CE combined with rolling circle amplification for sensitive DNA detection

Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.     

Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs

Highly sensitive digital nucleic acid techniques are of great significance for the prevention and control of epidemic diseases. Here we report the development of multiplexed droplet loop-mediated isothermal amplification (multiplexed dLAMP) with scorpion-shaped probes (SPs) and fluorescence microscopic counting for simultaneous quantification of multiple targets. A set of target-specific fluorescence-activable SPs are designed, which allows establishment of a novel multiplexed LAMP strategy for simultaneous detection of multiple cDNA targets. The digital multiplexed LAMP assay is thus developed by implementing the LAMP reaction using a droplet microfluidic chip coupled to a droplet counting microwell chip. The droplet counting system allows rapid and accurate counting of the numbers of total droplets and the positive droplets by collecting multi-color fluorescence images of the droplets in a microwell. The multiplexed dLAMP assay was successfully demonstrated for the quantification of HCV and HIV cDNA with high precision and detection limits as low as 4 copies per reaction. We also verified its potential for simultaneous digital assay of HCV and HIV RNA in clinical plasma samples. This multiplexed dLAMP technique can afford a useful platform for highly sensitive and specific detection of nucleic acids of viruses and other pathogens, enabling rapid diagnosis and prevention of infectious diseases.

The development of multiplexed dLAMP with scorpion-shaped probes and fluorescence microscopic counting affords simultaneous digital quantification of multiple virus RNAs.     

Instantaneous derivatization technology for the simultaneous and homogeneous detection of multiple double-stranded PCR amplicons

Herein we report on the development of instantaneous derivatization technology for the homogeneous and simultaneous detection of multiple PCR amplicons specific to the Hepatitis B Virus (HBV) by using three carriers: magnetic beads, polystyrene beads, and thermo-sensitive poly-N-isopropylacrylamide (PNIP). Briefly, PCR amplicons are labeled with digoxin, biotin or FITC via the modified up-stream primers respectively. After PCR amplification, the immunoreactions occur between a mixture of three target PCR amplicons and three modified carriers with anti-digoxin antibody, streptavidin or anti-FITC antibody in a single vessel, and then each carrier is separated from the others under different conditions based on their physio-chemical attributes. And then direct CL detection proceeds via the instantaneous derivatization reaction between intrinsic guanine nucleobases and 3,4,5-trimethoxylphenylglyoxal (TMPG). This new protocol directly measures the double-stranded DNA and therefore does not require a denaturing step, thus offering an enhanced sensitivity due to the absence of competitive hybridization, i.e., the detection limit had a 20-fold improvement on the conventional PCR measurement. Additionally, by comparison of previous guanine based detection formats, this protocol is easy to be used for the detection of any guanine containing targets without the use of guanine-free or inosine-substituted capture probes. Overall, the proposed technique takes the advantages of sensitivity, high-speed and cost-effectivity, which provides a promising alternative for the analysis of multiple PCR targets in a variety of clinical, environmental, and biodefense fields.     

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.     

Dual enzyme electrochemical coding for detecting DNA hybridization

Enzyme-based hybridization assays for the simultaneous electrochemical measurements of two DNA targets are described. Two encoding enzymes, alkaline phosphatase and beta-galactosidase, are used to differentiate the signals of two DNA targets in connection to chronopotentiometric measurements of their electroactive phenol and alpha-naphthol products. These products yield well-defined and resolved peaks at +0.31 V (alpha-naphthol) and +0.63 V (phenol) at the graphite working electrode (vs. Ag/AgCl reference). The position and size of these peaks reflect the identity and level of the corresponding target. The dual target detection capability is coupled to the amplification feature of enzyme tags (to yield fmol detection limits) and with an efficient magnetic removal of non-hybridized nucleic acids. Proper attention is given to the choice of the substrates (for attaining well resolved peaks), to the activity of the enzymes (for obtaining similar sensitivities), and to the selection of the enzymes (for minimizing cross interferences). The new bioassay is illustrated for the simultaneous detection of two DNA sequences related to the BCRA1 breast-cancer gene in a single sample in connection to magnetic beads bearing the corresponding oligonucleotide probes. Prospects for electrochemical coding of multiple DNA targets are discussed.     

Advances in molecular techniques for the detection and quantification of genetically modified organisms

Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.     

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.     

