Rapid and sensitive detection of Mycobacterium tuberculosis based on strand displacement amplification and magnetic beads

Tuberculosis is one of the main infectious diseases threatening public health, and the development of simple, rapid, and cost‐saving methods for tuberculosis diagnosis is of profound importance for tuberculosis prevention and treatment. The bacterium Mycobacterium tuberculosis (MTB) is the pathogen that causes tuberculosis, and assaying for MTB is the only criterion for tuberculosis diagnosis. A new enzyme‐free method based on strand displacement amplification and magnetic beads was developed for simple, rapid, and cost‐saving MTB detection. Under optimum conditions, a good linear relationship could be observed between fluorescence and MTB specific DNA concentration ranging from 0.05 to 150 nM with a correlation coefficient of 0.993 (n = 8) and a detection limit of 47 pM (3σ/K). The present method also distinguished a one base mismatch from MTB specific DNA, showing great promise for MTB genome single base polymorphism analysis. MTB specific DNA content in polymerase chain reaction samples was successfully detected using the new method, and recoveries were 97.8–100.8%, indicating that the present method had high accuracy and shows good potential for the early diagnosis of tuberculosis.     

Detection of microarray-hybridized oligonucleotides with magnetic beads

The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarray-hybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.     

Different approaches for the detection of thrombin by an electrochemical aptamer-based assay coupled to magnetic beads

Different assay formats based on the coupling of magnetic beads with electrochemical transduction were compared here for the detection of thrombin by using a thrombin specific aptamer. By using the thrombin-binding aptamer, a direct and an indirect competitive assay for thrombin have been developed by immobilising the aptamer or the protein, respectively. Moreover, another strategy was based on the direct measurement of the enzymatic product of thrombin captured by the immobilised aptamer. All the assays were developed by coupling the electrochemical transduction with the innovative and advantageous use of magnetic beads.

The assays based on the immobilisation of the protein were not successful since no binding was recorded between thrombin and its aptamer. With the direct competitive assay, when the aptamer was immobilised onto the magnetic beads, a detection limit of 430 nM for thrombin was achieved. A lower detection limit for the protein (175 nM) was instead obtained by detecting the product of the enzymatic reaction catalysed by thrombin. All these assays were finally compared with a sandwich assay which reached a detection limit of 0.45 nM of thrombin demonstrating the best analytical performances.

With this comparison the importance of a deep study on the different analytical approaches for thrombin detection to reach the performances of the best assay configuration has been demonstrated.     

磁珠法快速提取基因组DNA的实验研究

针对临床标本基因组DNA的提取方法缺乏广泛适用性,提取步骤繁琐,需要进行离心操作,并且提取过程中会用到苯酚、氯仿等有机试剂,会对操作人员有一定的危害性等不足,本研究拟建立一种适用于临床标本的基因组DNA快速提取方法。在传统的基因组DNA提取试剂和方法的基础上,本研究采用二氧化硅修饰的超顺磁珠设计了一种基因组DNA快速提取方法;探讨磁珠用量、裂解液p H、盐酸胍浓度等因素对基因组DNA提取效率的影响并采用凝胶电泳实验进行验证。当磁珠用量在50~100μg/100 mg样品,裂解液p H约为6,盐酸胍的浓度为6 mol/L时基因组DNA提取效果好、效率高,且磁珠的用量与吸附表面积成正比,但达到一定用量后不会增加提取量,对于100 mg肺组织,适宜的磁珠用量为80μg。此基因组DNA提取方法高效省时,简便快捷,性价比高,适用临床大量样本基因组DNA提取。     

Homogeneous amplified single-molecule detection: Characterization of key parameters

We recently presented a method that enables single-molecule enumeration by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target-specific padlock probe ligation, followed by rolling circle amplification (RCA), resulting in the creation of one rolling circle product (RCP) for each recognized target. The transformation makes optical detection and quantification possible using standard fluorescence microscopy by counting the number of generated RCPs in a sample pumped through a microfluidic channel. In this study, we demonstrate that confocal volume definition is crucial to achieve high-precision measurements in the microfluidic quantification (coefficient of variance typically 3%). We further demonstrate that complementary sequence motifs between RCPs is only a weak inducer of aggregates and that all detection sites of the RCPs are occupied at detection oligonucleotide concentrations greater than 5 nM if hybridized in the proper buffer conditions. Therefore, the signal/noise ratio is limited by the number of detection sites. By increasing the density of detection sites in the RCP by a factor of 1.9, we show that the optical signal/noise level can be increased from 42 to 75.     

Identification of functional nuclear export sequences in human topoisomerase IIalpha and beta

Nuclear localization of topoisomerase IIalpha and beta is important for normal cell function as well as being a determinant of tumour cell sensitivity to topoisomerase II-targeting chemotherapeutic agents. However, topoisomerase II is cytoplasmic under certain circumstances, indicating that it may undergo active nuclear export. We have examined the ability of Leu-rich potential nuclear export signal (NES) sequences present in human topoisomerase IIalpha and beta to direct the export of a green fluorescent protein-glutathione-S-transferase fusion protein following microinjection into HeLa cell nuclei. Of 12 sequences tested, only one potential NES sequence from the comparable location in each isoform (alphaNES(1018-1028) and betaNES(1034-1044)) was active. Mutation of hydrophobic residues in alphaNES(1018-1028) and betaNES(1034-1044) substantially reduced their nuclear export activity as did leptomycin B treatment of microinjected cells. Our results provide the first evidence of active nuclear export of topoisomerase II and suggest it is mediated by a CRM1-dependent pathway.     

