Detection and prevention of mycoplasma hominis infection

DOEpatents

DelVecchio, Vito G.; Gallia, Gary L.; McCleskey, Ferne K.

1997-01-21

The present invention is directed to a rapid and sensitive method for detecting Mycoplasma hominis using M. hominis-specific probes, oligonucleotides or antibodies. In particular a target sequence can be amplified by in vitro nucleic acid amplification techniques, detected by nucleic acid hybridization using the subject probes and oligonucleotides or detected by immunoassay using M. hominis-specific antibodies. M. hominis-specific nucleic acids which do not recognize or hybridize to genomic nucleic acid of other Mycoplasma species are also provided.

A rapid, ratiometric, enzyme-free, and sensitive single-step miRNA detection using three-way junction based FRET probes

NASA Astrophysics Data System (ADS)

Luo, Qingying; Liu, Lin; Yang, Cai; Yuan, Jing; Feng, Hongtao; Chen, Yan; Zhao, Peng; Yu, Zhiqiang; Jin, Zongwen

2018-03-01

MicroRNAs (miRNAs) are single stranded endogenous molecules composed of only 18-24 nucleotides which are critical for gene expression regulating the translation of messenger RNAs. Conventional methods based on enzyme-assisted nucleic acid amplification techniques have many problems, such as easy contamination, high cost, susceptibility to false amplification, and tendency to have sequence mismatches. Here we report a rapid, ratiometric, enzyme-free, sensitive, and highly selective single-step miRNA detection using three-way junction assembled (or self-assembled) FRET probes. The developed strategy can be operated within the linear range from subnanomolar to hundred nanomolar concentrations of miRNAs. In comparison with the traditional approaches, our method showed high sensitivity for the miRNA detection and extreme selectivity for the efficient discrimination of single-base mismatches. The results reveal that the strategy paved a new avenue for the design of novel highly specific probes applicable in diagnostics and potentially in microscopic imaging of miRNAs in real biological environments.

Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes.

PubMed

Gadkar, Vijay J; Goldfarb, David M; Gantt, Soren; Tilley, Peter A G

2018-04-03

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification (iNAAT) technique known for its simplicity, sensitivity and speed. Its low-cost feature has resulted in its wide scale application, especially in low resource settings. The major disadvantage of LAMP is its heavy reliance on indirect detection methods like turbidity and non-specific dyes, which often leads to the detection of false positive results. In the present work, we have developed a direct detection approach, whereby a labelled loop probe quenched in its unbound state, fluoresces only when bound to its target (amplicon). Henceforth, referred to as Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP), it allows for the sequence-specific detection of LAMP amplicons. The FLOS-LAMP concept was validated for rapid detection of the human pathogen, Varicella-zoster virus, from clinical samples. The FLOS-LAMP had a limit of detection of 500 copies of the target with a clinical sensitivity and specificity of 96.8% and 100%, respectively. The high level of specificity is a major advance and solves one of the main shortcomings of the LAMP technology, i.e. false positives. Self-quenching/de-quenching probes were further used with other LAMP primer sets and different fluorophores, thereby demonstrating its versatility and adaptability.

Highly sensitive MicroRNA 146a detection using a gold nanoparticle-based CTG repeat probing system and isothermal amplification.

PubMed

Le, Binh Huy; Seo, Young Jun

2018-01-25

We have developed a gold nanoparticle (AuNP)-based CTG repeat probing system displaying high quenching capability and combined it with isothermal amplification for the detection of miRNA 146a. This method of using a AuNP-based CTG repeat probing system with isothermal amplification allowed the highly sensitive (14 aM) and selective detection of miRNA 146a. A AuNP-based CTG repeat probing system having a hairpin structure and a dT F fluorophore exhibited highly efficient quenching because the CTG repeat-based stable hairpin structure imposed a close distance between the AuNP and the dT F residue. A small amount of miRNA 146a induced multiple copies of the CAG repeat sequence during rolling circle amplification; the AuNP-based CTG repeat probing system then bound to the complementary multiple-copy CAG repeat sequence, thereby inducing a structural change from a hairpin to a linear structure with amplified fluorescence. This AuNP-based CTG probing system combined with isothermal amplification could also discriminate target miRNA 146a from one- and two-base-mismatched miRNAs (ORN 1 and ORN 2, respectively). This simple AuNP-based CTG probing system, combined with isothermal amplification to induce a highly sensitive change in fluorescence, allows the detection of miRNA 146a with high sensitivity (14 aM) and selectivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Subtelomeric Rearrangements and Copy Number Variations in People with Intellectual Disabilities

ERIC Educational Resources Information Center

Christofolini, D. M.; De Paula Ramos, M. A.; Kulikowski, L. D.; Da Silva Bellucco, F. T.; Belangero, S. I. N.; Brunoni, D.; Melaragno, M. I.

2010-01-01

Background: The most prevalent type of structural variation in the human genome is represented by copy number variations that can affect transcription levels, sequence, structure and function of genes. Method: In the present study, we used the multiplex ligation-dependent probe amplification (MLPA) technique and quantitative PCR for the detection…

Weak Value Amplification of a Post-Selected Single Photon

NASA Astrophysics Data System (ADS)

Hallaji, Matin

Weak value amplification (WVA) is a measurement technique in which the effect of a pre- and post-selected system on a weakly interacting probe is magnified. In this thesis, I present the first experimental observation of WVA of a single photon. We observed that a signal photon --- sent through a polarization interferometer and post-selected by photodetection in the almost-dark port --- can act like eight photons. The effect of this single photon is measured as a nonlinear phase shift on a separate laser beam. The interaction between the two is mediated by a sample of laser- cooled 85Rb atoms. Electromagnetically induced transparency (EIT) is used to enhance the nonlinearity and overcome resonant absorption. I believe this work to be the first demonstration of WVA where a deterministic interaction is used to entangle two distinct optical systems. In WVA, the amplification is contingent on discarding a large portion of the original data set. While amplification increases measurement sensitivity, discarding data worsens it. Questioning whether these competing effects conspire to improve or diminish measurement accuracy has resulted recently in controversy. I address this question by calculating the maximum amount of information achievable with the WVA technique. By comparing this information to that achievable by the standard technique, where no post-selection is employed, I show that the WVA technique can be advantageous under a certain class of noise models. Finally, I propose a way to optimally apply the WVA technique.

Nonisotopic detection of human papillomavirus DNA in clinical specimens using a consensus PCR and a generic probe mix in an enzyme-linked immunosorbent assay format.

PubMed

Kornegay, J R; Shepard, A P; Hankins, C; Franco, E; Lapointe, N; Richardson, H; Coutleé, F

2001-10-01

We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR-enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated beta-globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses more objective interpretation criteria, making it useful in studies of HPV natural history.

Molecular alignment and orientation with a hybrid Raman scattering technique

NASA Astrophysics Data System (ADS)

Bustard, Philip J.; Lausten, R.; Sussman, Benjamin J.

2012-11-01

We demonstrate a scheme for the preparation of molecular alignment and angular momentum orientation using a hybrid combination of two limits of Raman scattering. First a weak, impulsive pump pulse initializes the system via the nonresonant dynamic Stark effect. Then, having overcome the influence of the vacuum fluctuations, an amplification pulse selectively enhances the initial coherences by transient stimulated Raman scattering, generating alignment and angular momentum orientation of molecular hydrogen. The amplitude and phase of the resulting coherent dynamics are experimentally probed, indicating an amplification factor of 4.5. An analytic theory is developed to model the dynamics.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology.

PubMed

Binga, Erik K; Lasken, Roger S; Neufeld, Josh D

2008-03-01

Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called multiple displacement amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a 'power tool' in the molecular toolbox of microbial ecologists.

The Microbial Detection Array Combined with Random Phi29-Amplification Used as a Diagnostic Tool for Virus Detection in Clinical Samples

PubMed Central

Erlandsson, Lena; Rosenstierne, Maiken W.; McLoughlin, Kevin; Jaing, Crystal; Fomsgaard, Anders

2011-01-01

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples. PMID:21853040

Evaluation of two molecular techniques for rapid detection of the main dermatophytic agents of tinea capitis.

PubMed

Deng, S; Zhou, Z; de Hoog, G S; Wang, X; Abliz, P; Sun, J; Najafzadeh, M J; Pan, W; Lei, W; Zhu, S; Hasimu, H; Zhang, P; Guo, Y; Deng, D; Liao, W

2015-12-01

Tinea capitis is very common in Western China, with the most widespread aetiological agent being Trichophyton violaceum, while Microsporum canis is prevalent in the remainder of China. Conventional diagnostics and internal transcribed spacer (ITS) sequencing analyses have proven relatively limited due to the close phylogenetic relationship of anthropophilic dermatophytes. Therefore, alternative molecular tools with sufficient specificity, reproducibility and sensitivity are necessary. To evaluate two molecular techniques [multiplex ligation-dependent probe amplification (MLPA) and rolling circle amplification (RCA)] for rapid detection of the aetiological agents of tinea capitis, T. violaceum and M. canis. Probes of RCA and MLPA were designed with target sequences in the rDNA ITS gene region. Strains tested consist of 31 T. violaceum, 22 M. canis and 24 reference strains of species that are taxonomically close to the target species. The specificity and reproducibility of RCA and MLPA in detection of T. violaceum and M. canis were both 100% in both species. Sensitivity testing showed that RCA was positive at concentrations down to 1·68 × 10(6) copies of DNA in the TvioRCA probe, and 2·7 × 10(8) copies of DNA in McRCA. MLPA yielded positive results at concentrations of DNA down to 1·68 × 10(1) copies in the TvioMLPA probe and 2·7 × 10(2) in McMLPA. The two techniques were sufficiently specific and sensitive for discriminating the target DNA of T. violaceum and M. canis from that of closely related dermatophytes. RCA and MLPA are advantageous in their reliability and ease of operation compared with standard polymerase chain reaction and conventional methods. © 2015 British Association of Dermatologists.

Development and evaluation of probe based real time loop mediated isothermal amplification for Salmonella: A new tool for DNA quantification.

PubMed

Mashooq, Mohmad; Kumar, Deepak; Niranjan, Ankush Kiran; Agarwal, Rajesh Kumar; Rathore, Rajesh

2016-07-01

A one step, single tube, accelerated probe based real time loop mediated isothermal amplification (RT LAMP) assay was developed for detecting the invasion gene (InvA) of Salmonella. The probe based RT LAMP is a novel method of gene amplification that amplifies nucleic acid with high specificity and rapidity under isothermal conditions with a set of six primers. The whole procedure is very simple and rapid, and amplification can be obtained in 20min. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of DNA binding dye at the end of the reaction results in colour difference and can be visualized under normal day light and in UV. The sensitivity of developed assay was found 10 fold higher than taqman based qPCR. The specificity of the RT LAMP assay was validated by the absence of any cross reaction with other members of enterobacteriaceae family and other gram negative bacteria. These results indicate that the probe based RT LAMP assay is extremely rapid, cost effective, highly specific and sensitivity and has potential usefulness for rapid Salmonella surveillance. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Detection of Listeria monocytogenes by using the polymerase chain reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bessesen, M.T.; Luo, Q.; Blaser, M.J.

1990-09-01

A method was developed for detection of Listeria monocytogens by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with {sup 32}P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains.

High-speed femtosecond pump-probe spectroscopy with a smart pixel detector array.

PubMed

Bourquin, S; Prasankumar, R P; Kärtner, F X; Fujimoto, J G; Lasser, T; Salathé, R P

2003-09-01

A new femtosecond pump-probe spectroscopy technique is demonstrated that permits the high-speed, parallel acquisition of pump-probe measurements at multiple wavelengths. This is made possible by use of a novel, two-dimensional smart pixel detector array that performs amplitude demodulation in real time on each pixel. This detector array can not only achieve sensitivities comparable with lock-in amplification but also simultaneously performs demodulation of probe transmission signals at multiple wavelengths, thus permitting rapid time- and wavelength-resolved femtosecond pump-probe spectroscopy. Measurements on a thin sample of bulk GaAs are performed across 58 simultaneous wavelengths. Differential probe transmission changes as small as approximately 2 x 10(-4) can be measured over a 5-ps delay scan in only approximately 3 min. This technology can be applied to a wide range of pump-probe measurements in condensed matter, chemistry, and biology.

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

PubMed

Santiago-Felipe, S; Tortajada-Genaro, L A; Puchades, R; Maquieira, A

2014-02-06

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. Copyright © 2013 Elsevier B.V. All rights reserved.

Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes.

PubMed

Marras, Salvatore A E

2006-01-01

With the introduction of simple and relatively inexpensive methods for labeling nucleic acids with nonradioactive labels, doors have been opened that enable nucleic acid hybridization probes to be used for research and development, as well as for clinical diagnostic applications. The use of fluorescent hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. The use of hybridization probes that bind to the amplification products in real-time markedly improves the ability to obtain quantitative results. Furthermore, real-time nucleic acid amplification assays can be carried out in sealed tubes, eliminating carryover contamination. Because fluorescent hybridization probes are available in a wide range of colors, 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. It is therefore important to carefully select the labels of hybridization probes, 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 chapter outlines different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers.

Proximity-dependent isothermal cycle amplification for small-molecule detection based on surface enhanced Raman scattering.

PubMed

Li, Ying; Zeng, Yan; Mao, Yaning; Lei, Chengcun; Zhang, Shusheng

2014-01-15

A novel proximity-dependent isothermal cycle amplification (PDICA) strategy has been proposed and successfully used for the determination of cocaine coupled with surface enhanced Raman scattering (SERS). For enhancing the SERS signal, Raman dye molecules modified bio-barcode DNA and gold nanoparticles (AuNPs) are used to prepare the Raman probes. Magnetic beads (MBs) are used as the carrier of amplification template and signal output products for circumventing the problem of high background induced by excess bio-barcode DNA. In the presence of target molecules, two label-free proximity probes can hybridize with each other and subsequently opens the hairpin connector-probe to perform the PDICA reaction including the target recycling amplification and strand-displacement amplification. As a result, abundant AuNPs Raman probes can be anchored on the surface of MBs and a low detection limit of 0.1 nM for cocaine is obtained. This assay also exhibits an excellent selectivity and has been successfully performed in human serum, which confirms the reliability and practicality of this protocol. © 2013 Elsevier B.V. All rights reserved.

Detection of mycobacterium tuberculosis in paraffin-embedded pleural biopsy specimens by commercial ribosomal RNA and DNA amplification kits.

PubMed

Ruiz-Manzano, J; Manterola, J M; Gamboa, F; Calatrava, A; Monsó, E; Martínez, C; Ausina, V

2000-09-01

To evaluate the utility of two gene amplification systems in historical paraffin-embedded pleural biopsy (PEB) tissues from patients with pleural tuberculosis, and to compare the results to those obtained with conventional histologic and microbiological methods. A retrospective study. Seventy-four formalin-fixed PEB tissues collected and stored over 12 years (1984 through 1995) were retrieved. Gene amplifications were performed in 57 tissues from patients with diagnoses of pleural tuberculosis and in 17 from patients with carcinoma as controls, using the first version of the Amplified Mycobacterium tuberculosis Direct Test (AMTDT; Gen-Probe; San Diego, CA) and the LCx Mycobacterium tuberculosis Assay (LCxMTB; Abbott Laboratories; Abbott Park, IL). The sensitivities of the AMTDT and LCxMTB were 52.6% and 63.2%, respectively (p = not statistically significant). The specificity of both tests was 100%. Twenty tissue samples (35.1%) were positive by both systems, and 10 tissues (17.5%) were positive only by the AMTDT, while 16 tissues (28.1%) were positive only by the LCxMTB. Both tests gave negative results for 11 specimens (19.3%). When both tests were used, a positive diagnosis was achieved in 80.7% of the samples. Diagnosis of 73.7% of patient conditions had previously been made by smear examination of pleural biopsy and sputum, pleural liquid, or biopsy culture. The overall diagnostic yield with both culture and amplification techniques was 96.5% (55 of 57 patients) for pleural tuberculosis, with amplification techniques adding 22.8% of the diagnoses. Amplification techniques are useful in archival PEB tissues, providing additional diagnoses beyond culturing, although the sensitivity should be improved, possibly by standardizing protocols.

High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18.

PubMed

Kumvongpin, Ratchanida; Jearanaikool, Patcharee; Wilailuckana, Chotechana; Sae-Ung, Nattaya; Prasongdee, Prinya; Daduang, Sakda; Wongsena, Metee; Boonsiri, Patcharee; Kiatpathomchai, Wansika; Swangvaree, Sukumarn Sanersak; Sandee, Alisa; Daduang, Jureerut

2016-08-01

High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

PubMed Central

Girard, Laurie D.; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G.

2014-01-01

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets. PMID:25489607

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

PubMed

Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

2015-02-07

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.

Interactive fluorophore and quencher pairs for labeling fluorescent nucleic acid hybridization probes.

PubMed

Marras, Salvatore A E

2008-03-01

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.

Multiplex detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

PubMed

Liu, Yacui; Zhang, Jiangyan; Tian, Jingxiao; Fan, Xiaofei; Geng, Hao; Cheng, Yongqiang

2017-01-01

A simple, highly sensitive, and specific assay was developed for the homogeneous and multiplex detection of microRNAs (miRNAs) by combining molecular beacon (MB) probes and T7 exonuclease-assisted cyclic amplification. An MB probe with five base pairs in the stem region without special modification can effectively prevent the digestion by T7 exonuclease. Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then digested by T7 exonuclease in the 5' to 3' direction. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. The results show that the combination of T7 exonuclease-assisted cyclic amplification reaction and MB probe possesses higher sensitivity for miRNA detection. Moreover, multiplex detection of miRNAs was successfully achieved by designing two MB probes labeled with FAM and Cy3, respectively. As a result, the method opens a new pathway for the sensitive and multiplex detection of miRNAs as well as clinical diagnosis. Graphical Abstract A simple, highly sensitive, and specific assay was developed for the detection of microRNAs by combining molecular beacon probes with T7 exonuclease-assisted cyclic amplification reaction.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

PubMed

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-10-21

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

PubMed Central

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-01-01

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring. PMID:28335318

Detection and signal amplification in zebrafish RNA FISH.

PubMed

Hauptmann, Giselbert; Lauter, Gilbert; Söll, Iris

2016-04-01

In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts. Copyright © 2016 Elsevier Inc. All rights reserved.

Signal amplification of microRNAs with modified strand displacement-based cycling probe technology.

PubMed

Jia, Huning; Bu, Ying; Zou, Bingjie; Wang, Jianping; Kumar, Shalen; Pitman, Janet L; Zhou, Guohua; Song, Qinxin

2016-10-24

Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA. The products generated by SDA are captured by a molecular beacon (MB), which is designed to initiate the second amplification cycle, with a similar principle to the cycling probe technology (CPT), which is based on repeated digestion of the DNA-RNA hybrid by the RNase H. After one sample enrichment and two steps of signal amplification, 0.1 pM of let-7a can be detected. The miRNA assay exhibits a great dynamic range of over 100 orders of magnitude and high specificity to clearly discriminate a single base difference in miRNA sequences. This isothermal amplification does not require any special temperature control instrument. The assay is also about signal amplification rather than template amplification, therefore minimising contamination issues. In addition, there is no need for the reverse transcription (RT) process. Thus the amplification is suitable for miRNA detection.

Signal amplification of padlock probes by rolling circle replication.

PubMed Central

Banér, J; Nilsson, M; Mendel-Hartvig, M; Landegren, U

1998-01-01

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe. PMID:9801302

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

PubMed

Nugen, Sam R; Leonard, Barbara; Baeumner, Antje J

2007-05-15

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.

An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

PubMed

Xu, Jingguo; Guo, Jia; Maina, Sarah Wanjiku; Yang, Yumeng; Hu, Yimin; Li, Xuanxuan; Qiu, Jiarong; Xin, Zhihong

2018-05-15

An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H 2 O 2 . The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-10 4  CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry. Copyright © 2018 Elsevier Inc. All rights reserved.

Raman amplification in the coherent wave-breaking regime.

PubMed

Farmer, J P; Pukhov, A

2015-12-01

In regimes far beyond the wave-breaking threshold of Raman amplification, we show that significant amplification can occur after the onset of wave breaking, before phase mixing destroys the coherent coupling between pump, probe, and plasma wave. Amplification in this regime is therefore a transient effect, with the higher-efficiency "coherent wave-breaking" (CWB) regime accessed by using a short, intense probe. Parameter scans illustrate the marked difference in behavior between below wave breaking, in which the energy-transfer efficiency is high but total energy transfer is low, wave breaking, in which efficiency is low, and CWB, in which moderate efficiencies allow the highest total energy transfer.

Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques.

PubMed

Passamani, Paulo Z; Carvalho, Carlos R; Soares, Fernanda A F

2018-01-01

Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

A cascade amplification strategy based on rolling circle amplification and hydroxylamine amplified gold nanoparticles enables chemiluminescence detection of adenosine triphosphate.

PubMed

Wang, Ping; Zhang, Tonghuan; Yang, Taoyi; Jin, Nan; Zhao, Yanjun; Fan, Aiping

2014-08-07

A highly sensitive and selective chemiluminescent (CL) biosensor for adenosine triphosphate (ATP) was developed by taking advantage of the ATP-dependent enzymatic reaction (ATP-DER), the powerful signal amplification capability of rolling circle amplification (RCA), and hydroxylamine-amplified gold nanoparticles (Au NPs). The strategy relies on the ability of ATP, a cofactor of T4 DNA ligase, to trigger the ligation-RCA reaction. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe, producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The ATP-DER is eventually converted into a detectable CL signal after a series of processes, including gold probe hybridization, hydroxylamine amplification, and oxidative gold metal dissolution coupled with a simple and sensitive luminol CL reaction. The CL signal is directly proportional to the ATP level. The results showed that the detection limit of the assay is 100 pM of ATP, which compares favorably with those of other ATP detection techniques. In addition, by taking advantage of ATP-DER, the proposed CL sensing system exhibits extraordinary specificity towards ATP and could distinguish the target molecule ATP from its analogues. The proposed method provides a new and versatile platform for the design of novel DNA ligation reaction-based CL sensing systems for other cofactors. This novel ATP-DER based CL sensing system may find wide applications in clinical diagnosis as well as in environmental and biomedical fields.

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification.

PubMed

Zeng, Yan; Wan, Yi; Zhang, Dun; Qi, Peng

2015-01-01

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3'-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14 pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria. Copyright © 2014 Elsevier B.V. All rights reserved.

Impacts of Sampling and Handling Procedures on DNA- and RNA-based Microbial Characterization and Quantification of Groundwater and Saturated Soil

DTIC Science & Technology

2012-07-01

use of molecular biological techniques (MBTs) has allowed microbial ecologists and environmental engineers to determine microbial community...metabolic genes). The most common approaches used in bioremediation research are those based on the polymerase chain reaction (PCR) amplification of... bioremediation . Because of its sensitivity compared to direct hybridization/probing, PCR is increasingly used to analyze groundwater samples and soil samples

Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

PubMed

Hoser, Mark J; Mansukoski, Hannu K; Morrical, Scott W; Eboigbodin, Kevin E

2014-01-01

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

[Utilization of nylon membranes for specific isolation and characterization of verotoxin-producing Escherichia coli using DNA probes].

PubMed

Gallien, P; Klie, H; Perlberg, K W; Protz, D

1996-01-01

A method for specific isolation of VT(+)-strains in raw milk is given. DNA-hybridization technique with DIG-labeled PCR-amplificates as probes are the basis. No background is seen by using "DIG Easy Hyb" solution and nylon membranes for colony- and plaque-hybridization (Boehringer Mannheim GmbH). Marked colonies are visible on the membranes after detection. So it is possible to select these colonies from a masterplate. The results are available within one day (without enrichment and membrane preparation). After stripping the membranes can be used for a new hybridisation to detect another factor of virulence.

Sequence independent amplification of DNA

DOEpatents

Bohlander, S.K.

1998-03-24

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example, the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei. 25 figs.

Sequence independent amplification of DNA

DOEpatents

Bohlander, Stefan K.

1998-01-01

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei.

A novel sensitive pathogen detection system based on Microbead Quantum Dot System.

PubMed

Wu, Tzong-Yuan; Su, Yi-Yu; Shu, Wei-Hsien; Mercado, Augustus T; Wang, Shi-Kwun; Hsu, Ling-Yi; Tsai, Yow-Fu; Chen, Chung-Yung

2016-04-15

A fast and accurate detection system for pathogens can provide immediate measurements for the identification of infectious agents. Therefore, the Microbead Quantum-dots Detection System (MQDS) was developed to identify and measure target DNAs of pathogenic microorganisms and eliminated the need of PCR amplifications. This nanomaterial-based technique can detect different microorganisms by flow cytometry measurements. In MQDS, pathogen specific DNA probes were designed to form a hairpin structure and conjugated on microbeads. In the presence of the complementary target DNA sequence, the probes will compete for binding with the reporter probes but will not interfere with the binding between the probe and internal control DNA. To monitor the binding process by flow cytometry, both the reporter probes and internal control probes were conjugated with Quantum dots that fluoresce at different emission wavelengths using the click reaction. When MQDS was used to detect the pathogens in environmental samples, a high correlation coefficient (R=0.994) for Legionella spp., with a detection limit of 0.1 ng of the extracted DNAs and 10 CFU/test, can be achieved. Thus, this newly developed technique can also be applied to detect other pathogens, particularly viruses and other genetic diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification.

PubMed

Carinelli, S; Kühnemund, M; Nilsson, M; Pividori, M I

2017-07-15

This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries. Copyright © 2016 Elsevier B.V. All rights reserved.

Self-locked aptamer probe mediated cascade amplification strategy for highly sensitive and selective detection of protein and small molecule.

PubMed

Li, Wei; Jiang, Wei; Wang, Lei

2016-10-12

In this work, a novel self-locked aptamer probe mediated cascade amplification strategy has been constructed for highly sensitive and specific detection of protein. First, the self-locked aptamer probe was designed with three functions: one was specific molecular recognition attributed to the aptamer sequence, the second was signal transduction owing to the transduction sequence, and the third was self-locking through the hybridization of the transduction sequence and part of the aptamer sequence. Then, the aptamer sequence specific recognized the target and folded into a three-way helix junction, leading to the release of the transduction sequence. Next, the 3'-end of this three-way junction acted as primer to trigger the strand displacement amplification (SDA), yielding a large amount of primers. Finally, the primers initiated the dual-exponential rolling circle amplification (DE-RCA) and generated numerous G-quadruples sequences. By inserting the fluorescent dye N-methyl mesoporphyrin IX (NMM), enhanced fluorescence signal was achieved. In this strategy, the self-locked aptamer probe was more stable to reduce the interference signals generated by the uncontrollable folding in unbounded state. Through the cascade amplification of SDA and DE-RCA, the sensitivity was further improved with a detection limit of 3.8 × 10(-16) mol/L for protein detection. Furthermore, by changing the aptamer sequence of the probe, sensitive and selective detection of adenosine has been also achieved, suggesting that the proposed strategy has good versatility and can be widely used in sensitive and selective detection of biomolecules. Copyright © 2016 Elsevier B.V. All rights reserved.

Feasibility and Outcomes of Multiplex Ligation-Dependent Probe Amplification on Buccal Smears as a Screening Method for Microdeletions and Duplications among 300 Adults with an Intellectual Disability of Unknown Aetiology

ERIC Educational Resources Information Center

Peppink, D.; Douma-Kloppenburg, D. D.; de Rooij-Askes, E. S. P.; van Zoest, I. M.; Evenhuis, H. M.; Gille, J. J. P.; van Hagen, J. M.

2008-01-01

Background: Determining the aetiology of intellectual disability (ID) enables anticipation of specific comorbidity and can thus be beneficial. Blood sampling, however, is considered stressful for people with ID. Our aim was to evaluate the feasibility of a non-invasive screening technique of nine microdeletions/duplications among adults with ID of…

Construction of a microfluidic chip, using dried-down reagents, for LATE-PCR amplification and detection of single-stranded DNA.

PubMed

Jia, Yanwei; Mak, Pui-In; Massey, Conner; Martins, Rui P; Wangh, Lawrence J

2013-12-07

LATE-PCR is an advanced form of non-symmetric PCR that efficiently generates single-stranded DNA which can readily be characterized at the end of amplification by hybridization to low-temperature fluorescent probes. We demonstrate here for the first time that monoplex and duplex LATE-PCR amplification and probe target hybridization can be carried out in double layered PDMS microfluidics chips containing dried reagents. Addition of a set of reagents during dry down overcomes the common problem of single-stranded oligonucleotide binding to PDMS. These proof-of-principle results open the way to construction of inexpensive point-of-care devices that take full advantage of the analytical power of assays built using LATE-PCR and low-temperature probes.

Linear RNA amplification for the production of microarray hybridization probes.

PubMed

Klebes, Ansgar; Kornberg, Thomas B

2008-01-01

To understand Drosophila development and other genetically controlled processes, it is often desirable to identify differences in gene expression levels. An experimental approach to investigate these processes is to catalog the transcriptome by hybridization of mRNA to DNA microbar-rays. In these experiments mRNA-derived hybridization probes are produced and hybridized to an array of DNA spots on a solid support. The labeled cDNAs of the complex hybridization probe will bind to their complementary sequences and provide quantification of the relative concentration of the corresponding transcript in the starting material. However, such approaches are often limited by the scarcity of the experimental sample because standard methods of probe preparation require microgram quantities of mRNA template. Linear RNA amplification can alleviate such limitations to support the generation of microarray hybridization probes from a few 100 pg of mRNA. These smaller quantities can be isolated from a few 100 cells. Here, we present a linear amplification protocol designed to preserve both the relative abundance of transcripts as well as their sequence complexity.

Simple and Sensitive Quantification of MicroRNAs via PS@Au Microspheres-Based DNA Probes and DSN-Assisted Signal Amplification Platform.

PubMed

Zhao, Qian; Piao, Jiafang; Peng, Weipan; Wang, Yang; Zhang, Bo; Gong, Xiaoqun; Chang, Jin

2018-01-31

Identifying the microRNA (miRNA) expression level can provide critical information for early diagnosis of cancers or monitoring the cancer therapeutic efficacy. This paper focused on a kind of gold-nanoparticle-coated polystyrene microbeads (PS@Au microspheres)-based DNA probe as miRNA capture and duplex-specific nuclease (DSN) signal amplification platform based on an RGB value readout for detection of miRNAs. In virtue of the outstanding selectivity and simple experimental operation, 5'-fluorochrome-labeled molecular beacons (MBs) were immobilized on PS@Au microspheres via their 3'-thiol, in the wake of the fluorescence quenching by nanoparticle surface energy transfer (NSET). Target miRNAs were captured by the PS@Au microspheres-based DNA probe through DNA/RNA hybridization. DSN enzyme subsequently selectively cleaved the DNA to recycle the target miRNA and release of fluorophores, thereby triggering the signal amplification with more free fluorophores. The RGB value measurement enabled a detection limit of 50 fM, almost 4 orders of magnitude lower than PS@Au microspheres-based DNA probe detection without DSN. Meanwhile, by different encoding of dyes, miRNA-21 and miRNA-10b were simultaneously detected in the same sample. Considering the ability for quantitation, high sensitivity, and convenient merits, the PS@Au microspheres-based DNA probe and DSN signal amplification platform supplied valuable information for early diagnosis of cancers.

Fluorescent Quantification of DNA Based on Core-Shell Fe3O4@SiO2@Au Nanocomposites and Multiplex Ligation-Dependent Probe Amplification.

PubMed

Fan, Jing; Yang, Haowen; Liu, Ming; Wu, Dan; Jiang, Hongrong; Zeng, Xin; Elingarami, Sauli; Ll, Zhiyang; Li, Song; Liu, Hongna; He, Nongyue

2015-02-01

In this research, a novel method for relative fluorescent quantification of DNA based on Fe3O4@SiO2@Au gold-coated magnetic nanocomposites (GMNPs) and multiplex ligation- dependent probe amplification (MLPA) has been developed. With the help of self-assembly, seed-mediated growth and chemical reduction method, core-shell Fe3O4@SiO2@Au GMNPs were synthesized. Through modified streptavidin on the GMNPs surface, we obtained a bead chip which can capture the biotinylated probes. Then we designed MLPA probes which were tagged with biotin or Cy3 and target DNA on the basis of human APP gene sequence. The products from the thermostable DNA ligase induced ligation reactions and PCR amplifications were incubated with SA-GMNPs. After washing, magnetic separation, spotting, the fluorescent scanning results showed our method can be used for the relative quantitative analysis of the target DNA in the concentration range of 03004~0.5 µM.

DNA fingerprinting of Brassica juncea cultivars using microsatellite probes.

PubMed

Bhatia, S; Das, S; Jain, A; Lakshmikumaran, M

1995-09-01

The genetic variability in the Brassica juncea cultivars was detected by employing in-gel hybridization of restricted DNA to simple repetitive sequences such as (GATA)4, (GACA)4 and (CAC)5. The most informative probe/enzyme combination was (GATA)4/EcoRI, yielding highly polymorphic fingerprint patterns for the B. juncea cultivars. This technique was found to be dependable for establishing the variety specific patterns for most of the cultivars studied, a prerequisite for germplasm preservation. The results of the present study were compared with those reported in our earlier study in which random amplification of polymorphic DNA (RAPD) was used for assessing the genetic variability in the B. juncea cultivars.

In Situ Detection of MicroRNA Expression with RNAscope Probes.

PubMed

Yin, Viravuth P

2018-01-01

Elucidating the spatial resolution of gene transcripts provides important insight into potential gene function. MicroRNAs are short, singled-stranded noncoding RNAs that control gene expression through base-pair complementarity with target mRNAs in the 3' untranslated region (UTR) and inhibiting protein expression. However, given their small size of ~22- to 24-nt and low expression levels, standard in situ hybridization detection methods are not amendable for microRNA spatial resolution. Here, I describe a technique that employs RNAscope probe design and propriety amplification technology that provides simultaneous single molecule detection of individual microRNA and its target gene. This method allows for rapid and sensitive detection of noncoding RNA transcripts in frozen tissue sections.

Adiabatic Amplification of Plasmons and Demons in 2D Systems

NASA Astrophysics Data System (ADS)

Sun, Zhiyuan; Basov, D. N.; Fogler, M. M.

2016-08-01

We theoretically investigate charged collective modes in a two-dimensional conductor with hot electrons where the instantaneous mode frequencies gradually increase or decrease with time. We show that the loss compensation or even amplification of the modes may occur. We apply our theory to two types of collective modes in graphene, the plasmons and the energy waves, which can be probed in optical pump-probe experiments.

Adiabatic Amplification of Plasmons and Demons in 2D Systems.

PubMed

Sun, Zhiyuan; Basov, D N; Fogler, M M

2016-08-12

We theoretically investigate charged collective modes in a two-dimensional conductor with hot electrons where the instantaneous mode frequencies gradually increase or decrease with time. We show that the loss compensation or even amplification of the modes may occur. We apply our theory to two types of collective modes in graphene, the plasmons and the energy waves, which can be probed in optical pump-probe experiments.

Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

PubMed

Del Río, Jonathan Sabaté; Lobato, Ivan Magriñà; Mayboroda, Olena; Katakis, Ioanis; O'Sullivan, Ciara K

2017-05-01

Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10 -15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

A signal-on electrochemical aptasensor for ultrasensitive detection of endotoxin using three-way DNA junction-aided enzymatic recycling and graphene nanohybrid for amplification

NASA Astrophysics Data System (ADS)

Bai, Lijuan; Chai, Yaqin; Pu, Xiaoyun; Yuan, Ruo

2014-02-01

Endotoxin, also known as lipopolysaccharide (LPS), is able to induce a strong immune response on its internalization into mammalian cells. To date, aptamer-based biosensors for LPS detection have been rarely reported. This work describes a new signal-on electrochemical aptasensor for the ultrasensitive detection of LPS by combining the three-way DNA hybridization process and nanotechnology-based amplification. With the help of DNA1 (associated with the concentration of target LPS), the capture probe hybridizes with DNA1 and the assistant probe to open its hairpin structure and form a ternary ``Y'' junction structure. The DNA1 can be released from the structure in the presence of nicking endonuclease to initiate the next hybridization process. Then a great deal of cleaved capture probe produced in the cyclic process can bind with DNA2-nanocomposite, which contains the electroactive toluidine blue (Tb) with the amplification materials graphene (Gra) and gold nanoparticles (AuNPs). Thus, an enhanced electrochemical signal can be easily read out. With the cascade signal amplification, this newly designed protocol provides an ultrasensitive electrochemical detection of LPS down to the femtogram level (8.7 fg mL-1) with a linear range of 6 orders of magnitude (from 10 fg mL-1 to 50 ng mL-1). Moreover, the high sensitivity and specificity make this method versatile for the detection of other biomolecules by changing the corresponding sequences of the capture probe and the assistant probe.

[Quantitative PCR in the diagnosis of Leishmania].

PubMed

Mortarino, M; Franceschi, A; Mancianti, F; Bazzocchi, C; Genchi, C; Bandi, C

2004-06-01

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.

Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

PubMed

Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

2015-06-16

A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

High frequency of ribosomal protein gene deletions in Italian Diamond-Blackfan anemia patients detected by multiplex ligation-dependent probe amplification assay

PubMed Central

Quarello, Paola; Garelli, Emanuela; Brusco, Alfredo; Carando, Adriana; Mancini, Cecilia; Pappi, Patrizia; Vinti, Luciana; Svahn, Johanna; Dianzani, Irma; Ramenghi, Ugo

2012-01-01

Diamond-Blackfan anemia is an autosomal dominant disease due to mutations in nine ribosomal protein encoding genes. Because most mutations are loss of function and detected by direct sequencing of coding exons, we reasoned that part of the approximately 50% mutation negative patients may have carried a copy number variant of ribosomal protein genes. As a proof of concept, we designed a multiplex ligation-dependent probe amplification assay targeted to screen the six genes that are most frequently mutated in Diamond-Blackfan anemia patients: RPS17, RPS19, RPS26, RPL5, RPL11, and RPL35A. Using this assay we showed that deletions represent approximately 20% of all mutations. The combination of sequencing and multiplex ligation-dependent probe amplification analysis of these six genes allows the genetic characterization of approximately 65% of patients, showing that Diamond-Blackfan anemia is indisputably a ribosomopathy. PMID:22689679

Development and Comparison of a Rapid Isothermal Nucleic Acid Amplification Test for Typing of Herpes Simplex Virus Types 1 and 2 on a Portable Fluorescence Detector

PubMed Central

Tong, Yanhong; McCarthy, Kaitlin; Kong, Huimin; Lemieux, Bertrand

2013-01-01

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration–cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH). PMID:22951487

Can we rely on the multiplex ligation-dependent probe amplification method (MLPA) for prenatal diagnosis?

PubMed Central

Omrani, Mir Davood; Azizi, Faezeh; Rajabibazl, Masoumeh; Safavi Naini, Niloufar; Omrani, Sara; Abbasi, Arezo Mona; Saleh Gargari, Soraya

2014-01-01

Background: The major aneuploidies that are diagnosed prenatally involve the autosomal chromosomes 13, 18, and 21, as well as sex chromosomes, X and Y. Because multiplex ligation-dependent probe amplification (MLPA) is rapid and non-invasive, it has replaced traditional culture methods for the screening and diagnosis of common aneuploidies in some countries. Objective: To evaluate the sensitivity and specificity of MLPA in a cross-sectional descriptive study for the detection of chromosomal aneuploidies in comparison to other methods. Materials and Methods: Genomic DNA was extracted from the peripheral blood samples of 10 normal controls and the amniotic fluid of 55 patients. Aneuploidies screening of chromosomes 13, 18, 21, X and Y were carried out using specific MLPA probe mixes (P095-A2). For comparison purposes, samples were also tested by Quantitative Fluorescent-PCR (QF-PCR) and routine chromosomal culture method. Results: Using this specific MLPA technique and data-analyzing software (Genemarker v1.85), one case was diagnosed with 45, X (e.g. Monosomy X or Turner’s Syndrome), and the remaining 54 cases revealed normal karyotypes. These results were concordant with routine chromosomal culture and QF-PCR findings. Conclusion: The experiment demonstrates that MLPA can provide a rapid and accurate clinical method for prenatal identification of common chromosomal aneuploidies with 100% sensitivity and 100% specificity. PMID:24976821

Unlabeled probes for the detection and typing of herpes simplex virus.

PubMed

Dames, Shale; Pattison, David C; Bromley, L Kathryn; Wittwer, Carl T; Voelkerding, Karl V

2007-10-01

Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however. Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR. The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe. Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.