Electrochemical coding technology for simultaneous detection of multiple DNA targets

Nucleic-acid hybridization assays based on the use of different inorganic-colloid (quantum dots) nanocrystal tracers for the simultaneous electrochemical measurements of multiple DNA targets are described. Three encoding nanoparticles (zinc sulfide, cadmium sulfide, and lead sulfide) are used to differentiate the signals of three DNA targets in connection to stripping-voltammetric measurements of the heavy metal dissolution products. These products yield well-defined and resolved stripping peaks at -1.12 V (Zn), -0.68 V (Cd), and -0.53 V (Pb) at the mercury-coated glassy-carbon electrode (vs Ag/AgCl reference). The position and size of these peaks reflect the identity and level of the corresponding DNA target. The multi-target detection capability is coupled to the amplification feature of stripping voltammetry (to yield femtomole detection limits) and with an efficient magnetic removal of nonhybridized nucleic acids to offer high sensitivity and selectivity. The protocol is illustrated for the simultaneous detection of three DNA sequences related to the BCRA1 breast-cancer gene in a single sample in connection to magnetic beads bearing the corresponding oligonucleotide probes. The new electrochemical coding is expected to bring new capabilities for DNA diagnostics, and for bioanalysis, in general.     

Tuning single nucleotide discrimination in polymerase chain reactions (PCRs): synthesis of primer probes bearing polar 4'-C-modifications and their application in allele-specific PCR

There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele-specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR-amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes by using allele-specific primer probes. PCR amplification by a DNA polymerase from matched 3'-primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real-time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4'-C-modified nucleotide residues at their 3' termini, and their evaluation in real-time asPCR. We found that primer probes bearing a 4'-C-methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.     

Immobilisation of quantum dots by bio-orthogonal PCR amplification and labelling for direct gene detection and quantitation

A sensitive and versatile detection scheme based on quantum dot immobilisation on a solid support through bio-orthogonal PCR amplification and labelling has been developed for detection and quantification of gene targets in complex DNA mixtures.     

Target-triggered hairpin-free chain-branching growth of DNA dendrimers for contrast-enhanced imaging in living cells by avoiding signal dispersion

The development of amplification strategies is one of the central challenges for detection of lowabundance targets. One-to-many(1:M) amplification strategies in which one target lights many signal probes, has improved the detection sensitivity in bulk solution, but with discounted contrast in cell imaging, because the lighted probes are dissociative and dispersible. In this work, a one-to-large(1:L) signaling mechanism, in which the lighted probes were orderly connected to each other, was concep...     

One-step, multiplexed fluorescence detection of microRNAs based on duplex-specific nuclease signal amplification

Traditional molecular beacons, widely applied for detection of nucleic acids, have an intrinsic limitation on sensitivity, as one target molecule converts only one beacon molecule to its fluorescent form. Herein, we take advantage of the duplex-specific nuclease (DSN) to create a new signal-amplifying mechanism, duplex-specific nuclease signal amplification (DSNSA), to increase the detection sensitivity of molecular beacons (Taqman probes). DSN nuclease is employed to recycle the process of target-assisted digestion of Taqman probes, thus, resulting in a significant fluorescence signal amplification through which one target molecule cleaves thousands of probe molecules. We further demonstrate the efficiency of this DSNSA strategy for rapid direct quantification of multiple miRNAs in biological samples. Our experimental results showed a quantitative measurement of sequence-specific miRNAs with the detection limit in the femtomolar range, nearly 5 orders of magnitude lower than that of conventional molecular beacons. This amplification strategy also demonstrated a high selectivity for discriminating differences between miRNA family members. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method promises a great potential of becoming a routine tool for simultaneously quantitative analysis of multiple miRNAs in tissues or cells, and supplies valuable information for biomedical research and clinical early diagnosis.     

Comparative quantification of nucleic acids using single-molecule detection and molecular beacons

This paper reports a highly sensitive homogenous method for comparative quantification of nucleic acids based on single-molecule detection (SMD) and molecular beacons (MBs). Two different color MBs were used to perform a separation-free comparative hybridization assay for simultaneous quantification of both target and control strands. A fluorescent burst, emitted from a single hybrid when it passes through a minuscule laser-focused region, is detected with high signal-to-noise ratio (SNR) by using single-molecule fluorescence spectroscopy. Targets are quantified via counting of discrete fluorescent bursts. The high SNR achieved in both detection channels overcame the complications of fluorescent variability usually observed in dual-color ensemble measurements. In comparison with the conventional ensemble methods, this method improved the detection limit by 3 orders of magnitude and reduced the probe consumption by 6 orders of magnitude, facilitating a highly sensitive approach for comparative quantification of nucleic acids and offering great promise for genomic quantification without amplification.     

