy for highly sensitive and selective colorimatric assay for genotyping of single-nucleotide polymorphisms (SNPs). It is based on the use of a specific gap ligation reaction, horseradish peroxidase (HRP) for signal amplification, and magnetic beads for the easy separation of the ligated product. Briefly, oligonucleotide capture probe functionalized magnetic beads are first hybridized to a target DNA. Biotinylated oligonucleotide detection probes are then allowed to hybridize to the already captured target DNA. A subsequent ligation at the mutation point joins the two probes together. The introduction of streptavidin-conjugated HRP and a simple magnetic separation allow colorimetric genotyping of SNPs. The assay is able to discriminate one copy of mutant in 1000 copies of wild-type KRAS oncogene at 30 picomolar. The detection limit of the assay is further improved to 1 femtomolar by incorporating a ligation chain reaction amplification step, offering an excellent opportunity for the development of a simple and highly sensitive diagnostic tool.     

Sequencing-independent method to generate oligonucleotide probes targeting a variable region in bacterial 16S rRNA by PCR with detachable primers

Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3' end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of VIBRIO: The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.     

Gold nanoparticle probe-based gene expression analysis with unamplified total human RNA

Microarray-based gene expression analysis plays a pivotal role in modern biology and is poised to enter the field of molecular diagnostics. Current microarray-based gene expression systems typically require enzymatic conversion of mRNA into labeled cDNA or cRNA. Conversion to cRNA involves a target amplification step that overcomes the low sensitivity associated with commonly used fluorescent detection methods. Herein, we present a novel enzyme-free, microarray-based gene expression system that uses unamplified total human RNA sample as the target nucleic acid. The detection of microarray-bound RNA molecules is accomplished by targeting the poly-A tail with an oligo-dT₂₀ modified gold nanoparticle probe, signal amplification by autometallography, and subsequent measurement of nanoparticle-mediated light scattering. The high sensitivity afforded by the nanoparticle probes allows differential gene expression from as little as 0.5 μg unamplified total human RNA in a 2 h hybridization without the need for elaborate sample labeling steps.     

Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes

Nucleic acid diagnostics is dominated by fluorescence-based assays that use complex and expensive enzyme-based target or signal-amplification procedures. Many clinical diagnostic applications will require simpler, inexpensive assays that can be done in a screening mode. We have developed a 'spot-and-read' colorimetric detection method for identifying nucleic acid sequences based on the distance-dependent optical properties of gold nanoparticles. In this assay, nucleic acid targets are recognized by DNA-modified gold probes, which undergo a color change that is visually detectable when the solutions are spotted onto an illuminated glass waveguide. This scatter-based method enables detection of zeptomole quantities of nucleic acid targets without target or signal amplification when coupled to an improved hybridization method that facilitates probe-target binding in a homogeneous format. In comparison to a previously reported absorbance-based method, this method increases detection sensitivity by over four orders of magnitude. We have applied this method to the rapid detection of mecA in methicillin-resistant Staphylococcus aureus genomic DNA samples.     

DNA-probes for the highly sensitive identification of single nucleotide polymorphism using single-molecule spectroscopy

This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.     

DNA hybridization detection in a microfluidic channel using two fluorescently labelled nucleic acid probes

A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5'-CTGAT TAGAG AGAGAA-TAMRA-3' and 5'-TET-ATGTC TGAGC TGCAGG-3') and target DNA (3'-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5') were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 x 10(-6) to 1.0 x 10(-7)M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase.     

Use of bioluminescence in nucleic acid hybridization reactions

Luminescence reactions can be used to detect specific nucleic acid sequences hybridized with a nucleic probe. Different labels such as cytidine sulphone, fluorescein, and biotin can be incorporated into DNA or oligonucleotide molecules and detected by antibody or avidin conjugates coupled to glucose-6P dehydrogenase. On supports such as nitrocellulose filters, sensitivity is not greatly increased using luminescence, but detection is rapid and easy to perform using polaroid film. Moreover, hybridization can be performed with different labelled probes on the same sample. In solution, luminescence can be used to monitor sandwich reactions. The method is less sensitive than detection on filters but can easily be automated. The performance of these assays can be increased considerably by enzymatic amplification of the target catalysed by a thermostable polymerase.     

Enzymatic reporting of peste des petits ruminants virus genes ligating two specific probes on nanoparticles

An alternative strategy for the detection of nucleic acid derived from peste des petits ruminants virus was developed omitting amplification. The assay is based on two probes complementary to the target sequences, one conjugated to magnetic microparticles the second to gold nanoparticles labeled with horseradish peroxidase. In the presence of target gene the two particles ligate via the probes and the complex can be magnetically separated. Applying substrate and chromogen a color reaction results for a positive case. Under optimized conditions, the approach had a linear detection range from 10 fM to 1 μM for ssDNA corresponding to an RNA low detection limit of 17.6 ng/μl. The quick performance (45 min) and not requiring expensive instrumentations offer a new way of detecting nucleic acids for the clinical diagnosis in our case for peste des petits ruminants virus.     

纳米金标记-逐步银染法快速检测金黄色葡萄球菌

将纳米金探针应用于目的核酸的检测,具有与PCR相当的灵敏度和特异性.本研究建立了一种可以在微孔板上快速检测金黄色葡萄球菌的纳米金标记-逐步银染法.该方法利用已包被链霉亲和素的微孔板,将PCR扩增的金黄色葡萄球菌nuc基因与生物素探针、纳米金探针形成的三明治杂交结构锚定其上,然后在低温下逐步银染显色,通过酶标仪检测放大的银染信号.这种纳米金标记-逐步银染法可以在显著降低非特异性背景信号的同时放大银染信号,检测金黄色葡萄球菌nuc基因的灵敏度为1 pmol/L,比常温一步银染法的灵敏度提高约102倍. 51例临床标本的检测结果与PCR法一致,与培养生化鉴定法的检测结果之间无显著性差异(P >0.05). 综上所述,本研究成功构建了金黄色葡萄球菌的纳米金标记-逐步银染法,在病原微生物的快速检测领域表现出广阔的发展潜力.