Strand Displacement Amplification Reaction on Quantum Dot-Encoded Silica Bead for Visual Detection of Multiplex MicroRNAs.

PubMed

Qu, Xiaojun; Jin, Haojun; Liu, Yuqian; Sun, Qingjiang

2018-03-06

The combination of microbead array, isothermal amplification, and molecular signaling enables the continuous development of next-generation molecular diagnostic techniques. Herein we reported the implementation of nicking endonuclease-assisted strand displacement amplification reaction on quantum dots-encoded microbead (Qbead), and demonstrated its feasibility for multiplexed miRNA assay in real sample. The Qbead featured with well-defined core-shell superstructure with dual-colored quantum dots loaded in silica core and shell, respectively, exhibiting remarkably high optical encoding stability. Specially designed stem-loop-structured probes were immobilized onto the Qbead for specific target recognition and amplification. In the presence of low abundance of miRNA target, the target triggered exponential amplification, producing a large quantity of stem-G-quadruplexes, which could be selectively signaled by a fluorescent G-quadruplex intercalator. In one-step operation, the Qbead-based isothermal amplification and signaling generated emissive "core-shell-satellite" superstructure, changing the Qbead emission-color. The target abundance-dependent emission-color changes of the Qbead allowed direct, visual detection of specific miRNA target. This visualization method achieved limit of detection at the subfemtomolar level with a linear dynamic range of 4.5 logs, and point-mutation discrimination capability for precise miRNA analyses. The array of three encoded Qbeads could simultaneously quantify three miRNA biomarkers in ∼500 human hepatoma carcinoma cells. With the advancements in ease of operation, multiplexing, and visualization capabilities, the isothermal amplification-on-Qbead assay could potentially enable the development of point-of-care diagnostics.

Molecular identification of Neofabraea species associated with bull's-eye rot on apple using rolling-circle amplification of partial EF-1α sequence.

PubMed

Lin, Huijiao; Jiang, Xiang; Yi, Jianping; Wang, Xinguo; Zuo, Ranling; Jiang, Zide; Wang, Weifang; Zhou, Erxun

2018-01-01

A rolling-circle amplification (RCA) method with padlock probes targeted on EF-1α regions was developed for rapid detection of apple bull's-eye rot pathogens, including Neofabraea malicorticis, N. perennans, N. kienholzii, and N. vagabunda (synonym: N. alba). Four padlock probes (PLP-Nm, PLP-Np, PLP-Nk, and PLP-Nv) were designed and tested against 28 samples, including 22 BER pathogen cultures, 4 closely related species, and 2 unrelated species that may cause serious apple decays. The assay successfully identified all the bull's-eye rot pathogenic fungi at the level of species, while no cross-reaction was observed in all target species and no false-positive reaction was observed with all strains used for reference. This study showed that the use of padlock probes and the combination of probe signal amplification by RCA provided an effective and sensitive method for the rapid identification of Neofabraea spp. The method could therefore be a useful tool for monitoring bull's-eye rot pathogens in port quarantine and orchard epidemiological studies.

Single-Cell Protein Analysis

PubMed Central

Wu, Meiye; Singh, Anup K

2012-01-01

Heterogeneity of cellular systems has been widely recognized but only recently have tools become available that allow probing of genes and proteins in single cells to understand it. While the advancement in single cell genomic analysis has been greatly aided by the power of amplification techniques (e.g., PCR), analysis of proteins in single cells has proven to be more challenging. However, recent advances in multi-parameter flow cytometry, microfluidics and other techniques have made it possible to measure wide variety of proteins in single cells. In this review, we highlight key recent developments in analysis of proteins in a single cell, and discuss their significance in biological research. PMID:22189001

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

PubMed Central

Dreier, Jens; Störmer, Melanie; Kleesiek, Knut

2005-01-01

Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at −20°C, 4°C, and room temperature was demonstrated. PMID:16145106

Invasive reaction assisted strand-displacement signal amplification for sensitive DNA detection.

PubMed

Zou, Bingjie; Song, Qinxin; Wang, Jianping; Liu, Yunlong; Zhou, Guohua

2014-11-18

A novel DNA detection assay was proposed by invasive reaction coupled with molecular beacon assisted strand-displacement signal amplification (IRASA). Target DNAs are firstly hybridized to two probes to initiate invasive reaction to produce amplified flaps. Then these flaps are further amplified by strand-displacement signal amplification. The detection limit was around 0.2 pM.

Sensitive detection of point mutation using exponential strand displacement amplification-based surface enhanced Raman spectroscopy.

PubMed

Huang, Si-Qiang; Hu, Juan; Zhu, Guichi; Zhang, Chun-Yang

2015-03-15

Accurate identification of point mutation is particularly imperative in the field of biomedical research and clinical diagnosis. Here, we develop a sensitive and specific method for point mutation assay using exponential strand displacement amplification (SDA)-based surface enhanced Raman spectroscopy (SERS). In this method, a discriminating probe and a hairpin probe are designed to specifically recognize the sequence of human K-ras gene. In the presence of K-ras mutant target (C→T), the 3'-terminal of discriminating probe and the 5'-terminal of hairpin probe can be ligated to form a SDA template. Subsequently, the 3'-terminal of hairpin probe can function as a primer to initiate the SDA reaction, producing a large amount of triggers. The resultant triggers can further hybridize with the discriminating probes to initiate new rounds of SDA reaction, leading to an exponential amplification reaction. With the addition of capture probe-modified gold nanoparticles (AuNPs) and the Rox-labeled reporter probes, the amplified triggers can be assembled on the surface of AuNPs through the formation of sandwich hybrids of capture probe-trigger-reporter probe, generating a strong Raman signal. While in the presence of K-ras wild-type target (C), neither ligation nor SDA reaction can be initiated and no Raman signal is observed. The proposed method exhibits high sensitivity with a detection limit of 1.4pM and can accurately discriminate as low as 1% variant frequency from the mixture of mutant target and wild-type target. Importantly, this method can be further applied to analyze the mutant target in the spiked HEK293T cell lysate, holding great potential for genetic analysis and disease prognosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

PubMed

Reid, Michael S; Le, X Chris; Zhang, Hongquan

2018-04-27

Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and comparison of a rapid isothermal nucleic acid amplification test for typing of herpes simplex virus types 1 and 2 on a portable fluorescence detector.

PubMed

Tong, Yanhong; McCarthy, Kaitlin; Kong, Huimin; Lemieux, Bertrand

2012-11-01

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration-cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH). Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A split recognition mode combined with cascade signal amplification strategy for highly specific, sensitive detection of microRNA.

PubMed

Wang, Rui; Wang, Lei; Zhao, Haiyan; Jiang, Wei

2016-12-15

MicroRNAs (miRNAs) are vital for many biological processes and have been regarded as cancer biomarkers. Specific and sensitive detection of miRNAs is essential for cancer diagnosis and therapy. Herein, a split recognition mode combined with cascade signal amplification strategy is developed for highly specific and sensitive detection of miRNA. The split recognition mode possesses two specific recognition processes, which are based on toehold-mediated strand displacement reaction (TSDR) and direct hybridization reaction. Two recognition probes, hairpin probe (HP) with overhanging toehold domain and assistant probe (AP), are specially designed. Firstly, the toehold domain of HP and AP recognize part of miRNA simultaneously, accompanied with TSDR to unfold the HP and form the stable DNA Y-shaped junction structure (YJS). Then, the AP in YJS can further act as primer to initiate strand displacement amplification, releasing numerous trigger sequences. Finally, the trigger sequences hybridize with padlock DNA to initiate circular rolling circle amplification and generate enhanced fluorescence responses. In this strategy, the dual recognition effect of split recognition mode guarantees the excellent selectivity to discriminate let-7b from high-homology sequences. Furthermore, the high amplification efficiency of cascade signal amplification guarantees a high sensitivity with the detection limit of 3.2 pM and the concentration of let-7b in total RNA sample extracted from Hela cells is determined. These results indicate our strategy will be a promising miRNA detection strategy in clinical diagnosis and disease treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

Amplification of chromosomal DNA in situ

DOEpatents

Christian, Allen T.; Coleman, Matthew A.; Tucker, James D.

2002-01-01

Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

Microfluidic electrochemical assay for rapid detection and quantification of Escherichia coli.

PubMed

Safavieh, Mohammadali; Ahmed, Minhaz Uddin; Tolba, Mona; Zourob, Mohammed

2012-01-15

Microfluidic electrochemical biosensor for performing Loop-mediated isothermal amplification (LAMP) was developed for the detection and quantification of Escherichia coli. The electrochemical detection for detecting the DNA amplification was achieved using Hoechst 33258 redox molecule and linear sweep voltametry (LSV). The DNA aggregation and minor groove binding with redox molecule cause a significant drop in the anodic oxidation of LSV. Unlike other electrochemical techniques, this method does not require the probe immobilization and the detection of the bacteria can be accomplished in a single chamber without DNA extraction and purification steps. The isothermal amplification time has a major role in the quantification of the bacteria. We have shown that we could detect and quantify 24 CFU/ml of bacteria and 8.6 fg/μl DNA in 60 min and 48 CFU/ml of bacteria in 35 min in LB media and urine samples. We believe that this microfluidic chip has great potential to be used as a point of care diagnostic (POC) device in the clinical/hospital application. Copyright © 2011 Elsevier B.V. All rights reserved.

Genus Level Identification of Mycobacteria from Clinical Specimens by Using an Easy-To-Handle Mycobacterium-Specific PCR Assay

PubMed Central

Stauffer, Fritz; Haber, Heinrich; Rieger, Armin; Mutschlechner, Robert; Hasenberger, Petra; Tevere, Vincent J.; Young, Karen K. Y.

1998-01-01

An easy-to-handle Mycobacterium-specific PCR assay for detection of the presence of a wide range of mycobacterial species in clinical samples was evaluated. The performance of the genus probe was compared with the performance of probes specific for Mycobacterium tuberculosis and Mycobacterium avium and with that of standard culture. In addition, the utility of an internal control in monitoring amplification inhibitors was studied. Of 545 respiratory and 325 nonrespiratory specimens (a total of 870 specimens), 58 (6.7%) showed the presence of amplification inhibitors, as determined by a negative result for the internal control. Of these 58 specimens, 31 (53%) were stool specimens; other material, even citrate blood after lysis of erythrocytes, did not pose a problem with regard to inhibition of PCR amplification. Eighty-one of the remaining 812 specimens had a positive Mycobacterium culture result. Of these culture-positive specimens, 58 (71.6%) showed a positive result with the Mycobacterium genus-specific probe. Seventy-two samples had a positive result with the Mycobacterium-specific probe but a negative culture result. Of these 72 samples, 26 samples were regarded as true positive, either because the M. tuberculosis- or M. avium-specific probe was also positive at the same time or because other specimens from the same patient taken at the same time were culture positive. The sensitivity of the Mycobacterium-specific probe was 78.5% and the specificity was 93.5%. This study showed that pretesting of clinical specimens for mycobacteria to the genus level with a Mycobacterium-specific probe offers the routine clinical laboratory the possibility of detecting tuberculous and nontuberculous mycobacteria with one test. Furthermore, specimens testing positive with the genus-specific probe can be immediately identified with species-specific probes. PMID:9508282

Enhancement of signal-to-noise ratio in Brillouin optical time domain analyzers by dual-probe detection

NASA Astrophysics Data System (ADS)

Iribas, Haritz; Loayssa, Alayn; Sauser, Florian; Llera, Miguel; Le Floch, Sébastien

2017-04-01

We demonstrate a simple technique to enhance the signal-to-noise ratio (SNR) in Brillouin optical time-domain analysis sensors by the addition of gain and loss processes. The technique is based on the shift of the pump pulse optical frequency in a double-sideband probe system, so that the gain and loss processes take place at different frequencies. In this manner, the loss and the gain do not cancel each other out, and it makes possible to take advantage of both informations at the same time, obtaining an improvement of 3 dB on the SNR. Furthermore, the technique does not need an optical filtering, so that larger improvement on SNR and a simplification of the setup are obtained. The method is experimentally demonstrated in a 101 km fiber spool, obtaining a measurement uncertainty of 2.6 MHz (2σ) at the worst-contrast position for 2 m spatial resolution. This leads, to the best of our knowledge, to the highest figure-of-merit in a BOTDA without using coding or raman amplification.

Reflective Amplification without Population Inversion from a Strongly Driven Superconducting Qubit

NASA Astrophysics Data System (ADS)

Wen, P. Y.; Kockum, A. F.; Ian, H.; Chen, J. C.; Nori, F.; Hoi, I.-C.

2018-02-01

Amplification of optical or microwave fields is often achieved by strongly driving a medium to induce population inversion such that a weak probe can be amplified through stimulated emission. Here we strongly couple a superconducting qubit, an artificial atom, to the field in a semi-infinite waveguide. When driving the qubit strongly on resonance such that a Mollow triplet appears, we observe a 7% amplitude gain for a weak probe at frequencies in between the triplet. This amplification is not due to population inversion, neither in the bare qubit basis nor in the dressed-state basis, but instead results from a four-photon process that converts energy from the strong drive to the weak probe. We find excellent agreement between the experimental results and numerical simulations without any free fitting parameters. Since our device consists of a single two-level artificial atom, the simplest possible quantum system, it can be viewed as the most fundamental version of a four-wave-mixing parametric amplifier.

Development of microbial genome-probing microarrays using digital multiple displacement amplification of uncultivated microbial single cells.

PubMed

Chang, Ho-Won; Sung, Youlboong; Kim, Kyoung-Ho; Nam, Young-Do; Roh, Seong Woon; Kim, Min-Soo; Jeon, Che Ok; Bae, Jin-Woo

2008-08-15

A crucial problem in the use of previously developed genome-probing microarrays (GPM) has been the inability to use uncultivated bacterial genomes to take advantage of the high sensitivity and specificity of GPM in microbial detection and monitoring. We show here a method, digital multiple displacement amplification (MDA), to amplify and analyze various genomes obtained from single uncultivated bacterial cells. We used 15 genomes from key microbes involved in dichloromethane (DCM)-dechlorinating enrichment as microarray probes to uncover the bacterial population dynamics of samples without PCR amplification. Genomic DNA amplified from single cells originating from uncultured bacteria with 80.3-99.4% similarity to 16S rRNA genes of cultivated bacteria. The digital MDA-GPM method successfully monitored the dynamics of DCM-dechlorinating communities from different phases of enrichment status. Without a priori knowledge of microbial diversity, the digital MDA-GPM method could be designed to monitor most microbial populations in a given environmental sample.

Amplifications of chromosomal region 20q13 as a prognostic indicator breast cancer

DOEpatents

Gray, Joe W.; Collins, Colin; Pinkel, Daniel; Kallioniemi, Olli-Pekka; Tanner, Minna M.

2001-01-01

The present invention relates to in situ hybridization methods for the identification of new chromosomal abnormalities associated with various diseases. In particular, it provides probes which are specific to a region of amplification in chromosome 20.

Ligation with Nucleic Acid Sequence–Based Amplification

PubMed Central

Ong, Carmichael; Tai, Warren; Sarma, Aartik; Opal, Steven M.; Artenstein, Andrew W.; Tripathi, Anubhav

2012-01-01

This work presents a novel method for detecting nucleic acid targets using a ligation step along with an isothermal, exponential amplification step. We use an engineered ssDNA with two variable regions on the ends, allowing us to design the probe for optimal reaction kinetics and primer binding. This two-part probe is ligated by T4 DNA Ligase only when both parts bind adjacently to the target. The assay demonstrates that the expected 72-nt RNA product appears only when the synthetic target, T4 ligase, and both probe fragments are present during the ligation step. An extraneous 38-nt RNA product also appears due to linear amplification of unligated probe (P3), but its presence does not cause a false-positive result. In addition, 40 mmol/L KCl in the final amplification mix was found to be optimal. It was also found that increasing P5 in excess of P3 helped with ligation and reduced the extraneous 38-nt RNA product. The assay was also tested with a single nucleotide polymorphism target, changing one base at the ligation site. The assay was able to yield a negative signal despite only a single-base change. Finally, using P3 and P5 with longer binding sites results in increased overall sensitivity of the reaction, showing that increasing ligation efficiency can improve the assay overall. We believe that this method can be used effectively for a number of diagnostic assays. PMID:22449695

An Engineered Kinetic Amplification Mechanism for Single Nucleotide Variant Discrimination by DNA Hybridization Probes.

PubMed

Chen, Sherry Xi; Seelig, Georg

2016-04-20

Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology.

Design of a sensitive aptasensor based on magnetic microbeads-assisted strand displacement amplification and target recycling.

PubMed

Li, Ying; Ji, Xiaoting; Song, Weiling; Guo, Yingshu

2013-04-03

A cross-circular amplification system for sensitive detection of adenosine triphosphate (ATP) in cancer cells was developed based on aptamer-target interaction, magnetic microbeads (MBs)-assisted strand displacement amplification and target recycling. Here we described a new recognition probe possessing two parts, the ATP aptamer and the extension part. The recognition probe was firstly immobilized on the surface of MBs and hybridized with its complementary sequence to form a duplex. When combined with ATP, the probe changed its conformation, revealing the extension part in single-strand form, which further served as a toehold for subsequent target recycling. The released complementary sequence of the probe acted as the catalyst of the MB-assisted strand displacement reaction. Incorporated with target recycling, a large amount of biotin-tagged MB complexes were formed to stimulate the generation of chemiluminescence (CL) signal in the presence of luminol and H2O2 by incorporating with streptavidin-HRP, reaching a detection limit of ATP as low as 6.1×10(-10)M. Moreover, sample assays of ATP in Ramos Burkitt's lymphoma B cells were performed, which confirmed the reliability and practicality of the protocol. Copyright © 2013 Elsevier B.V. All rights reserved.

Enzyme- and label-free electrochemical aptasensor for kanamycin detection based on double stir bar-assisted toehold-mediated strand displacement reaction for dual-signal amplification.

PubMed

Hong, Feng; Chen, Xixue; Cao, Yuting; Dong, Youren; Wu, Dazhen; Hu, Futao; Gan, Ning

2018-07-30

It is critically important to detect antibiotic residues for monitoring food safety. In this study, an enzyme- and label-free electrochemical aptasensor for antibiotics, with kanamycin (Kana) as a typical analyte, was developed based on a double stir bar-assisted toehold-mediated strand displacement reaction (dSB-TMSDR) for dual-signal amplification. First, we modified two gold electrodes (E-1 and E-2) with different DNA probes (S1/S2 hybrid probe in E-1 and DNA fuel strand S3 in E-2). In the presence of Kana, an S1/S2 probe can be disassembled from E-1 to form an S2/Kana complex in supernatant. The S2/Kana could react with S3 on E-2 to form S2/S3 hybrid and release Kana through TMSDR. After then, the target recycling was triggered. Subsequently, the formed S2/S3 hybrid can also trigger a hybridization chain reaction (HCR). Consequently, the dual-signal amplification strategy was established, which resulted in many long dsDNA chains on E-2. The chains can associate with methylene blue (MB) as redox probes to produce a current response for the quantification of Kana. The assay exhibited high sensitivity and specificity with a detection limit at 16 fM Kana due to the dual-signal amplification. The double stir bars system can both increase phase separation and prevent leakage of DNA fuel to reduce background interference. Moreover, it allows flexible sequence design of the TMSDR probes. The assay was successfully employed to detect Kana residues in food and showed potential application value in food safety detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Long-lived states to sustain SABRE hyperpolarised magnetisation.

PubMed

Roy, Soumya S; Rayner, Peter J; Norcott, Philip; Green, Gary G R; Duckett, Simon B

2016-09-14

The applicability of the magnetic resonance (MR) technique in the liquid phase is limited by poor sensitivity and short nuclear spin coherence times which are insufficient for many potential applications. Here we illustrate how it is possible to address both of these issues simultaneously by harnessing long-lived hyperpolarised spin states that are formed by adapting the Signal Amplification by Reversible Exchange (SABRE) technique. We achieve more than 4% net 1 H-polarisation in a long-lived form that remains detectable for over ninety seconds by reference to proton pairs in the biologically important molecule nicotinamide and a pyrazine derivative whose in vivo imaging will offer a new route to probe disease in the future.

Amplifications of chromosomal region 20q13 as a prognostic indicator in breast cancer

DOEpatents

Gray, Joe W.; Collins, Colin; Pinkel, Daniel; Kallioniemi, Olli-Pekka; Tanner, Minna M.