Protein detection via direct enzymatic amplification of short DNA aptamers

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Here we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. Because both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 microM to 10 pM).     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

Design,synthesis and antiproliferative evaluation of fluorenone analogs with DNA topoisomerase I inhibitory properties

A series of 2,7-diamidofluorenones were designed, synthesized, and screened by SRB assay. Some synthesized compounds exhibited antitumor activities in submicromolar range. Ten compounds (3a, 3b, 3c, 3g, 3j, 3l, 4a, 4h, 4i, and 4j) were also selected by NCI screening system and 3c (GI₅₀ = 1.66 μM) appeared to be the most active agent of this series. Furthermore, 3c attenuated topoisomerase I-mediated DNA relaxation at low micromolar concentrations. These results indicated that fluorenones have potential to be further developed into anticancer drugs.     

Hyperbranched rolling circle amplification as a novel method for rapid and sensitive detection of Amphidinium carterae

High quality of coastal water is critical to marine ecosystems, marine fisheries, public health, and aquatic environment. Specially, bio-toxin derived from toxic microalgae is currently threatening many coastal countries. Therefore, development of rapid and sensitive methods for the detection of toxin-producing microalgae is necessary for warning of water quality. In this paper, we established a novel method for rapid and sensitive detection of Amphidinium carterae by hyperbranched rolling circle amplification (HRCA). The partial large subunit rDNA (LSU D1–D2) of A. carterae was sequenced to design species-specific padlock probe (PLP). The PLP-coupled with two amplification primers were employed for HRCA. The optimized HRCA conditions were as follows: padlock concentration, 20 pM; ligation temperature, 65 °C; ligation time, 15 min; amplification temperature, 61 °C; and amplification time, 15 min. The developed HRCA was confirmed to be specific for A. carterae by tests with other algae. The sensitivity of HRCA was 100-fold higher than regular PCR, exhibiting a detection limit of 1 fg/μL representing 283 copies for the recombinant plasmid containing the target LSU D1–D2, and 1 cell for target species. Finally, a simplified protocol was applied to the simulated field and environmental materials, and exhibited a good performance. The whole detection could be completed within 1.5 h, displaying a repeated detection limit of 1 cell. The positive HRCA results could be visualized through coloration reaction by adding the fluorescent dye SYBR Green I to the amplification products. The HRCA provides a useful tool to quickly screen large sample sets for A. carterae, as well as other toxic species.     

Fragment responsible for translocation in the N-terminal domain of human topoisomerase I

The N-terminal domain is a fragment that binds proteins and anchors topoisomerase I in the nucleolus. As a separate polypeptide, it translocates from the nucleolus to nucleoplasm upon camptothecin treatment. In this paper, we show that the translocation depends on the short fragment of the domain (residues from 1 to 67). We also present a list of proteins that specifically bind to the fragment responsible for translocation.     

Quantification of telomerase activity by direct scintillation counting

An improved telomerase assay was developed that allows direct quantification of the enzyme activity by scintillation counting of the labeled telomerase product. The assay measures the incorporation of ³²P-dGTP into telomeric repeats synthesized at the 3′ end of a biotinylated primer. Telomerase reaction product is separated from the reaction mix by streptavidin-coated magnetic beads and counted. The assay can be used for quantitative studies of human telomerase and its inhibitors.     

Detection and identification of opportunistic Exophiala species using the rolling circle amplification of ribosomal internal transcribed spacers

Deep infections by melanized fungi deserve special attention because of a potentially fatal, cerebral or disseminated course of disease in otherwise healthy patients. Timely diagnostics are a major problem with these infections. Rolling circle amplification (RCA) is a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of microorganisms. RCA-based diagnostics are characterized by good reproducibility, with few amplification errors compared to PCR. The method is applied here to species of Exophiala known to cause systemic infections in humans. The ITS rDNA region of five Exophiala species (E. dermatitidis, E. oligosperma, E. spinifera, E. xenobiotica, and E. jeanselmei) was sequenced and aligned in view of designing specific padlock probes to be used for the detection of single nucleotide polymorphisms (SNPs) of the Exophiala species concerned. The assay proved to successfully amplify DNA of the target fungi at the level of species; while no cross-reactivity was observed. Amplification products were visualized on 1% agarose gels to verify the specificity of probe-template binding. Amounts of reagents were minimized to avoid the generation of false positive results. The sensitivity of RCA may help to improve early diagnostics of these difficult to diagnose infections.     