单细胞核酸编码扩增成像分析

单细胞成像可在单细胞水平观测目标物位置、 确定目标物含量, 在生命科学与临床医学研究领域应用广泛. 核酸编码扩增技术利用特定分子反应将待测目标识别转化为核酸条码的扩增, 具有探针种类多、 易编程、 反应条件温和及信号放大效率高等特点, 在单细胞低丰度、 高灵敏、 多目标物成像中优势显著, 为理解细胞状态、 探索生命过程提供了新思路. 本文综合评述了核酸编码扩增在单细胞荧光成像领域的研究进展, 以目标物的编码方式为分类依据, 系统阐述了固定细胞原位成像和活细胞成像中不同目标物编码与扩增成像方式的区别, 并对活细胞成像中多重检测面临的问题以及未来发展前景进行了展望.     

Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.     

Express and sensitive detection of multiple miRNAs via DNA cascade reactors functionalized photonic crystal array

Array based detection techniques with fluorescence signal reading is a powerful tool for multiple targets analysis. However,when applied fluorescence array for micro RNA detection, time-consuming multi-steps surface signal amplification is usually required due to the low abundance of micro RNA in total RNA expressions, which impairs detection efficiency and limits its application in point of care test(POCT) manner. Herein, DNA cascade reactors(DCRs) functionalized photonic crystal(PC)array was fabricated for express and sensitive detections of mi RNA-21 and mi RNA-155. DCRs were assembled by interval conjugation of self-quenched hairpin DNA probes to single strand DNA nanowire synthesized by rolling circle amplification,which generated cascade DNA hybridization reactions in response to target mi RNAwith instant fluorescence recovery signal. PC array patterns with multi-structure colors further amplified fluorescence with their respective photonic bandgaps(PBGs)matching with the emission peaks of fluorescence molecules labelled on DCRs. The as-prepared DCRs functionalized PC array demonstrated express and sensitive simultaneous detections of mi RNA-21 and mi RNA-155 with hundreds f M detection limits only in 15 min, and was successfully applied in fast quantifications of low abundance mi RNAs from cell lysates and spiked mi RNAs from human serum, which would hold great potential for disease diagnosis and therapeutic effect monitoring with a POCT manner.     

Colloidal gold-polystyrene bead hybrid for chemiluminescent detection of sequence-specific DNA

A sensitive chemiluminescent (CL) detection of sequence-specific DNA has been developed by taking advantage of a magnetic separation/mixing process and the amplification feature of colloidal gold labels. In this protocol, the target oligonucleotides are hybridized with magnetic bead-linked capture probes, followed by the hybridization of the biotin-terminated amplifying DNA probes and the binding of streptavidin-coated gold nanoparticles; the nanometer-sized gold tags are then dissolved and quantified by a simple and sensitive luminol CL reaction. The proposed CL protocol is evaluated for a 30-base model DNA sequence, and the amount as low as 0.01 pmol of DNA is determined, which exhibits a 150 x enhancement in sensitivity over previous gold dissolution-based electrochemical formats and an enhancement of 20 x over the ICPMS detection. Further signal amplification is achieved by the assembly of biotinylated colloidal gold onto the surface of streptavidin-coated polystyrene beads. Such amplified CL transduction allows detection of DNA targets down to the 100 amol level, and offers great promise for ultrasensitive detection of other biorecognition events.     

Associating and Dissociating Nanodimer Analysis for Quantifying Ultrasmall Amounts of DNA

The amplification‐ and enzyme‐free quantification of DNA at ultralow concentrations, on the order of 10–1000 targets, is highly beneficial but extremely challenging. To address this challenge, true detection signals must be reliably discriminated from false or noise signals. Herein, we describe the development of associating and dissociating nanodimer analysis (ADNA) as a method that enables a maximum number of detection signals to be collected from true target‐binding events while keeping nonspecific signals at a minimum level. In the ADNA assay for ultralow target concentrations, Au nanoprobes on a lipid micropattern were monitored and analyzed in situ, and newly defined dissociating dimers, which are eventually decoupled into monomers again, were incorporated into the detection results. Tens to thousands of DNA copies can be reliably quantified with excellent single‐base‐mismatch differentiation capability by this non‐enzymatic, amplification‐free ADNA method.     

Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP–HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L⁻¹, with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.