1998-01-01

The present invention relates to in situ hybridization methods for the identification of new chromosomal abnormalities associated with various diseases. In particular, it provides probes which are specific to a region of amplification in chromosome 20.

Detection of the free-living forms of sulfide-oxidizing gill endosymbionts in the lucinid habitat (Thalassia testudinum environment).

PubMed

Gros, Olivier; Liberge, Martine; Heddi, Abdelaziz; Khatchadourian, Chaqué; Felbeck, Horst

2003-10-01

Target DNA from the uncultivable Codakia orbicularis endosymbiont was PCR amplified from sea-grass sediment. To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment. Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of Codakia orbicularis is present in the environment as a free-living uncultivable form.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification.

PubMed

Ma, Youlong; Teng, Feiyue; Libera, Matthew

2018-06-05

Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA - amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency; (c) inhibition of T7 RNA polymerase activity by AMV-RT.

Electrochemical sensor for glutathione detection based on mercury ion triggered hybridization chain reaction signal amplification.

PubMed

Wang, Yonghong; Jiang, Lun; Leng, Qinggang; Wu, Yaohui; He, Xiaoxiao; Wang, Kemin

2016-03-15

In this work, we design a new simple and highly sensitive strategy for electrochemical detection of glutathione (GSH) via mercury ion (Hg(2+)) triggered hybridization chain reaction (HCR) signal amplification. It is observed that in the absence of GSH, a specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination can fold into hairpin structures. While in the presence of GSH, it thus can be chelated with Hg(2+), resulting in Hg(2+) released from the T-Hg(2+)-T hairpin complex which then forms into ssDNA structure to further hybridize with the surface-immobilized capture DNA probe on the gold electrode with a sticky tail left. The presence of two hairpin helper probes through HCR leads to the formation of extended dsDNA superstructure on the electrode surface, which therefore causes the intercalation of numerous electroactive species ([Ru(NH3)6](3+)) into the dsDNA grooves, followed by a significantly amplified signal output whose intensity is related to the concentration of the GSH. Taking advantage of merits of enzyme-free amplification power of the HCR, the inherent high sensitivity of the electrochemical technique, and label-free detection which utilizes an electroactive species as a signaling molecule that binds to the anionic phosphate backbone of DNA strands via electrostatic force, not only does the proposed strategy enable sensitive detection of GSH, but show high selectivity against other amino acid, making our method a simple and sensitive addition to the amplified GSH detection. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification.

PubMed

Cheng, Wei; Zhang, Wei; Yan, Yurong; Shen, Bo; Zhu, Dan; Lei, Pinhua; Ding, Shijia

2014-12-15

A novel electrochemical biosensing strategy was developed for ultrasensitive and specific detection of target DNA using a cascade signal amplification based on molecular beacon (MB) mediated circular strand displacement (CSD), rolling circle amplification (RCA), biotin-strepavidin system, and enzymatic amplification. The target DNA hybridized with the loop portion of MB probe immobilized on the gold electrode and triggered the CSD, leading to multiple biotin-tagged DNA duplex. Furthermore, via biotin-streptavidin interaction, the RCA was implemented, producing long massive tandem-repeat DNA sequences for binding numerous biotinylated detection probes. This enabled an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor showed very high sensitivity and selectivity with a dynamic response range from 1 fM to 100 pM. The proposed strategy could have the potential for applying in clinical molecular diagnostics and environmental monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of Two-Photon Pump Polarization Spectroscopy Probe Technique Tpp-Psp for Measurements of Atomic Hydrogen .

NASA Astrophysics Data System (ADS)

Satija, Aman; Lucht, Robert P.

2015-06-01

Atomic hydrogen (H) is a key radical in combustion and plasmas. Accurate knowledge of its concentration can be used to better understand transient phenomenon such as ignition and extinction in combustion environments. Laser induced polarization spectroscopy is a spatially resolved absorption technique which we have adapted for quantitative measurements of H atom. This adaptation is called two-photon pump, polarization spectroscopy probe technique (TPP-PSP) and it has been implemented using two different laser excitation schemes. The first scheme involves the two-photon excitation of 1S-2S transitions using a linearly polarized 243-nm beam. An anisotropy is created amongst Zeeman states in 2S-3P levels using a circularly polarized 656-nm pump beam. This anisotropy rotates the polarization of a weak, linearly polarized probe beam at 656 nm. As a result, the weak probe beam "leaks" past an analyzer in the detection channel and is measured using a PMT. This signal can be related to H atom density in the probe volume. The laser beams were created by optical parametric generation followed by multiple pulse dye amplification stages. This resulted in narrow linewidth beams which could be scanned in frequency domain and varied in energy. This allowed us to systematically investigate saturation and Stark effect in 2S-3P transitions with the goal of developing a quantitative H atom measurement technique. The second scheme involves the two-photon excitation of 1S-2S transitions using a linearly polarized 243-nm beam. An anisotropy is created amongst Zeeman states in 2S-4P transitions using a circularly polarized 486-nm pump beam. This anisotropy rotates the polarization of a weak, linearly polarized probe beam at 486 nm. As a result the weak probe beam "leaks" past an analyzer in the detection channel and is measured using a PMT. This signal can be related to H atom density in the probe volume. A dye laser was pumped by third harmonic of a Nd:YAG laser to create a laser beam at 486 nm. The 486-nm beam was frequency doubled to a 243-nm beam. Use of the second scheme simplifies the TPP-PSP technique making it more convenient for diagnostics in practical systems.

Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification.

PubMed Central

Wimpee, C F; Nadeau, T L; Nealson, K H

1991-01-01

By using two highly conserved region of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria. Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies. The fourth probe, generated from Vibrio harveyi DNA, cross-reacted with DNAs from two closely related species, V. orientalis and V. vulnificus. When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies. Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency. A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species, Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies. Images PMID:1854194

Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

PubMed

Miles, Timothy D; Martin, Frank N; Coffey, Michael D

2015-02-01

Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing, thereby reducing the time necessary for accurate diagnostics and making management decisions.

A PCR-Based Method for RNA Probes and Applications in Neuroscience.

PubMed

Hua, Ruifang; Yu, Shanshan; Liu, Mugen; Li, Haohong

2018-01-01

In situ hybridization (ISH) is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes. However, in most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. Probes originating from plasmid vectors are time-consuming and not suitable for the rapid gene mapping. Here, we introduce a simplified method to prepare digoxigenin (DIG)-labeled non-radioactive RNA probes based on polymerase chain reaction (PCR) amplification and applications in free-floating mouse brain sections. Employing a transgenic reporter line, we investigate the expression of the somatostatin (SST) mRNA in the adult mouse brain. The method can be applied to identify the colocalization of SST mRNA and proteins including corticotrophin-releasing hormone (CRH) and protein kinase C delta type (PKC-δ) using double immunofluorescence, which is useful for understanding the organization of complex brain nuclei. Moreover, the method can also be incorporated with retrograde tracing to visualize the functional connection in the neural circuitry. Briefly, the PCR-based method for non-radioactive RNA probes is a useful tool that can be substantially utilized in neuroscience studies.

A modified multiplex ligation-dependent probe amplification method for the detection of 22q11.2 copy number variations in patients with congenital heart disease.

PubMed

Zhang, Xiaoqing; Xu, Yuejuan; Liu, Deyuan; Geng, Juan; Chen, Sun; Jiang, Zhengwen; Fu, Qihua; Sun, Kun

2015-05-08

Copy number variations (CNVs) of chromosomal region 22q11.2 are associated with a subset of patients with congenital heart disease (CHD). Accurate and efficient detection of CNV is important for genetic analysis of CHD. The aim of the study was to introduce a novel approach named CNVplex®, a high-throughput analysis technique designed for efficient detection of chromosomal CNVs, and to explore the prevalence of sub-chromosomal imbalances in 22q11.2 loci in patients with CHD from a single institute. We developed a novel technique, CNVplex®, for high-throughput detection of sub-chromosomal copy number aberrations. Modified from the multiplex ligation-dependent probe amplification (MLPA) method, it introduced a lengthening ligation system and four universal primer sets, which simplified the synthesis of probes and significantly improved the flexibility of the experiment. We used 110 samples, which were extensively characterized with chromosomal microarray analysis and MLPA, to validate the performance of the newly developed method. Furthermore, CNVplex® was used to screen for sub-chromosomal imbalances in 22q11.2 loci in 818 CHD patients consecutively enrolled from Shanghai Children's Medical Center. In the methodology development phase, CNVplex® detected all copy number aberrations that were previously identified with both chromosomal microarray analysis and MLPA, demonstrating 100% sensitivity and specificity. In the validation phase, 22q11.2 deletion and 22q11.2 duplication were detected in 39 and 1 of 818 patients with CHD by CNVplex®, respectively. Our data demonstrated that the frequency of 22q11.2 deletion varied among sub-groups of CHD patients. Notably, 22q11.2 deletion was more commonly observed in cases with conotruncal defect (CTD) than in cases with non-CTD (P<0.001). With higher resolution and more probes against selected chromosomal loci, CNVplex® also identified several individuals with small CNVs and alterations in other chromosomes. CNVplex® is sensitive and specific in its detection of CNVs, and it is an alternative to MLPA for batch screening of pathogenetic CNVs in known genomic loci.

Multiplex ligation-dependent probe amplification analysis on capillary electrophoresis instruments for a rapid gene copy number study.

PubMed

Jankowski, Stéphane; Currie-Fraser, Erica; Xu, Licen; Coffa, Jordy

2008-09-01

Annotated DNA samples that had been previously analyzed were tested using multiplex ligation-dependent probe amplification (MLPA) assays containing probes targeting BRCA1, BRCA2, and MMR (MLH1/MSH2 genes) and the 9p21 chromosomal region. MLPA polymerase chain reaction products were separated on a capillary electrophoresis platform, and the data were analyzed using GeneMapper v4.0 software (Applied Biosystems, Foster City, CA). After signal normalization, loci regions that had undergone deletions or duplications were identified using the GeneMapper Report Manager and verified using the DyeScale functionality. The results highlight an easy-to-use, optimal sample preparation and analysis workflow that can be used for both small- and large-scale studies.

A copy number variation genotyping method for aneuploidy detection in spontaneous abortion specimens.

PubMed

Chen, Songchang; Liu, Deyuan; Zhang, Junyu; Li, Shuyuan; Zhang, Lanlan; Fan, Jianxia; Luo, Yuqin; Qian, Yeqing; Huang, Hefeng; Liu, Chao; Zhu, Huanhuan; Jiang, Zhengwen; Xu, Chenming

2017-02-01

Chromosomal abnormalities such as aneuploidy have been shown to be responsible for causing spontaneous abortion. Genetic evaluation of abortions is currently underperformed. Screening for aneuploidy in the products of conception can help determine the etiology. We designed a high-throughput ligation-dependent probe amplification (HLPA) assay to examine aneuploidy of 24 chromosomes in miscarriage tissues and aimed to validate the performance of this technique. We carried out aneuploidy screening in 98 fetal tissue samples collected from female subjects with singleton pregnancies who experienced spontaneous abortion. The mean maternal age was 31.6 years (range: 24-43), and the mean gestational age was 10.2 weeks (range: 4.6-14.1). HLPA was performed in parallel with array comparative genomic hybridization, which is the gold standard for aneuploidy detection in clinical practices. The results from the two platforms were compared. Forty-nine out of ninety-eight samples were found to be aneuploid. HLPA showed concordance with array comparative genomic hybridization in diagnosing aneuploidy. High-throughput ligation-dependent probe amplification is a rapid and accurate method for aneuploidy detection. It can be used as a cost-effective screening procedure in clinical spontaneous abortions. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification.

PubMed

Oishi, Motoi

2015-05-01

An enzyme-free and isothermal microRNA (miRNA) detection method has been developed based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction (HCR) on magnetic beads (MBs). The click-chemical ligation between an azide-modified probe DNA and a dibenzocyclooctyne-modified probe DNA occurred through the hybridization of target miRNA (miR-141). HCR on MBs was performed by the addition of DNA hairpin monomers (H1 and H2). After magnetic separation and denaturation/rehybridization of HCR products ([H1/H2] n ), the resulting HCR products were analyzed by the fluorescence emitted from an intercalative dye, allowing amplification of the fluorescent signal. The proposed assay had a limit of detection of 0.55 fmol, which was 230-fold more sensitive than that of the HCR on the MBs coupled with a conventional sandwich hybridization assay (without click-chemical ligation) (limit of detection 127 fmol). Additionally, the proposed assay could discriminate between miR-141 and other miR-200 family members. In contrast to quantitative reverse transcription polymerase chain reaction techniques using enzymes and thermal cycling, this is an enzyme-free assay that can be conducted under isothermal conditions and can specifically detect miR-141 in fetal bovine serum.

A comparison of restriction fragment length polymorphism, tetra primer amplification refractory mutation system PCR and unlabeled probe melting analysis for LTA+252 C>T SNP genotyping.

PubMed

Soler, Stephan; Rittore, Cécile; Touitou, Isabelle; Philibert, Laurent

2011-02-20

From the wide range of methods currently available for genotyping, we wished to identify a quick, reliable and affordable approach for routine use in our laboratory for LTA+252 C>T SNP screening. We set up and compared three genotyping methods for SNP detection: restriction fragment length polymorphism (RFLP), tetra primer amplification refractory mutation system PCR (TPAP) and unlabeled probe melting analysis (UPMA). The SNP model used was LTA+252 C>T, a cytokine gene polymorphism that has been associated with response to treatment in rheumatoid arthritis. The study was performed using 46 samples from healthy Caucasian volunteers. Allele and genotype distribution was similar to that previously described in the same population. All three genotyping methods showed good reproducibility and are suitable for a medium scale throughput molecular platform. UPMA was the most cost effective, reliable and safe method since it required the shortest technician time, could be performed in a single closed tube and involved automatic data analysis. This work is the first to compare these three genotyping techniques and provides evidence for UPMA being the method of choice for LTA+252 C>T SNP genotyping. Copyright © 2010 Elsevier B.V. All rights reserved.

Analysis of genomic alterations in neuroblastoma by multiplex ligation-dependent probe amplification and array comparative genomic hybridization: a comparison of results.

PubMed

Combaret, Valérie; Iacono, Isabelle; Bréjon, Stéphanie; Schleiermacher, Gudrun; Pierron, Gäelle; Couturier, Jérôme; Bergeron, Christophe; Blay, Jean-Yves

2012-12-01

In cases of neuroblastoma, recurring genetic alterations--losses of the 1p, 3p, 4p, and 11q and/or gains of 1q, 2p, and 17q chromosome arms--are currently used to define the therapeutic strategy in therapeutic protocols for low- and intermediate-risk patients. Different genome-wide analysis techniques, such as array comparative genomic hybridization (aCGH) or multiplex ligation-dependent probe amplification (MLPA), have been suggested for detecting chromosome segmental abnormalities. In this study, we compared the results of the two technologies in the analyses of the DNA of tumor samples from 91 neuroblastoma patients. Similar results were obtained with the two techniques for 75 samples (82%). In five cases (5.5%), the MLPA results were not interpretable. Discrepancies between the aCGH and MLPA results were observed in 11 cases (12%). Among the discrepancies, a 18q21.2-qter gain and 16p11.2 and 11q14.1-q14.3 losses were detected only by aCGH. The MLPA results showed that the 7p, 7q, and 14q chromosome arms were affected in six cases, while in two cases, 2p and 17q gains were observed; these results were confirmed by neither aCGH nor fluorescence in situ hybridization (FISH) analysis. Because of the higher sensitivity and specificity of genome-wide information, reasonable cost, and shorter time of aCGH analysis, we recommend the aCGH procedure for the analysis of genomic alterations in neuroblastoma. Copyright © 2012 Elsevier Inc. All rights reserved.

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection.

PubMed

Takahashi, Hirokazu; Ohkawachi, Masahiko; Horio, Kyohei; Kobori, Toshiro; Aki, Tsunehiro; Matsumura, Yukihiko; Nakashimada, Yutaka; Okamura, Yoshiko

2018-05-17

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

Improved Multiplex Ligation-dependent Probe Amplification (i-MLPA) for rapid copy number variant (CNV) detection.

PubMed

Saxena, Sonal; Gowdhaman, Kavitha; Kkani, Poornima; Vennapusa, Bhavyasri; Rama Subramanian, Chellamuthu; Ganesh Kumar, S; Mohan, Kommu Naga

2015-10-23

In Multiplex Ligation-dependent Probe Amplification (MLPA), copy number variants (CNVs) for specific genes are identified after normalization of the amounts of PCR products from ligated reference probes hybridized to genomic regions that are ideally free from normal variation. However, we observed ambiguous calls for two reference probes in an investigation of the human 15q11.2 region by MLPA among 20 controls, due to the presence of single nucleotide polymorphisms (SNPs) in the probe-binding regions. Further in silico analysis revealed that 18 out of 19 reference probes hybridize to regions subject to variation, underlining the requirement for designing new reference probes against variation-free regions. An improved MLPA (i-MLPA) method was developed by generating a new set of reference probes to reduce the chances of ambiguous calls and new reagents that reduce hybridization times to 30 min from 16h to obtain MLPA ratio data within 6h. Using i-MLPA, we screened 240 schizophrenia patients for CNVs in 15q11.2 region. Three deletions and two duplications were identified among the 240 schizophrenia patients. No variation was observed for the new reference probes. Taken together, i-MLPA procedure helps obtaining non-ambiguous CNV calls within 6h without compromising accuracy. Copyright © 2015 Elsevier B.V. All rights reserved.

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

PubMed

Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

2018-06-29

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Youyu; Tang, Zhiwen; Wang, Jun

2010-08-01

A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate duemore » to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.« less

Electrochemical current rectification-a novel signal amplification strategy for highly sensitive and selective aptamer-based biosensor.

PubMed

Feng, Lingyan; Sivanesan, Arumugam; Lyu, Zhaozi; Offenhäusser, Andreas; Mayer, Dirk

2015-04-15

Electrochemical aptamer-based (E-AB) sensors represent an emerging class of recently developed sensors. However, numerous of these sensors are limited by a low surface density of electrode-bound redox-oligonucleotides which are used as probe. Here we propose to use the concept of electrochemical current rectification (ECR) for the enhancement of the redox signal of E-AB sensors. Commonly, the probe-DNA performs a change in conformation during target binding and enables a nonrecurring charge transfer between redox-tag and electrode. In our system, the redox-tag of the probe-DNA is continuously replenished by solution-phase redox molecules. A unidirectional electron transfer from electrode via surface-linked redox-tag to the solution-phase redox molecules arises that efficiently amplifies the current response. Using this robust and straight-forward strategy, the developed sensor showed a substantial signal amplification and consequently improved sensitivity with a calculated detection limit of 114nM for ATP, which was improved by one order of magnitude compared with the amplification-free detection and superior to other previous detection results using enzymes or nanomaterials-based signal amplification. To the best of our knowledge, this is the first demonstration of an aptamer-based electrochemical biosensor involving electrochemical rectification, which can be presumably transferred to other biomedical sensor systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH).

PubMed

Kubota, Kengo; Ohashi, Akiyoshi; Imachi, Hiroyuki; Harada, Hideki

2006-09-01

Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.

Multiplex Ligation-Dependent Probe Amplification Analysis on Capillary Electrophoresis Instruments for a Rapid Gene Copy Number Study

PubMed Central

Jankowski, Stéphane; Currie-Fraser, Erica; Xu, Licen; Coffa, Jordy

2008-01-01

Annotated DNA samples that had been previously analyzed were tested using multiplex ligation-dependent probe amplification (MLPA) assays containing probes targeting BRCA1, BRCA2, and MMR (MLH1/MSH2 genes) and the 9p21 chromosomal region. MLPA polymerase chain reaction products were separated on a capillary electrophoresis platform, and the data were analyzed using GeneMapper v4.0 software (Applied Biosystems, Foster City, CA). After signal normalization, loci regions that had undergone deletions or duplications were identified using the GeneMapper Report Manager and verified using the DyeScale functionality. The results highlight an easy-to-use, optimal sample preparation and analysis workflow that can be used for both small- and large-scale studies. PMID:19137113

Development of a Loop Mediated Isothermal Amplification (LAMP) - Surface Enhanced Raman spectroscopy (SERS) Assay for the Detection of Salmonella Enterica Serotype Enteritidis.