End-labeling of long DNA fragments with biotin and detection of DNA immobilized on magnetic beads

To immobilize DNA fragments onto magnetic beads coated with streptavidin for isolation purpose, it is important to label one biotin molecule at one terminus of DNA fragment. After failure to label long DNA with biotin by PCR and filling-in reaction, a 9.2 kb DNA was labeled with biotin by a modified ligation strategy. A simple method is also reported to detect the quantity and integrity of DNA immobilized on the magnetic beads.     

Homogeneous and label-free fluorescence detection of single-nucleotide polymorphism using target-primed branched rolling circle amplification

We present a simple, sensitive, and cost-effective fluorescent assay of single-nucleotide polymorphism (SNP) with target-primed branched rolling circle amplification (TPBRCA). Designed padlock probe is circularized after perfect hybridization to mutant DNA. Then rolling circle amplification (RCA) reaction can be initiated from the mutant DNA that acts as primer and generates a long tandem single-stranded DNA (ssDNA) product. At the same time, the introduction of a reverse primer complementary to the target-primed RCA products leads to the branched RCA and eventually generates the various lengths of ssDNA and double-stranded DNA products, which are sensitively detected using SYBR Green I (SG) fluorescence dye. In contrast, the wild DNA contains a single mismatched base with the padlock probe and primes only a limited extension with the unligated padlock probe, generating weak background fluorescence with the addition of SG. Due to the excellent specificity and powerful amplification of TPBRCA reaction, the mutant DNA was distinctively differentiated from the wild DNA in a homogeneous and label-free manner. The assay is sensitive and specific enough to detect 5-amol (8.6-fM) mutant DNA strands. It was possible to accurately determine the mutant allele frequency as low as 1.0%.     

Recyclable chaperone-conjugated magnetic beads for in vitro refolding of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> lipase

Polymer-coated magnetic beads have become widely used in biological applications because of their facile recovery and easily modifiable surface. Herein, we report the application of magnetic beads to in vitro refolding of B. cepacia lipase. Magnetic particles (Fe₃O₄) prepared by co-precipitation of Fe²⁺ and Fe³⁺ ions under basic conditions were subsequently coated with carboxylic acid-containing polystyrene by emulsion polymerization. The polymer-coated magnetic beads were then conjugated with molecular chaperone proteins to assist with refolding. The chaperone-conjugated magnetic beads efficiently refolded B. cepacia lipase and were easily reused. The beads showed comparable refolding activity to the soluble chaperone, and retained more than 95% of their refolding activity after five cycles of refolding B. cepacia lipase.     

Rapid and prolonged stalling of human DNA topoisomerase I in UVA-irradiated genomic areas

DNA topoisomerase I appears to be involved in DNA damage and repair in a complex manner. The enzyme is required for DNA maintenance and repair, but it may also damage DNA through its covalently DNA-bound, catalytic intermediate. The latter mechanism plays a role in tumor cell killing by camptothecins, but seems also involved in oxidative cell killing and certain stages of apoptosis. Stalling and/or suicidal DNA cleavage of topoisomerase I adjacent to nicks and modified DNA bases has been demonstrated in vitro. Here, we investigate the enzyme's interactions with UVA-induced DNA lesions inside living cells. We irradiated cells expressing GFP-tagged topoisomerase I with an UVA laser focused through a confocal microscope at confined areas of the nuclei. At irradiated sites, topoisomerase I accumulated within seconds, and accumulation lasted for more than 90 min. This effect was apparently due to reduced mobility, although the enzyme was not immobilized at the irradiated nuclear sites. Similar observations were made with mutant versions of topoisomerase I lacking the active site tyrosine or the N-terminal domain, but not with the N-terminal domain alone. Thus, accumulation of topoisomerase I at UVA-modified DNA sites is most likely due to non-covalent binding to damaged DNA, and not suicidal cleavage of such lesions. The rapid onset of accumulation suggests that topoisomerase I functions in this context as a component of DNA damage recognition and/or a cofactor of fast DNA-repair processes. However, the prolonged duration of accumulation suggests that it is also involved in more long-termed processes.     

Efficient capture of infectious H5 avian influenza virus utilizing magnetic beads coated with anionic polymer

The possible emergence of a pandemic influenza virus from the avian influenza virus (AIV) has become a serious threat. The isolation of viruses will be crucial for further virological analysis and the development of vaccines. However, currently, there is no simple method for facilitating the isolation of infectious AIV. Here, we have developed a simple method of capturing AIV using anionic magnetic beads. The method employed the capture of AIV (H5N1, H5N2, and H5N3) from liquid samples such as allantoic fluid (AF) and cell culture medium (CM) using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydride). After their incubation with AIV-containing samples, the magnetic beads were separated from the supernatant by applying a magnetic field. The absorption of AIV on the beads was confirmed by immunochromatography and an enzyme-linked immunosorbent assay, which indicated the presence of hemagglutinin, neuraminidase, and nucleoprotein of AIV. Furthermore, the infectivity in chicken eggs of AIV captured by magnetic beads was similar to that of the starting materials. The capture of AIV using magnetic beads coated with anionic polymers will contribute to the sufficient recovery of infectious AIV and approach for potential pandemic influenza viruses.