PubMed

Draz, Mohamed Shehata; Lu, Xiaonan

2016-01-01

As a major foodborne pathogen, Salmonella enterica serotype Enteritidis is increasingly rising as a global health concern. Here, we developed an integrated assay that combines loop mediated isothermal amplification (LAMP) and surface enhanced Raman spectroscopy (SERS) for DNA detection of S. Enteritidis using specifically designed Raman active Au-nanoprobes. The target DNA was amplified by LAMP and then labeled with Au-nanoprobes comprised of gold nanoparticle-modified with specific cy5/DNA probes to allow the detection by SERS. The sensitivity of the developed LAMP-SERS detection assay (66 CFU/mL) was ~100-fold higher than the conventional polymerase chain reaction (PCR) method. Significantly, this technique allowed highly specific detection of the target DNA of S. Enteritidis and could differentiate it from the DNA of closely related bacterial species or non-specific contamination, making it more accurate and reliable than the standard LAMP technique. The applicability of detection of S. Enteritidis in milk samples using LAMP-SERS assay was validated as well. In sum, the developed LAMP-SERS assay is highly specific and sensitive, and has the potential to be applied for rapid detection of different foodborne pathogens and other microbial contaminants.

An innovative Yb-based ultrafast deep ultraviolet source for time-resolved photoemission experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boschini, F.; Hedayat, H.; Dallera, C.

2014-12-15

Time- and angle-resolved photoemission spectroscopy is a powerful technique to study ultrafast electronic dynamics in solids. Here, an innovative optical setup based on a 100-kHz Yb laser source is presented. Exploiting non-collinear optical parametric amplification and sum-frequency generation, ultrashort pump (hν = 1.82 eV) and ultraviolet probe (hν = 6.05 eV) pulses are generated. Overall temporal and instrumental energy resolutions of, respectively, 85 fs and 50 meV are obtained. Time- and angle-resolved measurements on BiTeI semiconductor are presented to show the capabilities of the setup.

Detection of the Free-Living Forms of Sulfide-Oxidizing Gill Endosymbionts in the Lucinid Habitat (Thalassia testudinum Environment)

PubMed Central

Gros, Olivier; Liberge, Martine; Heddi, Abdelaziz; Khatchadourian, Chaqué; Felbeck, Horst

2003-01-01

Target DNA from the uncultivable Codakia orbicularis endosymbiont was PCR amplified from sea-grass sediment. To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment. Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of Codakia orbicularis is present in the environment as a free-living uncultivable form. PMID:14532089

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

PubMed

Buh Gasparic, Meti; Tengs, Torstein; La Paz, Jose Luis; Holst-Jensen, Arne; Pla, Maria; Esteve, Teresa; Zel, Jana; Gruden, Kristina

2010-03-01

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.

Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

NASA Astrophysics Data System (ADS)

Cheglakov, Zoya

Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide a beneficial strategy for simultaneous tracking readily accomplished by multicolor imaging with diverse fluorescent tags. The third method in this thesis will demonstrate the advantage of DNAzymes probes amplification, and offers potential strategy to monitor miRNAs in mammalian live cells.

Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification.

PubMed

Ebai, Tonge; Souza de Oliveira, Felipe Marques; Löf, Liza; Wik, Lotta; Schweiger, Caroline; Larsson, Anders; Keilholtz, Ulrich; Haybaeck, Johannes; Landegren, Ulf; Kamali-Moghaddam, Masood

2017-09-01

Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals. Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader. We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor 15 (GDF-15) by PLARCA and conventional sandwich ELISA or immuno-RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared to ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA. PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories. © 2017 American Association for Clinical Chemistry.

An exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus.

PubMed

Wang, Jian-Chang; Liu, Li-Bing; Han, Qing-An; Wang, Jin-Feng; Yuan, Wan-Zhe

2017-10-01

Recombinase polymerase amplification (RPA), an isothermal amplification technology, has been developed as an alternative to PCR in pathogen detection. A real-time RPA assay (rt-RPA) was developed to detect the porcine parvovirus (PPV) using primers and exo probe specific for the VP2 gene. The amplification was performed at 39°C for 20min. There was no cross-reaction with other pathogens tested. Using the recombinant plasmid pPPV-VP2 as template, the analytical sensitivity was 103 copies. The assay performance was evaluated by testing 115 field samples by rt-RPA and a real-time PCR assay. The diagnostic agreement between assays was 100%, and PPV DNA was detected in 94 samples. The R 2 value of rt-RPA and real-time PCR was 0.909 by linear regression analysis. The developed rt-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of PPV in diagnostic laboratories and at point-of-care, especially in remote and rural areas. Copyright © 2017 Elsevier B.V. All rights reserved.

Ultrasensitive and selective signal-on electrochemical DNA detection via exonuclease III catalysis and hybridization chain reaction amplification.

PubMed

Ren, Wang; Gao, Zhong Feng; Li, Nian Bing; Luo, Hong Qun

2015-01-15

This work reported a novel, ultrasensitive, and selective platform for electrochemical detection of DNA, employing an integration of exonuclease III (Exo-III) assisted target recycling and hybridization chain reaction (HCR) for the dual signal amplification strategy. The hairpin capture probe DNA (C-DNA) with an Exo-III 3' overhang end was self-assembled on a gold electrode. In the presence of target DNA (T-DNA), C-DNA hybridized with the T-DNA to form a duplex region, exposing its 5' complementary sequence (initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue, facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0 fM to 1.0 nM, and a detection limit as low as 0.2 fM. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Anomalous amplification of a homodyne signal via almost-balanced weak values.

PubMed

Liu, Wei-Tao; Martínez-Rincón, Julián; Viza, Gerardo I; Howell, John C

2017-03-01

We propose precision measurements of ultra-small angular velocities of a mirror within a modified Sagnac interferometer, where the counter-propagating beams are spatially separated, using the recently proposed technique of almost-balanced weak values amplification (ABWV) [Phys. Rev. Lett.116, 100803 (2016)PRLTAO0031-900710.1103/PhysRevLett.116.100803]. The separation between the two beams provides additional amplification with respect to using collinear beams in a Sagnac interferometer. Within the same setup, the weak-value amplification technique is also performed for comparison. Much higher amplification factors can be obtained using the almost-balanced weak values technique, with the best one achieved in our experiments being as high as 1.2×10⁷. In addition, the amplification factor monotonically increases with decreasing of the post-selection phase for the ABWV case in our experiments, which is not the case for weak-value amplification (WVA) at small post-selection phases. Both techniques consist of measuring the angular velocity. The sensitivity of the ABWV technique is ∼38  nrad/s per averaged pulse for a repetition rate of 1 Hz and ∼33  nrad/s per averaged pulse for the WVA technique.

Graphene Nanoprobes for Real-Time Monitoring of Isothermal Nucleic Acid Amplification.

PubMed

Li, Fan; Liu, Xiaoguo; Zhao, Bin; Yan, Juan; Li, Qian; Aldalbahi, Ali; Shi, Jiye; Song, Shiping; Fan, Chunhai; Wang, Lihua

2017-05-10

Isothermal amplification is an efficient way to amplify DNA with high accuracy; however, the real-time monitoring for quantification analysis mostly relied on expensive and precisely designed probes. In the present study, a graphene oxide (GO)-based nanoprobe was used to real-time monitor the isothermal amplification process. The interaction between GO and different DNA structures was systematically investigated, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), DNA 3-helix, and long rolling circle amplification (RCA) and hybridization chain reaction (HCR) products, which existed in one-, two-, and three-dimensional structures. It was found that the high rigid structures exhibited much lower affinity with GO than soft ssDNA, and generally the rigidity was dependent on the length of targets and the hybridization position with probe DNA. On the basis of these results, we successfully monitored HCR amplification process, RCA process, and the enzyme restriction of RCA products with GO nanoprobe; other applications including the detection of the assembly/disassembly of DNA 3-helix structures were also performed. Compared to the widely used end-point detection methods, the GO-based sensing platform is simple, sensitive, cost-effective, and especially in a real-time monitoring mode. We believe such studies can provide comprehensive understandings and evocation on design of GO-based biosensors for broad application in various fields.

New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips.

PubMed

Powell, Michael L; Bowler, Frank R; Martinez, Aurore J; Greenwood, Catherine J; Armes, Niall; Piepenburg, Olaf

2018-02-15

Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings. Copyright © 2017. Published by Elsevier Inc.

Homogenous assay for protein detection based on proximity DNA hybridization and isothermal circular strand displacement amplification reaction.

PubMed

Zhang, Manjun; Li, Ruimin; Ling, Liansheng

2017-06-01

This work proposed a homogenous fluorescence assay for proteins, based on the target-triggered proximity DNA hybridization in combination with strand displacement amplification (SDA). It benefited from target-triggered proximity DNA hybridization to specifically recognize the target and SDA making recycling signal amplification. The system included a molecular beacon (MB), an extended probe (EP), and an assistant probe (AP), which were not self-assembly in the absence of target proteins, due to the short length of the designed complementary sequence among MB, EP, and AP. Upon addition of the target proteins, EP and AP are bound to the target proteins, which induced the occurrence of proximity hybridization between MB, EP, and AP and followed by strand displacement amplification. Through the primer extension, a tripartite complex of probes and target was displaced and recycled to hybridize with another MB, and the more opened MB enabled the detection signal to amplify. Under optimum conditions, it was used for the detection of streptavidin and thrombin. Fluorescence intensity was proportional to the concentration of streptavidin and thrombin in the range of 0.2-30 and 0.2-35 nmol/L, respectively. Furthermore, this fluorescent method has a good selectivity, in which the fluorescence intensity of thrombin was ~37-fold or even larger than that of the other proteins at the same concentration. It is a new and simple method for SDA-involved target protein detection and possesses a great potential for other protein detection in the future. Graphical abstract A homogenous assay for protein detection is based on proximity DNA hybridization and strand displacement amplification reaction.

Colorimetric detection of mercury ion based on unmodified gold nanoparticles and target-triggered hybridization chain reaction amplification

NASA Astrophysics Data System (ADS)

Wang, Qing; Yang, Xiaohan; Yang, Xiaohai; Liu, Pei; Wang, Kemin; Huang, Jin; Liu, Jianbo; Song, Chunxia; Wang, Jingjing

2015-02-01

A novel unmodified gold nanoparticles (AuNPs)-based colorimetric strategy for label-free, specific and sensitive mercury ion (Hg2+) detection was demonstrated by using thymine-Hg2+-thymine (T-Hg2+-T) recognition mechanism and hybridization chain reaction (HCR) amplification strategy. In this protocol, a structure-switching probe (H0) was designed to recognize Hg2+ and then propagated a chain reaction of hybridization events between two other hairpin probes (H1 and H2). In the absence of Hg2+, all hairpin probes could stably coexist in solution, the exposed sticky ends of hairpin probes were capable of stabilizing AuNPs. As a result, salt-induced AuNPs aggregation could be effectively prevented. In the presence of Hg2+, thymine bases of H0 could specifically interact with Hg2+ to form stable T-Hg2+-T complex. Consequently, the hairpin structure of H0 probe was changed. As H1/H2 probes were added, the HCR process could be triggered and nicked double-helixes were formed. Since it was difficult for the formed nicked double-helixes to inhibit salt-induced AuNPs aggregation, a red-to-blue color change was observed in the colloid solution as the salt concentration increased. With the elegant amplification effect of HCR, a detection limit of around 30 nM was achieved (S/N = 3), which was about 1-2 orders of magnitudes lower than that of previous unmodified AuNPs-based colorimetric methods. By using the T-Hg2+-T recognition mechanism, high selectivity was also obtained. As an unmodified AuNPs-based colorimetric strategy, the system was simple in design, convenient in operation, and eliminated the requirements of separation processes, chemical modifications, and sophisticated instrumentations.

Modified beacon probe assisted dual signal amplification for visual detection of microRNA.

PubMed

Sun, Xiuwei; Ying, Na; Ju, Chuanjing; Li, Zhongyi; Xu, Na; Qu, Guijuan; Liu, Wensen; Wan, Jiayu

2018-06-01

In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity. Copyright © 2018 Elsevier Inc. All rights reserved.

Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays.

PubMed

Durtschi, Jacob D; Stevenson, Jeffery; Hymas, Weston; Voelkerding, Karl V

2007-02-01

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein-Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.

Determination for Enterobacter cloacae based on a europium ternary complex labeled DNA probe

NASA Astrophysics Data System (ADS)

He, Hui; Niu, Cheng-Gang; Zeng, Guang-Ming; Ruan, Min; Qin, Pin-Zhu; Liu, Jing

2011-11-01

The fast detection and accurate diagnosis of the prevalent pathogenic bacteria is very important for the treatment of disease. Nowadays, fluorescence techniques are important tools for diagnosis. A two-probe tandem DNA hybridization assay was designed for the detection of Enterobacter cloacae based on time-resolved fluorescence. In this work, the authors synthesized a novel europium ternary complex Eu(TTA) 3(5-NH 2-phen) with intense luminescence, high fluorescence quantum yield and long lifetime before. We developed a method based on this europium complex for the specific detection of original extracted DNA from E. cloacae. In the hybridization assay format, the reporter probe was labeled with Eu(TTA) 3(5-NH 2-phen) on the 5'-terminus, and the capture probe capture probe was covalent immobilized on the surface of the glutaraldehyde treated glass slides. The original extracted DNA of samples was directly used without any DNA purification and amplification. The detection was conducted by monitoring the fluorescence intensity from the glass surface after DNA hybridization. The detection limit of the DNA was 5 × 10 -10 mol L -1. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment.

Development of the polymerase chain reaction for diagnosis of chancroid.

PubMed Central

Chui, L; Albritton, W; Paster, B; Maclean, I; Marusyk, R

1993-01-01

The published nucleotide sequences of the 16S rRNA gene of Haemophilus ducreyi were used to develop primer sets and probes for the diagnosis of chancroid by polymerase chain reaction (PCR) DNA amplification. One set of broad specificity primers yielded a 303-bp PCR product from all bacteria tested. Two 16-base probes internal to this sequence were species specific for H. ducreyi when tested with 12 species of the families Pasteurellaceae and Enterobacteriaceae. The two probes in combination with the broad specificity primers were 100% sensitive with 51 strains of H. ducreyi isolated from six continents over a 15-year period. The direct detection of H. ducreyi from 100 clinical specimens by PCR showed a sensitivity of 83 to 98% and a specificity of 51 to 67%, depending on the number of amplification cycles. Images PMID:8458959

Method to detect the end-point for PCR DNA amplification using an ionically labeled probe and measuring impedance change

DOEpatents

Miles, Robin R [Danville, CA; Belgrader, Phillip [Severna Park, MD; Fuller, Christopher D [Oakland, CA

2007-01-02

Impedance measurements are used to detect the end-point for PCR DNA amplification. A pair of spaced electrodes are located on a surface of a microfluidic channel and an AC or DC voltage is applied across the electrodes to produce an electric field. An ionically labeled probe will attach to a complementary DNA segment, and a polymerase enzyme will release the ionic label. This causes the conductivity of the solution in the area of the electrode to change. This change in conductivity is measured as a change in the impedance been the two electrodes.

In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

PubMed Central

1986-01-01

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick- translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling. PMID:3084498

Real-time electrochemical LAMP: a rational comparative study of different DNA intercalating and non-intercalating redox probes.

PubMed

Martin, Alexandra; Bouffier, Laurent; Grant, Kathryn B; Limoges, Benoît; Marchal, Damien

2016-06-20

We present a comparative study of ten redox-active probes for use in real-time electrochemical loop-mediated isothermal amplification (LAMP). Our main objectives were to establish the criteria that need to be fulfilled for minimizing some of the current limitations of the technique and to provide future guidelines in the search for ideal redox reporters. To ensure a reliable comparative study, each redox probe was tested under similar conditions using the same LAMP reaction and the same entirely automatized custom-made real-time electrochemical device (designed for electrochemically monitoring in real-time and in parallel up to 48 LAMP samples). Electrochemical melt curve analyses were recorded immediately at the end of each LAMP reaction. Our results show that there are a number of intercalating and non-intercalating redox compounds suitable for real-time electrochemical LAMP and that the best candidates are those able to intercalate strongly into ds-DNA but not too much to avoid inhibition of the LAMP reaction. The strongest intercalating redox probes were finally shown to provide higher LAMP sensitivity, speed, greater signal amplitude, and cleaner-cut DNA melting curves than the non-intercalating molecules.

Advanced yellow fever virus genome detection in point-of-care facilities and reference laboratories.

PubMed

Domingo, Cristina; Patel, Pranav; Yillah, Jasmin; Weidmann, Manfred; Méndez, Jairo A; Nakouné, Emmanuel Rivalyn; Niedrig, Matthias

2012-12-01

Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

PubMed

Kimura, Yasumasa; Soma, Takahiro; Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J L; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

2016-01-01

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays

PubMed Central

Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J. L.; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

2016-01-01

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download. PMID:26863543

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

USDA-ARS?s Scientific Manuscript database

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

Nucleic acid probes in diagnostic medicine

NASA Technical Reports Server (NTRS)

Oberry, Phillip A.

1991-01-01

The need for improved diagnostic procedures is outlined and variations in probe technology are briefly reviewed. A discussion of the application of probe technology to the diagnosis of disease in animals and humans is presented. A comparison of probe versus nonprobe diagnostics and isotopic versus nonisotopic probes is made and the current state of sequence amplification is described. The current market status of nucleic acid probes is reviewed with respect to their diagnostic application in human and veterinary medicine. Representative product examples are described and information on probes being developed that offer promise as future products is discussed.

[Molecular diagnosis of spinal muscular atrophy by multiplex ligation-dependent probe amplification].

PubMed

Zeng, Jian; Ke, Long-feng; Deng, Xiao-jun; Cai, Mei-ying; Tu, Xiang-dong; Lan, Feng-hua

2008-12-16

To investigate the effect of multiplex ligation-dependent probe amplification (MLPA) in molecular diagnosis of spinal muscular atrophy (SMA). Peripheral blood samples were collected from 13 SMA patients, 31 parents of SMA patients, 50 healthy individuals without family history of SMA, and 10 specimens of amniotic fluid from these families were collected too. Genomic DNA was analyzed by MLPA, conventional PCR-RFLP, and allele-specific PCR. In complete agreement with the results of conventional PCR-RFLP and allele-specific PCR, MLPA analysis showed that all of the 13 patients had homozygous deletion of the survival of motor neuron 1 (SMN1) gene, and there was significant difference between the SMA severity (type I to type III) and SMN2 copy number (P < 0.05). Of the 31 parents 29 (93.5%) had 1 copy of SMN1, 2 (6.5%) had 2 copies of SMN1. Of the 50 healthy individuals, 1 (2.0%) had 1 copy of SMN1, 48 (96.0%) had 2 copies of SMN1, and 1 (2.0%) had 3 copies. The SMN1 copy number of the parents was significantly higher than that of the healthy individuals (P < 0.01). Two of the 10 fetuses had homozygous deletion of SMN1. The MLPA technique has proved to be an accurate and reliable tool for the molecular diagnosis of SMA, both in patients and in healthy carriers.

MYC and MYCN amplification can be reliably assessed by aCGH in medulloblastoma.

PubMed

Bourdeaut, Franck; Grison, Camille; Maurage, Claude-Alain; Laquerriere, Annie; Vasiljevic, Alexandre; Delisle, Marie-Bernadette; Michalak, Sophie; Figarella-Branger, Dominique; Doz, François; Richer, Wilfrid; Pierron, Gaelle; Miquel, Catherine; Delattre, Olivier; Couturier, Jérôme

2013-04-01

As prognostic factors, MYC and MYCN amplifications are routinely assessed in medulloblastomas. Fluorescence in situ hybridization (FISH) is currently considered as the technique of reference. Recently, array comparative genomic hybridization (aCGH) has been developed as an alternative technique to evaluate genomic abnormalities in other tumor types; however, this technique has not been widely adopted as a replacement for FISH in medulloblastoma. In this study, 34 tumors were screened by both FISH and aCGH. In all cases showing amplification by FISH, aCGH also unambiguously revealed the abnormality. The aCGH technique was also performed on tumors showing no amplification by FISH, and the absence of amplification was confirmed in all cases. Interestingly, one tumor showed a subclonal MYC amplification by FISH. This subclonal amplification was observed in approximately 20% of tumor cells and was clearly evident on aCGH. In conclusion, our analysis confirms that aCGH is as safe as FISH for the detection of MYC/MYCN gene amplification. Given its cost efficiency in comparison to two FISH tests and the global genomic information additionally provided by an aCGH experiment, this reproducible technique can be safely retained as an alternative to FISH for routine investigation of medulloblastoma. Copyright © 2013 Elsevier Inc. All rights reserved.

Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

PubMed

Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

2016-05-01

India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

Invading stacking primer: A trigger for high-efficiency isothermal amplification reaction with superior selectivity for detecting microRNA variants.

PubMed

Liu, Weipeng; Zhu, Minjun; Liu, Hongxing; Wei, Jitao; Zhou, Xiaoming; Xing, Da

2016-07-15

Searching for a strategy to enhance the efficiency of nucleic acid amplification and achieve exquisite discrimination of nucleic acids at the single-base level for biological detection has become an exciting research direction in recent years. Here, we have developed a simple and universal primer design strategy which produces a fascinating effect on isothermal strand displacement amplification (iSDA). We refer to the resultant primer as "invading stacking primer (IS-Primer)" which is based on contiguous stacking hybridization and toehold-mediated exchange reaction and function by merely changing the hybridization location of the primer. Using the IS-Primer, the sensitivity in detecting the target miR-21 is improved approximately five fold compared with the traditional iSDA reaction. It was further demonstrated that the IS-Primer acts as an invading strand to initiate branch migration which can increase the efficiency of the untwisting of the hairpin probe. This effect is equivalent to reducing the free energy of the stem, and the technique shows superior selectivity for single-base mismatches. By demonstrating the enhanced effect of the IS-Primer in the iSDA reaction, this work may provide a potentially new avenue for developing more sensitive and selective nucleic acids assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Sensitive Immunofluorescent Staining of Cells via Generation of Fluorescent Nanoscale Polymer Films in Response to Biorecognition

PubMed Central

Avens, Heather J.; Berron, Brad J.; May, Allison M.; Voigt, Katerina R.; Seedorf, Gregory J.; Balasubramaniam, Vivek; Bowman, Christopher N.

2011-01-01

Immunofluorescent staining is central to nearly all cell-based research, yet only a few fluorescent signal amplification approaches for cell staining exist, each with distinct limitations. Here, the authors present a novel, fluorescent polymerization-based amplification (FPBA) method that is shown to enable similar signal intensities as the highly sensitive, enzyme-based tyramide signal amplification (TSA) approach. Being non-enzymatic, FPBA is not expected to suffer from nonspecific staining of endogenous enzymes, as occurs with enzyme-based approaches. FPBA employs probes labeled with photopolymerization initiators, which lead to the controlled formation of fluorescent polymer films only at targeted biorecognition sites. Nuclear pore complex proteins (NPCs; in membranes), vimentin (in filaments), and von Willebrand factor (in granules) were all successfully immunostained by FPBA. Also, FPBA was demonstrated to be capable of multicolor immunostaining of multiple antigens. To assess relative sensitivity, decreasing concentrations of anti-NPC antibody were used, indicating that both FPBA and TSA stained NPC down to a 1:100,000 dilution. Nonspecific, cytoplasmic signal resulting from NPC staining was found to be reduced up to 5.5-fold in FPBA as compared to TSA, demonstrating better signal localization with FPBA. FPBA’s unique approach affords a combination of preferred attributes, including high sensitivity and specificity not otherwise available with current techniques. PMID:21339175

Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content.

PubMed

Ishihara, Satoru; Kotomura, Naoe; Yamamoto, Naoki; Ochiai, Hiroshi

2017-08-15

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

PubMed Central

Qi, Xiaoquan; Bakht, Saleha; Devos, Katrien M.; Gale, Mike D.; Osbourn, Anne

2001-01-01

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays. PMID:11713336

A cascade signal amplification strategy for surface enhanced Raman spectroscopy detection of thrombin based on DNAzyme assistant DNA recycling and rolling circle amplification.

PubMed

Gao, Fenglei; Du, Lili; Tang, Daoquan; Lu, Yao; Zhang, Yanzhuo; Zhang, Lixian

2015-04-15

A sensitive protocol for surface enhanced Raman spectroscopy (SERS) detection of thrombin is designed with R6G-Ag NPs as a signal tag by combining DNAzyme assistant DNA recycling and rolling circle amplification (RCA). Molecular beacon (MB) as recognition probe immobilizes on the glass slides and performs the amplification procedure. After thrombin-induced structure-switching DNA hairpins of probe 1, the DNAzyme is liberated from the caged structure, which hybridizes with the MB for cleavage of the MB in the presence of cofactor Zn(2+) and initiates the DNA recycling process, leading to the cleavage of a large number of MB and the generation of numerous primers for triggering RCA reaction. The long amplified RCA product which contained hundreds of tandem-repeat sequences, which can bind with oligonucleotide functionalized Ag NPs reporters. The attached signal tags can be easily read out by SERS. Because of the cascade signal amplification, these newly designed protocols provides a sensitive SERS detection of thrombin down to the femolar level (2.3fM) with a linear range of 5 orders of magnitude (from 10(-14) to 10(-9)M) and have high selectivity toward its target protein. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

NASA Technical Reports Server (NTRS)

Fan, Wenhong (Inventor); Han, Jie (Inventor); Cassell, Alan M. (Inventor)

2006-01-01

Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

Amplification and chromosomal dispersion of human endogenous retroviral sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steele, P.E.; Martin, M.A.; Rabson, A.B.

1986-09-01

Endogenous retroviral sequences have undergone amplification events involving both viral and flanking cellular sequences. The authors cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification andmore » dispersion events may be linked.« less

Application of an oligonucleotide microarray-based nano-amplification technique for the detection of fungal pathogens.

PubMed

Lu, Weiping; Gu, Dayong; Chen, Xingyun; Xiong, Renping; Liu, Ping; Yang, Nan; Zhou, Yuanguo

2010-10-01

The traditional techniques for diagnosis of invasive fungal infections in the clinical microbiology laboratory need improvement. These techniques are prone to delay results due to their time-consuming process, or result in misidentification of the fungus due to low sensitivity or low specificity. The aim of this study was to develop a method for the rapid detection and identification of fungal pathogens. The internal transcribed spacer two fragments of fungal ribosomal DNA were amplified using a polymerase chain reaction for all samples. Next, the products were hybridized with the probes immobilized on the surface of a microarray. These species-specific probes were designed to detect nine different clinical pathogenic fungi including Candida albicans, Candida tropocalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida lusitaniae, Candida guilliermondii, Candida keyfr, and Cryptococcus neoformans. The hybridizing signals were enhanced with gold nanoparticles and silver deposition, and detected using a flatbed scanner or visually. Fifty-nine strains of fungal pathogens, including standard and clinically isolated strains, were correctly identified by this method. The sensitivity of the assay for Candida albicans was 10 cells/mL. Ten cultures from clinical specimens and 12 clinical samples spiked with fungi were also identified correctly. This technique offers a reliable alternative to conventional methods for the detection and identification of fungal pathogens. It has higher efficiency, specificity and sensitivity compared with other methods commonly used in the clinical laboratory.

Droplet microfluidics for amplification-free genetic detection of single cells.

PubMed

Rane, Tushar D; Zec, Helena C; Puleo, Chris; Lee, Abraham P; Wang, Tza-Huei

2012-09-21

In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.

A microRNA detection system based on padlock probes and rolling circle amplification

PubMed Central

Jonstrup, Søren Peter; Koch, Jørn; Kjems, Jørgen

2006-01-01

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19–24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA. PMID:16888321

A microRNA detection system based on padlock probes and rolling circle amplification.

PubMed

Jonstrup, Søren Peter; Koch, Jørn; Kjems, Jørgen

2006-09-01

The differential expression and the regulatory roles of microRNAs (miRNAs) are being studied intensively these years. Their minute size of only 19-24 nucleotides and strong sequence similarity among related species call for enhanced methods for reliable detection and quantification. Moreover, miRNA expression is generally restricted to a limited number of specific cells within an organism and therefore requires highly sensitive detection methods. Here we present a simple and reliable miRNA detection protocol based on padlock probes and rolling circle amplification. It can be performed without specialized equipment and is capable of measuring the content of specific miRNAs in a few nanograms of total RNA.

Comparison of the rolling circle amplification and ligase-dependent reaction methods for the identification of opportunistic Exophiala species.

PubMed

Kaplan, Engin; Ilkit, Macit; de Hoog, G Sybren

2017-10-26

We developed two ligase-dependent probe amplification assays based on rolling circle amplification (RCA) and the ligase-dependent reaction (LDR) to differentiate species of Exophiala targeting the rDNA internal transcribed spacer region. We focused on Exophiala dermatitidis and E. phaeomuriformis, two opportunistic inhabitants of indoor wet cells, and further detected E. heteromorpha, E. xenobiotica, and E. crusticola; 57 reference isolates representing the five species were tested. Depending on the RCA probes used, the sensitivity was 100%, and the specificity ranged from 3.7% to 88.6% (median: 46.1%). In contrast, the sensitivity and specificity of the LDR probes targeting the same isolates were 88.6-100% (median: 95.8%) and 95.4-100% (median: 97.7%), respectively. We analyzed 198 additional environmental isolates representing the same Exophiala species. Overall, the sensitivity and specificity of LDR ranged from 89.7% to 100% (median: 94.1%) and from 93.9% to 100% (median: 96.9%), respectively. The assessment of performance and validation of LDR probes using SYBR Green quantitative polymerase chain reaction revealed high reproducibility and an acceptable range limit, in line with the guidelines of the European Network of GMO Laboratories. In conclusion, the LDR assay was more reliable and less expensive than RCA for species-level identification of Exophiala isolates. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

PubMed

Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

2017-09-01

Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (<5min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4°C for 2min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B.V. All rights reserved.

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

PubMed

Bassler, H A; Flood, S J; Livak, K J; Marmaro, J; Knorr, R; Batt, C A

1995-10-01

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.

Optomechanical transistor with mechanical gain

NASA Astrophysics Data System (ADS)

Zhang, X. Z.; Tian, Lin; Li, Yong

2018-04-01

We study an optomechanical transistor, where an input field can be transferred and amplified unidirectionally in a cyclic three-mode optomechanical system. In this system, the mechanical resonator is coupled simultaneously to two cavity modes. We show that it only requires a finite mechanical gain to achieve the nonreciprocal amplification. Here the nonreciprocity is caused by the phase difference between the linearized optomechanical couplings that breaks the time-reversal symmetry of this system. The amplification arises from the mechanical gain, which provides an effective phonon bath that pumps the mechanical mode coherently. This effect is analogous to the stimulated emission of atoms, where the probe field can be amplified when its frequency is in resonance with that of the anti-Stokes transition. We show that by choosing optimal parameters, this optomechanical transistor can reach perfect unidirectionality accompanied with strong amplification. In addition, the presence of the mechanical gain can result in ultralong delay in the phase of the probe field, which provides an alternative to controlling light transport in optomechanical systems.

A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.

PubMed

Han, Yang; Hou, Shao-Yang; Ji, Shang-Zhi; Cheng, Juan; Zhang, Meng-Yue; He, Li-Juan; Ye, Xiang-Zhong; Li, Yi-Min; Zhang, Yi-Xuan

2017-11-15

A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

Ligation-rolling circle amplification combined with γ-cyclodextrin mediated stemless molecular beacon for sensitive and specific genotyping of single-nucleotide polymorphism.

PubMed

Zou, Zhen; Qing, Zhihe; He, Xiaoxiao; Wang, Kemin; He, Dinggeng; Shi, Hui; Yang, Xue; Qing, Taiping; Yang, Xiaoxiao

2014-07-01

A novel approach for highly sensitive and selective genotyping of single-nucleotide polymorphism (SNP) has been developed based on ligation-rolling circle amplification (L-RCA) and stemless molecular beacon. In this approach, two tailored DNA probes were involved. The stemless molecular beacon, formed through the inclusion interactions of γ-cyclodextrin (γ-CD) and bis-pyrene labeled DNA fragment, was served as signal probe. In the absence of mutant target, the two pyrene molecules were bound in the γ-CD cavity to form an excimer and showed a strong fluorescence at 475 nm. It was here named γ-CD-P-MB. The padlock DNA probe was designed as recognition probe. Upon the recognition of a point mutation DNA targets, the padlock probe was ligated to generate a circular template. An RCA amplification was then initiated using the circular template in the presence of Phi29 polymerase and dNTPs. The L-RCA products, containing repetitive sequence units, subsequently hybridized with the γ-CD-P-MB. This made pyrene molecules away from γ-CD cavity and caused a decrease of excimer fluorescence. As a proof-of-concept, SNP typing of β-thalassemia gene at position -28 was investigated using this approach. The detection limit of mutated target was determined to be 40 fM. In addition, DNA ligase offered high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant target even when the ratio of the wildtype to the mutant is 999:1. Given these attractive characteristics, the developed approach might provide a great genotyping platform for pathogenic diagnosis and genetic analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Thermal diffusivity measurement in thin metallic filaments using the mirage method with multiple probe beams and a digital camera

NASA Astrophysics Data System (ADS)

Vargas, E.; Cifuentes, A.; Alvarado, S.; Cabrera, H.; Delgado, O.; Calderón, A.; Marín, E.

2018-02-01

Photothermal beam deflection is a well-established technique for measuring thermal diffusivity. In this technique, a pump laser beam generates temperature variations on the surface of the sample to be studied. These variations transfer heat to the surrounding medium, which may be air or any other fluid. The medium in turn experiences a change in the refractive index, which will be proportional to the temperature field on the sample surface when the distance to this surface is small. A probe laser beam will suffer a deflection due to the refractive index periodical changes, which is usually monitored by means of a quadrant photodetector or a similar device aided by lock-in amplification. A linear relationship that arises in this technique is that given by the phase lag of the thermal wave as a function of the distance to a punctual heat source when unidimensional heat diffusion can be guaranteed. This relationship is useful in the calculation of the sample's thermal diffusivity, which can be obtained straightforwardly by the so-called slope method, if the pump beam modulation frequency is well-known. The measurement procedure requires the experimenter to displace the probe beam at a given distance from the heat source, measure the phase lag at that offset, and repeat this for as many points as desired. This process can be quite lengthy in dependence of the number points. In this paper, we propose a detection scheme, which overcomes this limitation and simplifies the experimental setup using a digital camera that substitutes all detection hardware utilizing motion detection techniques and software digital signal lock-in post-processing. In this work, the method is demonstrated using thin metallic filaments as samples.

Thermal diffusivity measurement in thin metallic filaments using the mirage method with multiple probe beams and a digital camera.

PubMed

Vargas, E; Cifuentes, A; Alvarado, S; Cabrera, H; Delgado, O; Calderón, A; Marín, E

2018-02-01

Photothermal beam deflection is a well-established technique for measuring thermal diffusivity. In this technique, a pump laser beam generates temperature variations on the surface of the sample to be studied. These variations transfer heat to the surrounding medium, which may be air or any other fluid. The medium in turn experiences a change in the refractive index, which will be proportional to the temperature field on the sample surface when the distance to this surface is small. A probe laser beam will suffer a deflection due to the refractive index periodical changes, which is usually monitored by means of a quadrant photodetector or a similar device aided by lock-in amplification. A linear relationship that arises in this technique is that given by the phase lag of the thermal wave as a function of the distance to a punctual heat source when unidimensional heat diffusion can be guaranteed. This relationship is useful in the calculation of the sample's thermal diffusivity, which can be obtained straightforwardly by the so-called slope method, if the pump beam modulation frequency is well-known. The measurement procedure requires the experimenter to displace the probe beam at a given distance from the heat source, measure the phase lag at that offset, and repeat this for as many points as desired. This process can be quite lengthy in dependence of the number points. In this paper, we propose a detection scheme, which overcomes this limitation and simplifies the experimental setup using a digital camera that substitutes all detection hardware utilizing motion detection techniques and software digital signal lock-in post-processing. In this work, the method is demonstrated using thin metallic filaments as samples.

Highly sensitive and selective microRNA detection based on DNA-bio-bar-code and enzyme-assisted strand cycle exponential signal amplification.

PubMed

Dong, Haifeng; Meng, Xiangdan; Dai, Wenhao; Cao, Yu; Lu, Huiting; Zhou, Shufeng; Zhang, Xueji

2015-04-21

Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNP-DNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

Specificity Re-evaluation of Oligonucleotide Probes for the Detection of Marine Picoplankton by Tyramide Signal Amplification-Fluorescent In Situ Hybridization.

PubMed

Riou, Virginie; Périot, Marine; Biegala, Isabelle C

2017-01-01

Oligonucleotide probes are increasingly being used to characterize natural microbial assemblages by Tyramide Signal Amplification-Fluorescent in situ Hybridization (TSA-FISH, or CAtalysed Reporter Deposition CARD-FISH). In view of the fast-growing rRNA databases, we re-evaluated the in silico specificity of eleven bacterial and eukaryotic probes and competitor frequently used for the quantification of marine picoplankton. We performed tests on cell cultures to decrease the risk for non-specific hybridization, before they are used on environmental samples. The probes were confronted to recent databases and hybridization conditions were tested against target strains matching perfectly with the probes, and against the closest non-target strains presenting one to four mismatches. We increased the hybridization stringency from 55 to 65% formamide for the Eub338+EubII+EubIII probe mix to be specific to the Eubacteria domain. In addition, we found that recent changes in the Gammaproteobacteria classification decreased the specificity of Gam42a probe, and that the Roseo536R and Ros537 probes were not specific to, and missed part of the Roseobacter clade. Changes in stringency conditions were important for bacterial probes; these induced, respectively, a significant increase, in Eubacteria and Roseobacter and no significant changes in Gammaproteobacteria concentrations from the investigated natural environment. We confirmed the eukaryotic probes original conditions, and propose the Euk1209+NChlo01+Chlo02 probe mix to target the largest picoeukaryotic diversity. Experiences acquired through these investigations leads us to propose the use of seven steps protocol for complete FISH probe specificity check-up to improve data quality in environmental studies.

Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.

PubMed

Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

2014-09-15

Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Single palindromic molecular beacon-based amplification for genetic analysis of cancers.

PubMed

Li, Feng; Zhao, Hui; Wang, Zheng-Yong; Wu, Zai-Sheng; Yang, Zhe; Li, Cong-Cong; Xu, Huo; Lyu, Jian-Xin; Shen, Zhi-Fa

2017-05-15

The detection of biomarkers is of crucial importance in reducing the morbidity and mortality of complex diseases. Thus, there is a great desire to develop highly efficient and simple sensing methods to fulfill the different diagnostic and therapeutic purposes. Herein, using tumor suppressor p53 gene as model target DNA, we developed a novel palindromic fragment-incorporated molecular beacon (P-MB) that can perform multiple functions, including recognition element, signal reporter, polymerization template and primer. Upon specific hybridization with target DNA, P-MBs can interact with each other and are extended by polymerase without any additional probes. As a result, hybridized targets are peeled off from P-MBs and initiate the next round of reactions, leading to the unique strand displacement amplification (SDA). The newly-proposed enzymatic amplification displays the detection limit as low as 100pM and excellent selectivity in distinguishing single-base mutation with the linear response range from 100pM to 75nM. This is the simplest SDA sensing system so far because of only involving one type of DNA probe. This impressive sensing paradigm is expected to provide new insight into developing new-type of DNA probes that hold tremendous potential with important applications in molecular biology research and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

PubMed Central

Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

2016-01-01

Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification. PMID:26729209

Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

NASA Astrophysics Data System (ADS)

Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

2016-01-01

Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification.

PubMed

Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

2016-01-05

Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

PubMed

Dmitrienko, E V; Khomiakova, E A; Pyshnaia; Bragin, A G; Vedernikov, V E; Pyshnyĭ, D V

2010-01-01

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

A Smart Detection System Based on Specific Magnetic and Rolling Cycle Amplification Signal-Amplified Dual-Aptamers to Accurately Monitor Minimal Residual Diseases in Patients with T-ALL.

PubMed

Li, Xa; Zhou, Bo; Zhao, Zilong; Hu, Zixi; Zhou, Sufang; Yang, Nuo; Huang, Yong; Zhang, Zhenghua; Su, Jing; Lan, Dan; Qin, Xue; Meng, Jinyu; Zheng, Duo; He, Jian; Huang, Xianing; Zhao, Jing; Zhang, Zhiyong; Tan, Weihong; Lu, Xiaoling; Zhao, Yongxiang

2016-12-01

It is a major clinical challenge for clinicians how to early find out minimal residual diseases (MRD) of leukemia. Here, we developed a smart detection system for MRD involving magnetic aptamer sgc8 probe (M-sgc8 probe) to capture CEM cells and rolling cycle amplification probe (RCA-sgc8 probe) to initiate RCA, producing a single-stranded tandem repeated copy of the circular template. The DNA products were hybridized with molecular beacon to generate the amplified fluorescence signal. An in vitro model to mimic MRD was established to evaluate the sensitivity of the smart detection system. The smart detection system was used to detect MRD in patients with T-ALL peri-chemotherapy, which could not only specifically captured T-ALL cells, but also significantly amplified fluorescence signals on them. The sensitivity was 1/20,000. These results indicate that the smart detection system with high specificity and sensitivity could more efficiently monitor the progress of T-ALL peri-chemotherapy.

[Application of isothermal amplification technology for pathogen detection in parasitic and other diseases].

PubMed

Ting, Li; Kun, Yang

2018-04-16

The in vitro nucleic acid amplification technique based on polymerase chain reaction (PCR) has been successfully applied to scientific researches. In recent years, the emergence of isothermal amplification technology is increasingly applied in the molecular diagnosis and disease detection because of its advantages of constant temperature, high efficiency, short time-consuming, and less reliance on equipment and instruments. The principle, characteristics and application of the partial isothermal amplification technique in the pathogen detection in parasitic and other diseases are reviewed in this paper, and the prospects of the wide development of the technique are also discussed.

Development of PCR for screening of enteroaggregative Escherichia coli.

PubMed Central

Schmidt, H; Knop, C; Franke, S; Aleksic, S; Heesemann, J; Karch, H

1995-01-01

In this study, we determined the sequence of the EcoRI-PstI fragment of the plasmid pCVD432, also termed the enteroaggregative Escherichia coli (EAggEC) probe. A primer pair complementary to this probe was designed for PCR amplification of a 630-bp region. Comparison of the analysis of the EAggEC probe sequence with those in database libraries revealed no significant similarity to any known bacterial gene. Pure cultures of E. coli cells, as well as mixed cultures from stool specimens, were investigated with the PCR assay, the EAggEC probe test, and the adherence test. Of 50 E. coli strains which demonstrated aggregative adherence to HEp-2 cells, 43 (86%) were positive with the EAggEC PCR. All 43 of these strains reacted with the EAggEC probe. Six EAggEC strains gave negative results by both molecular techniques. In contrast, only 4 of 418 (0.96%) strains representing other categories of diarrheagenic E. coli demonstrated a positive PCR result. The PCR was also successful in screening for the presence of EAggEC in enriched cultures grown from stool specimens. Compared with cell culture assays and colony hybridization, our findings revealed that the PCR assay was more rapid, simple, and highly sensitive and can therefore be recommended as a screening method for EAggEC in the clinical laboratory. PMID:7751380

Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

DOEpatents

Gray, Joe W.; Weier, Heinz-Ulrich

1998-01-01

A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences.

Y chromosome specific nucleic acid probe and method for identifying the Y chromosome in SITU

DOEpatents

Gray, Joe W.; Weier, Heinz-Ulrich

1999-01-01

A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences.

Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

DOEpatents

Gray, Joe W.; Weier, Heinz-Ulrich

2001-01-01

A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences.

Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

DOEpatents

Gray, J.W.; Weier, H.U.

1998-11-24

A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences. 9 figs.

Y chromosome specific nucleic acid probe and method for identifying the Y chromosome in SITU

DOEpatents

Gray, J.W.; Weier, H.U.

1999-03-30

A method for producing a Y chromosome specific probe selected from highly repeating sequences on that chromosome is described. There is little or no nonspecific binding to autosomal and X chromosomes, and a very large signal is provided. Inventive primers allowing the use of PCR for both sample amplification and probe production are described, as is their use in producing large DNA chromosome painting sequences. 9 figs.

Sensitive detection of T4 polynucleotide kinase activity based on multifunctional magnetic probes and polymerization nicking reactions mediated hyperbranched rolling circle amplification.

PubMed

Li, Xia; Xu, Xiaowen; Song, Juan; Xue, Qingwang; Li, Chenzhong; Jiang, Wei

2017-05-15

T4 polynucleotide kinase (PNK) plays critical roles in regulating DNA phosphorylation modes during the repair of DNA lesions. The aberrant activity of T4 PNK has been proven to be associated with a variety of human pathologies. Sensitive detection of T4 PNK activity is critical to both clinical diagnosis and therapeutics. Herein, a background-eliminated fluorescence assay for sensitive detection of T4 PNK activity has been developed by multifunctional magnetic probes and polymerization nicking reactions mediated hyperbranched rolling circle amplification (HRCA). First, the streptavidin-magnetic nanobeads (MBs) were functionalized with the biotin modified hairpin probe (HP) with 3'-phosphoryl, forming multifunctional magnetic probes (HP-MBs). Then, in the presence of T4 PNK, the 3'-phosphoryl of HP-MBs was hydrolyzed to 3'-hydroxyl, thus serving as primers to initiate the polymerization extension and nicking endonuclease cleavage reaction. Next, the primers released from above "polymerization-nicking" cycles were separated out to trigger the subsequently HRCA process, producing plenty of dsDNA. Finally, the intercalating dye SYBR Green I (SG) was inserted into the dsDNA, generating enhanced fluorescence signals. In our design, the HP-MBs here serve together as the T4 PNK, DNA polymerase, and endonuclease recognition probe, and thus avoid the demands of utilizing multiple probes design. Moreover, it performed primary "polymerization-nicking" amplification and mediate secondary HRCA. In addition to, performing the separation function, the binding of HP-MBs and SG could be avoided while a low background was acquired. This method showed excellent sensitivity with a detection limit of 0.0436 mU/mL, and accomplished exceptional characterization T4 PNK activity in cell extracts, offering a powerful tool for biomedical research and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Enzyme-free detection of sequence-specific microRNAs based on nanoparticle-assisted signal amplification strategy.

PubMed

Li, Ru-Dong; Wang, Qian; Yin, Bin-Cheng; Ye, Bang-Ce

2016-03-15

Developing direct and convenient methods for microRNAs (miRNAs) analysis is of great significance in understanding biological functions of miRNAs, and early diagnosis of cancers. We have developed a rapid, enzyme-free method for miRNA detection based on nanoparticle-assisted signal amplification coupling fluorescent metal nanoclusters as signal output. The proposed method involves two processes: target miRNA-mediated nanoparticle capture, which consists of magnetic microparticle (MMP) probe and CuO nanoparticle (NP) probe, and nanoparticle-mediated amplification for signal generation, which consists of fluorescent DNA-Cu/Ag nanocluster (NC) and 3-mercaptopropionic acid (MPA). In the presence of target miRNA, MMP probe and NP probe sandwich-capture the target miRNA via their respective complementary sequence. The resultant sandwich complex (MMP probe-miRNA-CuO NP probe) is separated using a magnetic field and further dissolved by acidolysis to turn CuO NP into a great amount of copper (II) ions (Cu(2+)). Cu(2+) could disrupt the interactions between thiol moiety of MPA and the fluorescent Cu/Ag NCs by preferentially reacting with MPA to form a disulfide compound as intermediate. By this way, the fluorescence emission of the DNA-Cu/Ag NCs in the presence of MPA increases upon the increasing concentration of Cu(2+), which is directly proportional to the amount of target miRNA. The proposed method allows quantitative detection of a liver-specific miR-221-5p in the range of 5 pM to 1000 pM with a detection limit of ~0.73 pM, and shows a good ability to discriminate single-base difference. Moreover, the detection assay can be applied to detect miRNA in cancerous cell lysates in excellent agreement with that from a commercial miRNA detection kit. Copyright © 2015 Elsevier B.V. All rights reserved.

Lidar using the backscatter amplification effect

NASA Astrophysics Data System (ADS)

Razenkov, Igor A.; Banakh, Victor A.

2018-04-01

Experimental data proving the possibility of lidar measurement of the refractive turbulence strength based on the effect of backscatter amplification (BSA) are reported. It is shown that the values of the amplification factor correlate with the variance of random jitter of optical image of an incoherent light source depending on the value of the structure constant of the air refractive index turbulent fluctuations averaged over the probing path. This paper presents the results of measurements of the BSA factor in comparison with the simultaneous measurements of the BSA peak, which is very narrow and only occurs on the laser beam axis. It is constructed the range-time images of the derivative of the amplification factor gives a comprehensive picture of the location of turbulent zones and their temporal dynamics.

Water-Soluble Conjugated Polymers: Self-Assembly and Biosensor Applications

NASA Astrophysics Data System (ADS)

Bazan, Guillermo

2005-03-01

Homogeneous assays can be designed which take advantage of the optical amplification of conjugated polymers and the self-assembly characteristic of aqueous polyelectrolytes. For example, a ssDNA sequence sensor comprises an aqueous solution containing a cationic water soluble conjugated polymer such as poly(9,9-bis(trimethylammonium)-hexyl)-fluorene phenylene) with a peptide nucleic acid (PNA) labeled with a dye (PNA-C*). Signal transduction is controlled by hybridization of the neutral PNA-C* probe and the negative ssDNA target, resulting in favorable electrostatic interactions between the hybrid complex and the cationic polymer. Distance requirements for Förster energy transfer are thus met only when ssDNA of complementary sequence to the PNA-C* probe is present. Signal amplification by the conjugated polymer provides fluorescein emission >25 times higher than that of the directly excited dye. Transduction by electrostatic interactions followed by energy transfer is a general strategy. Examples involving other biomolecular recognition events, such as DNA/DNA, RNA/protein and RNA/RNA, will also be provided. The mechanism of biosensing will be discussed, with special attention to the varying contributions of hydrophobic and electrostatic forces, polymer conformation, charge density, local concentration of C*s and tailored defect sites for aggregation-induced optical changes. Finally, the water solubility of these conjugated polymers opens possibilities for spin casting onto organic materials, without dissolving the underlying layers. This property is useful for fabricating multilayer organic optoelectronic devices by simple solution techniques.

Label-free genotyping of cytochrome P450 2D6*10 using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection.

PubMed

Wang, Hong-Qi; Wu, Zhan; Zhang, Yan; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

2012-01-13

Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies. Copyright © 2011 Elsevier B.V. All rights reserved.

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

PubMed Central

Rector, Annabel; Tachezy, Ruth; Van Ranst, Marc

2004-01-01

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information. PMID:15113879

A cascade autocatalytic strand displacement amplification and hybridization chain reaction event for label-free and ultrasensitive electrochemical nucleic acid biosensing.

PubMed

Chen, Zhiqiang; Liu, Ying; Xin, Chen; Zhao, Jikuan; Liu, Shufeng

2018-08-15

Herein, an autocatalytic strand displacement amplification (ASDA) strategy was proposed for the first time, which was further ingeniously coupled with hybridization chain reaction (HCR) event for the isothermal, label-free and multiple amplification toward nucleic acid detection. During the ASDA module, the target recognition opens the immobilized hairpin probe (IP) and initiates the annealing of the auxiliary DNA strand (AS) with the opened IP for the successive polymerization and nicking reaction in the presence of DNA polymerase and nicking endonuclease. This induces the target recycling and generation of a large amount of intermediate DNA sequences, which can be used as target analogy to execute the autocatalytic strand displacement amplification. Simultaneously, the introduced AS strand can propagate the HCR between two hairpins (H1 and H2) to form a linear DNA concatamer with cytosine (C)-rich loop region, which can facilitate the in-situ synthesis of silver nanoclusters (AgNCs) as electrochemical tags for further amplification toward target responses. With current cascade ASDA and HCR strategy, the detection of target DNA could be achieved with a low detection limit of about 0.16 fM and a good selectivity. The developed biosensor also exhibits the distinct advantages of flexibility and simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

PubMed

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

Helicase-dependent amplification of nucleic acids.

PubMed

Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

2013-10-11

Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

Multivalent Bifunctional Chelator Scaffolds for Gallium-68 Based Positron Emission Tomography Imaging Probe Design: Signal Amplification via Multivalency

PubMed Central

Singh, Ajay N.; Liu, Wei; Hao, Guiyang; Kumar, Amit; Gupta, Anjali; Öz, Orhan K.; Hsieh, Jer-Tsong; Sun, Xiankai

2011-01-01

The role of the multivalent effect has been well recognized in the design of molecular imaging probes towards the desired imaging signal amplification. Recently we reported a bifunctional chelator (BFC) scaffold design, which provides a simple and versatile approach to impart multivalency to radiometal based nuclear imaging probes. In this work, we report a series of BFC scaffolds (tBu3-1-COOH, tBu3-2-(COOH)2 and tBu3-3-(COOH)3) constructed on the framework of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for 68Ga-based PET probe design and signal amplification via multivalent effect. For proof of principle, a known integrin αvβ3 specific ligand (c(RGDyK)) was used to build the corresponding NOTA conjugates (H31, H32, and H33), which present 1 – 3 copies of c(RGDyK) peptide, respectively, in a systematic manner. Using the integrin αvβ3 binding affinities (IC50 values), the enhanced specific binding was observed for multivalent conjugates (H32: 43.9 ± 16.1 nM; H33: 14.7 ± 5.0 nM) as compared to their monovalent counterpart (H31: 171 ± 60 nM) and the intact c(RGDyK) peptide (204 ± 76 nM). The obtained conjugates were efficiently labeled with 68Ga3+ within 30 min at room temperature in high radiochemical yields (> 95%). The in vivo evaluation of the labeled conjugates, 68Ga-1, 68Ga-2 and 68Ga-3, was performed using male severe combined immunodeficiency (SCID) mice bearing integrin αvβ3 positive PC-3 tumor xenografts (n = 3). All 68Ga -labeled conjugates showed high in vivo stability with no detectable metabolites found by radio-HPLC within 2 h post-injection (p.i.). The PET signal amplification in PC-3 tumor by multivalent effect was clearly displayed by the tumor uptake of the 68Ga-labeled conjugates (68Ga-3: 2.55 ± 0.50%ID/g; 68Ga-2: 1.90 ± 0.10 %ID/g; 68Ga-1: 1.66 ± 0.15 %ID/g) at 2 h p.i. In summary, we have designed and synthesized a series of NOTA-based BFC scaffolds with signal amplification properties, which may find potential applications in diagnostic gallium radiopharmaceuticals. PMID:21740059

Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity.

PubMed

Wu, Yushu; Wang, Lei; Jiang, Wei

2017-03-15

Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP) sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7×10 -5 U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis. Copyright © 2016 Elsevier B.V. All rights reserved.

[Investigation of RNA viral genome amplification by multiple displacement amplification technique].

PubMed

Pang, Zheng; Li, Jian-Dong; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

2013-06-01

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

In situ rolling circle amplification detection of Crimean Congo hemorrhagic fever virus (CCHFV) complementary and viral RNA.

PubMed

Andersson, Cecilia; Henriksson, Sara; Magnusson, Karl-Eric; Nilsson, Mats; Mirazimi, Ali

2012-05-10

Crimean Congo hemorrhagic fever virus (CCHFV) is a human pathogen that causes a severe disease with high fatality rate for which there is currently no specific treatment. Knowledge regarding its replication cycle is also highly limited. In this study we developed an in situ technique for studying the different stages during the replication of CCHFV. By integrating reverse transcription, padlock probes, and rolling circle amplification, we were able to detect and differentiate between viral RNA (vRNA) and complementary RNA (cRNA) molecules, and to detect viral protein within the same cell. These data demonstrate that CCHFV nucleocapsid protein (NP) is detectable already at 6 hours post infection in vRNA- and cRNA-positive cells. Confocal microscopy showed that cRNA is enriched and co-localized to a large extent with NP in the perinuclear area, while vRNA has a more random distribution in the cytoplasm with only some co-localize with NP. However, vRNA and cRNA did not appear to co-localize directly. Copyright © 2012 Elsevier Inc. All rights reserved.

Enhanced detection of Rickettsia species in Ixodes pacificus using highly sensitive fluorescence in situ hybridization coupled with Tyramide Signal Amplification.

PubMed

Bagheri, Ghazaleh; Lehner, Jeremy D; Zhong, Jianmin

2017-10-01

Ixodes pacificus is a host of many bacteria including Rickettsia species phylotypes G021 and G022. As part of the overall goal of understanding interactions of phylotypes with their tick host, this study focused on molecular detection of rickettsiae in ovarian and midgut tissue of I. pacificus by fluorescent in situ hybridization (FISH), PCR, and ultrastructural analysis. Of three embedding media (Technovit 8100, Unicryl, and paraffin) tested for generating thin sections, tissues embedded in paraffin resulted in the visualization of bacteria with low autofluorescence in FISH. Digoxigenin-labeled probes were used in FISH to intensify bacterial hybridization signals using Tyramide Signal Amplification reaction. Using this technique, rickettsiae were detected in the cytoplasm of oocytes of I. pacificus. The presence of rickettsiae in the ovary and midgut was further confirmed by PCR and transmission electron microscopic analysis. Overall, the methods in this study can be used to identify locations of tick-borne bacteria in tick tissues and understand transmission routes of bacterial species in ticks. Copyright © 2017 Elsevier GmbH. All rights reserved.

Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification.

PubMed

Mekuria, Tefera A; Zhang, Shulu; Eastwell, Kenneth C

2014-09-01

Little cherry virus 2 (LChV2) (genus Ampelovirus) is the primary causal agent of little cherry disease (LCD) in sweet cherry (Prunus avium) in North America and other parts of the world. This mealybug-transmitted virus does not induce significant foliar symptoms in most sweet cherry cultivars, but does cause virus-infected trees to yield unevenly ripened small fruits with poor flavor. Most fruits from infected trees are unmarketable. In the present study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) technique was developed using LChV2 coat protein specific primers and probe. Detection of terminally labeled amplicons was achieved with a high affinity lateral flow strip. The RT-RPA is confirmed to be simple, fast, and specific. In comparison, although it retains the sensitivity of RT-PCR, it is a more cost-effective procedure. RT-RPA will be a very useful tool for detecting LChV2 from crude extracts in any growth stage of sweet cherry from field samples. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Homogeneous real-time detection of single-nucleotide polymorphisms by strand displacement amplification on the BD ProbeTec ET system.

PubMed

Wang, Sha-Sha; Thornton, Keith; Kuhn, Andrew M; Nadeau, James G; Hellyer, Tobin J

2003-10-01

The BD ProbeTec ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations. The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human beta(2)-adrenergic receptor (beta(2)AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing. Unprocessed whole blood was successfully genotyped with as little as 0.1-1 micro L of sample per reaction. All six beta(2)AR assays were able to accommodate >/==" BORDER="0">20 micro L of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six beta(2)AR assays agreed with DNA sequencing results. SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing.

PubMed

Su, Huilan; Yuan, Ruo; Chai, Yaqin; Mao, Li; Zhuo, Ying

2011-07-15

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H₂O₂. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL. Copyright © 2011 Elsevier B.V. All rights reserved.

Signal amplification strategies for DNA and protein detection based on polymeric nanocomposites and polymerization: A review.

PubMed

Zhou, Shaohong; Yuan, Liang; Hua, Xin; Xu, Lingling; Liu, Songqin

2015-06-02

Demand is increasing for ultrasensitive bioassays for disease diagnosis, environmental monitoring and other research areas. This requires novel signal amplification strategies to maximize the signal output. In this review, we focus on a series of significant signal amplification strategies based on polymeric nanocomposites and polymerization. Some common polymers are used as carriers to increase the local concentration of signal probes and/or biomolecules on their surfaces or in their interiors. Some polymers with special fluorescence and optical properties can efficiently transfer the excitation energy from a single site to the whole polymer backbone. This results in superior fluorescence signal amplification due to the resulting collective effort (integration of signal). Recent polymerization-based signal amplification strategies that employ atom transfer radical polymerization (ATRP) and photo-initiated polymerization are also summarized. Several distinctive applications of polymers in ultrasensitive bioanalysis are highlighted. Copyright © 2015 Elsevier B.V. All rights reserved.

An enzyme-free flow cytometric bead assay for the sensitive detection of microRNAs based on click nucleic acid ligation-mediated signal amplification.

PubMed

Qi, Yan; Qiu, Liying; Fan, Wenjiao; Liu, Chenghui; Li, Zhengping

2017-08-07

A versatile flow cytometric bead assay (FCBA) coupled with a completely enzyme-free signal amplification mechanism is developed for the sensitive detection of microRNAs (miRNAs). This new strategy integrates click chemistry-mediated ligation chain reaction (CLCR) with hybridization chain reaction (HCR) for enzyme-free signal amplification on magnetic beads (MBs), and a flow cytometer for the robust fluorescence readout of the MBs. Firstly, target miRNA can initiate CLCR on the surface of MBs based on the click chemical ligation between dibenzocyclooctyne (DBCO)- and azide-modified single-stranded DNA (ssDNA) probes, and the amount of ligated ssDNA sequences on the MBs will be proportional to the dosage of target miRNA. Afterward, each of the ligated ssDNA products can trigger a cascade chain reaction of hybridization events between two alternating fluorophore-tagged hairpin probes, resulting in another signal amplification pathway with an amplified accumulation of fluorophores on the MBs. Finally, the fluorophore-anchored MBs are directly and rapidly analyzed by using a flow cytometer without any separation or elution processes. Herein, the click nucleic acid ligation only occurs on the surface of MBs, so the nonspecific ligations are greatly inhibited compared with that of ligation reaction performed in homogeneous solution. Furthermore, the signal amplification by CLCR-HCR is highly efficient but totally enzyme-free, which may overcome the potential drawbacks of conventional enzyme-catalyzed signal amplification protocols and lead to a high sensitivity. The CLCR-HCR-based FCBA has pushed the detection limit of let-7a miRNA down to the femtomolar (fM) level, showing great potential in miRNA-related biological studies and disease diagnosis.

Novel selection methods for DNA-encoded chemical libraries

PubMed Central

Chan, Alix I.; McGregor, Lynn M.; Liu, David R.

2015-01-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. PMID:25723146

Mimicking an Enzyme-Based Colorimetric Aptasensor for Antibiotic Residue Detection in Milk Combining Magnetic Loop-DNA Probes and CHA-Assisted Target Recycling Amplification.

PubMed

Luan, Qian; Gan, Ning; Cao, Yuting; Li, Tianhua

2017-07-19

A mimicking-enzyme-based colorimetric aptasensor was developed for the detection of kanamycin (KANA) in milk using magnetic loop-DNA-NMOF-Pt (m-L-DNA) probes and catalytic hairpin assembly (CHA)-assisted target recycling for signal amplification. The m-L-DNA probes were constructed via hybridization of hairpin DNA H1 (containing aptamer sequence) immobilized magnetic beads (m-H1) and signal DNA (sDNA, partial hybridization with H1) labeled nano Fe-MIL-88NH 2 -Pt (NMOF-Pt-sDNA). In the presence of KANA and complementary hairpin DNA H2, the m-L-DNA probes decomposed and formed an m-H1/KANA intermediate, which triggered the CHA reaction to form a stable duplex strand (m-H1-H2) while releasing KANA again for recycling. Consequently, numerous NMOF-Pt-sDNA as mimicking enzymes can synergistically catalyze 3,3',5,5'-tetramethylbenzidine (TMB) for color development. The aptasensor exhibited high selectivity and sensitivity for KANA in milk with a detection limit of 0.2 pg mL -1 within 30 min. The assay can be conveniently extended for on-site screening of other antibiotics in foods by simply changing the base sequence of the probes.

Apricot DNA as an indicator for persipan: detection and quantitation in marzipan using ligation-dependent probe amplification.

PubMed

Luber, Florian; Demmel, Anja; Hosken, Anne; Busch, Ulrich; Engel, Karl-Heinz

2012-06-13

The confectionery ingredient marzipan is exclusively prepared from almond kernels and sugar. The potential use of apricot kernels, so-called persipan, is an important issue for the quality assessment of marzipan. Therefore, a ligation-dependent probe amplification (LPA) assay was developed that enables a specific and sensitive detection of apricot DNA, as an indicator for the presence of persipan. The limit of detection was determined to be 0.1% persipan in marzipan. The suitability of the method was confirmed by the analysis of 20 commercially available food samples. The integration of a Prunus -specific probe in the LPA assay as a reference allowed for the relative quantitation of persipan in marzipan. The limit of quantitation was determined to be 0.5% persipan in marzipan. The analysis of two self-prepared mixtures of marzipan and persipan demonstrated the applicability of the quantitation method at concentration levels of practical relevance for quality control.

Detection of Legionella pneumophila by PCR-ELISA method in industrial cooling tower water.

PubMed

Soheili, Majid; Nejadmoghaddam, Mohammad Reza; Babashamsi, Mohammad; Ghasemi, Jamileh; Jeddi Tehrani, Mahmood

2007-11-15

Water supply and Cooling Tower Water (CTW) are among the most common sources of Legionella pneumophila (LP) contamination. A nonradio active method is described to detect LP in industrial CTW samples. DNA was purified and amplified by nested -PCR with amplimers specific for the 16s rRNA gene of LP. The 5' end biotinylated oligomer probe was immobilized on sterptavidin B coated microtiter plates. The nested-PCR product was labeled with digoxigenin and then hybridized with 5'-biotinylated probes. The amplification products were detected by using proxidase-labled anti dioxygenin antibody in a colorimetric reaction. The assay detected LP present in 1 L of 5 CTW samples examined. All of the samples were Legionella positive in both culture and PCR-ELISA methods. The PCR-ELISA assay appears to exhibit high specificity and is a more rapid technique in comparison with bacterial culture method. Thus could prove suitable for use in the routine examination of industrial CTW contamination.

Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries

PubMed Central

Zimmerman, Sandra G; Peters, Nathaniel C; Altaras, Ariel E; Berg, Celeste A

2014-01-01

In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)–RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase–conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform. PMID:24113787

In Situ Identification of Cyanobacteria with Horseradish Peroxidase-Labeled, rRNA-Targeted Oligonucleotide Probes

PubMed Central

Schönhuber, Wilhelm; Zarda, Boris; Eix, Stella; Rippka, Rosmarie; Herdman, Michael; Ludwig, Wolfgang; Amann, Rudolf

1999-01-01

Individual cyanobacterial cells are normally identified in environmental samples only on the basis of their pigmentation and morphology. However, these criteria are often insufficient for the differentiation of species. Here, a whole-cell hybridization technique is presented that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides for in situ identification of cyanobacteria. This indirect method, in which the probe-conferred enzyme has to be visualized in an additional step, was necessary since fluorescently monolabeled oligonucleotides were insufficient to overstain the autofluorescence of the target cells. Initially, a nonfluorescent detection assay was developed and successfully applied to cyanobacterial mats. Later, it was demonstrated that tyramide signal amplification (TSA) resulted in fluorescent signals far above the level of autofluorescence. Furthermore, TSA-based detection of HRP was more sensitive than that based on nonfluorescent substrates. Critical points of the assay, such as cell fixation and permeabilization, specificity, and sensitivity, were systematically investigated by using four oligonucleotides newly designed to target groups of cyanobacteria. PMID:10049892

Laser cooling and control of excitations in superfluid helium

NASA Astrophysics Data System (ADS)

Harris, G. I.; McAuslan, D. L.; Sheridan, E.; Sachkou, Y.; Baker, C.; Bowen, W. P.

2016-08-01

Superfluidity is a quantum state of matter that exists macroscopically in helium at low temperatures. The elementary excitations in superfluid helium have been probed with great success using techniques such as neutron and light scattering. However, measurements of phonon excitations have so far been limited to average thermodynamic properties or the driven response far out of thermal equilibrium. Here, we use cavity optomechanics to probe the thermodynamics of phonon excitations in real time. Furthermore, strong light-matter interactions allow both laser cooling and amplification. This represents a new tool to observe and control superfluid excitations that may provide insight into phonon-phonon interactions, quantized vortices and two-dimensional phenomena such as the Berezinskii-Kosterlitz-Thouless transition. The third sound modes studied here also offer a pathway towards quantum optomechanics with thin superfluid films, including the prospect of femtogram masses, high mechanical quality factors, strong phonon-phonon and phonon-vortex interactions, and self-assembly into complex geometries with sub-nanometre feature size.

A TaqMan real-time PCR-based assay for the identification of Fasciola spp.

PubMed

Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Jowers, Michael J; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

2011-06-30

Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the first qPCR-based assay for the identification of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus-specific primers for Fasciola ITS-2 amplification, and we designed species-specific internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid 'intermediate'Fasciola. These primers and probes were used for the highly specific, sensitive, and simple identification of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identification of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of real-time recombinase polymerase amplification assay for rapid and sensitive detection of canine parvovirus 2.

PubMed

Geng, Yunyun; Wang, Jianchang; Liu, Libing; Lu, Yan; Tan, Ke; Chang, Yan-Zhong

2017-11-06

Canine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species. Early detection of canine parvovirus (CPV-2) is crucial to initiating appropriate outbreak control strategies. Recombinase polymerase amplification (RPA), a novel isothermal gene amplification technique, has been developed for the molecular detection of diverse pathogens. In this study, a real-time RPA assay was developed for the detection of CPV-2 using primers and an exo probe targeting the CPV-2 nucleocapsid protein gene. The real-time RPA assay was performed successfully at 38 °C, and the results were obtained within 4-12 min for 10 5 -10 1 molecules of template DNA. The assay only detected CPV-2, and did not show cross-detection of other viral pathogens, demonstrating a high level of specificity. The analytical sensitivity of the real-time RPA was 10 1 copies/reaction of a standard DNA template, which was 10 times more sensitive than the common RPA method. The clinical sensitivity of the real-time RPA assay matched 100% (n = 91) to the real-time PCR results. The real-time RPA assay is a simple, rapid, reliable and affordable method that can potentially be applied for the detection of CPV-2 in the research laboratory and point-of-care diagnosis.

A functional glycoprotein competitive recognition and signal amplification strategy for carbohydrate-protein interaction profiling and cell surface carbohydrate expression evaluation

NASA Astrophysics Data System (ADS)

Wang, Yangzhong; Chen, Zhuhai; Liu, Yang; Li, Jinghong

2013-07-01

A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes.A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes. Electronic supplementary information (ESI) available: Experimental details; characterization of probes; the influence of electrolyte pH; probe concentration and glucose concentration on the electrode ECL effect. See DOI: 10.1039/c3nr01598j

Nonlinear X-Ray and Auger Spectroscopy at X-Ray Free-Electron Laser Sources

NASA Astrophysics Data System (ADS)

Rohringer, Nina

2015-05-01

X-ray free-electron lasers (XFELs) open the pathway to transfer non-linear spectroscopic techniques to the x-ray domain. A promising all x-ray pump probe technique is based on coherent stimulated electronic x-ray Raman scattering, which was recently demonstrated in atomic neon. By tuning the XFEL pulse to core-excited resonances, a few seed photons in the spectral tail of the XFEL pulse drive an avalanche of resonant inelastic x-ray scattering events, resulting in exponential amplification of the scattering signal by of 6-7 orders of magnitude. Analysis of the line profile of the emitted radiation permits to demonstrate the cross over from amplified spontaneous emission to coherent stimulated resonance scattering. In combination with statistical covariance mapping, a high-resolution spectrum of the resonant inelastic scattering process can be obtained, opening the path to coherent stimulated x-ray Raman spectroscopy. An extension of these ideas to molecules and a realistic feasibility study of stimulated electronic x-ray Raman scattering in CO will be presented. Challenges to realizing stimulated electronic x-ray Raman scattering at present-day XFEL sources will be discussed, corroborated by results of a recent experiment at the LCLS XFEL. Due to the small gain cross section in molecular targets, other nonlinear spectroscopic techniques such as nonlinear Auger spectroscopy could become a powerful alternative. Theory predictions of a novel pump probe technique based on resonant nonlinear Auger spectroscopic will be discussed and the method will be compared to stimulated x-ray Raman spectroscopy.

Ultrasensitive detection of nucleic acids and proteins using quartz crystal microbalance and surface plasmon resonance sensors based on target-triggering multiple signal amplification strategy.

PubMed

Sun, Wenbo; Song, Weiling; Guo, Xiaoyan; Wang, Zonghua

2017-07-25

In this study, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) sensors were combined with template enhanced hybridization processes (TEHP), rolling circle amplification (RCA) and biocatalytic precipitation (BCP) for ultrasensitive detection of DNA and protein. The DNA complementary to the aptamer was released by the specific binding of the aptamer to the target protein and then hybridized with the capture probe and the assistant DNA to form a ternary "Y" junction structure. The initiation chain was generated by the template-enhanced hybridization process which leaded to the rolling circle amplification reaction, and a large number of repeating unit sequences were formed. Hybridized with the enzyme-labeled probes, the biocatalytic precipitation reaction was further carried out, resulting in a large amount of insoluble precipitates and amplifying the detection signal. Under the optimum conditions, detection limits as low as 43 aM for target DNA and 53 aM for lysozyme were achieved. In addition, this method also showed good selectivity and sensitivity in human serum. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization▿ †

PubMed Central

Smolina, Irina; Lee, Charles; Frank-Kamenetskii, Maxim

2007-01-01

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes. PMID:17293504

Developments and advances concerning the hyperpolarisation technique SABRE.

PubMed

Mewis, Ryan E

2015-10-01

To overcome the inherent sensitivity issue in NMR and MRI, hyperpolarisation techniques are used. Signal Amplification By Reversible Exchange (SABRE) is a hyperpolarisation technique that utilises parahydrogen, a molecule that possesses a nuclear singlet state, as the source of polarisation. A metal complex is required to break the singlet order of parahydrogen and, by doing so, facilitates polarisation transfer to analyte molecules ligated to the same complex through the J-coupled network that exists. The increased signal intensities that the analyte molecules possess as a result of this process have led to investigations whereby their potential as MRI contrast agents has been probed and to understand the fundamental processes underpinning the polarisation transfer mechanism. As well as discussing literature relevant to both of these areas, the chemical structure of the complex, the physical constraints of the polarisation transfer process and the successes of implementing SABRE at low and high magnetic fields are discussed. Copyright © 2015 John Wiley & Sons, Ltd.

Costs of genetic testing: Supporting Brazilian Public Policies for the incorporating of molecular diagnostic technologies

PubMed Central

Schlatter, Rosane Paixão; Matte, Ursula; Polanczyk, Carisi Anne; Koehler-Santos, Patrícia; Ashton-Prolla, Patricia

2015-01-01

This study identifies and describes the operating costs associated with the molecular diagnosis of diseases, such as hereditary cancer. To approximate the costs associated with these tests, data informed by Standard Operating Procedures for various techniques was collected from hospital software and a survey of market prices. Costs were established for four scenarios of capacity utilization to represent the possibility of suboptimal use in research laboratories. Cost description was based on a single site. The results show that only one technique was not impacted by rising costs due to underutilized capacity. Several common techniques were considerably more expensive at 30% capacity, including polymerase chain reaction (180%), microsatellite instability analysis (181%), gene rearrangement analysis by multiplex ligation probe amplification (412%), non-labeled sequencing (173%), and quantitation of nucleic acids (169%). These findings should be relevant for the definition of public policies and suggest that investment of public funds in the establishment of centralized diagnostic research centers would reduce costs to the Public Health System. PMID:26500437

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L.

PubMed

Cano, I; Ferro, P; Alonso, M C; Sarasquete, C; Garcia-Rosado, E; Borrego, J J; Castro, D

2009-02-01

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.

Tailored pump compensation for Brillouin optical time-domain analysis with distributed Brillouin amplification.

PubMed

Kim, Young Hoon; Song, Kwang Yong

2017-06-26

A Brillouin optical time domain analysis (BOTDA) system utilizing tailored compensation for the propagation loss of the pump pulse is demonstrated for long-range and high-resolution distributed sensing. A continuous pump wave for distributed Brillouin amplification (DBA pump) of the pump pulse co-propagates with the probe wave, where gradual variation of the spectral width is additionally introduced to the DBA pump to obtain a uniform Brillouin gain along the position. In the experimental confirmation, a distributed strain measurement along a 51.2 km fiber under test is presented with a spatial resolution of 20 cm, in which the measurement error (σ) of less than 1.45 MHz and the near-constant Brillouin gain of the probe wave are maintained throughout the fiber.

Long-lived states to sustain SABRE hyperpolarised magnetisation† †Electronic supplementary information (ESI) available: Experimental details, sample specification, substrate characterisation, SABRE studies, pulse sequence details, simulations. See DOI: 10.1039/c6cp02844f Click here for additional data file.

PubMed Central

Roy, Soumya S.; Rayner, Peter J.; Norcott, Philip; Green, Gary G. R.

2016-01-01

The applicability of the magnetic resonance (MR) technique in the liquid phase is limited by poor sensitivity and short nuclear spin coherence times which are insufficient for many potential applications. Here we illustrate how it is possible to address both of these issues simultaneously by harnessing long-lived hyperpolarised spin states that are formed by adapting the Signal Amplification by Reversible Exchange (SABRE) technique. We achieve more than 4% net 1H-polarisation in a long-lived form that remains detectable for over ninety seconds by reference to proton pairs in the biologically important molecule nicotinamide and a pyrazine derivative whose in vivo imaging will offer a new route to probe disease in the future. PMID:27711398

Mapping of single-copy genes by TSA-FISH in the codling moth, Cydia pomonella.

PubMed

Carabajal Paladino, Leonela Z; Nguyen, Petr; Síchová, Jindra; Marec, František

2014-01-01

We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth. Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome. We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms.

Mapping of single-copy genes by TSA-FISH in the codling moth, Cydia pomonella

PubMed Central

2014-01-01

Background We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth. Results Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome. Conclusions We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms. PMID:25471491

Improving colorimetric assays through protein enzyme-assisted gold nanoparticle amplification.

PubMed

Xie, Xiaoji; Xu, Wei; Liu, Xiaogang

2012-09-18

The discovery of the DNA-mediated assembly of gold nanoparticles was a great moment in the history of science; this understanding and chemical control enabled the rational design of functional nanomaterials as novel probes in biodetection. In contrast with conventional probes such as organic dyes, gold nanoparticles exhibit high photostability and unique size-dependent optical properties. Because of their high extinction coefficients and strong distance dependent optical properties, these nanoparticles have emerged over the past decade as a promising platform for rapid, highly sensitive colorimetric assays that allow for the visual detection of low concentrations of metal ions, small molecules, and biomacromolecules. These discoveries have deepened our knowledge of biological phenomena and facilitated the development of many new diagnostic and therapeutic tools. Despite these many advances and continued research efforts, current nanoparticle-based colorimetric detection systems still suffer from several drawbacks, such as limited sensitivity and selectivity. This Account describes the recent development of colorimetric assays based on protein enzyme-assisted gold nanoparticle amplification. The benefits of such detection systems include significantly improved detection sensitivity and selectivity. First, we discuss the general design of enzyme-modified nanoparticle systems in colorimetric assays. We show that a quantitative understanding of the unique properties of different enzymes is paramount for effective biological assays. We then examine the assays for nucleic acid detection based on different types of enzymes, including endonucleases, ligases, and polymerases. For each of these assays, we identify the underlying principles that contribute to the enhanced detection capability of nanoparticle systems and illustrate them with selected examples. Furthermore, we demonstrate that the combination of gold nanoparticles and specific enzymes can probe enzyme dynamics and function with high specificity, offering substantial advantages in both sensitivity and specificity over conventional detection methods. The screening of nuclease, methyltransferase, protease, and kinase activities can be colorimetrically performed in a straightforward manner. Finally, we discuss examples of colorimetric assays for metal ions and small molecules that constitute important advances toward visual monitoring of enzyme catalytic functions and gene expression. Although these enzyme-assisted assay methods hold great promise for myriad applications in biomedicine and bioimaging, the application of the described techniques in vivo faces formidable challenges. In addition, researchers do not fully understand the interactions of gold nanoparticles with enzyme molecules. This understanding will require the development of new techniques to probe enzyme substrate dynamics at the particle interface with higher spatial resolution and chemical specificity.

Can Anomalous Amplification be Attained without Postselection?

PubMed

Martínez-Rincón, Julián; Liu, Wei-Tao; Viza, Gerardo I; Howell, John C

2016-03-11

We present a parameter estimation technique based on performing joint measurements of a weak interaction away from the weak-value-amplification approximation. Two detectors are used to collect full statistics of the correlations between two weakly entangled degrees of freedom. Without discarding of data, the protocol resembles the anomalous amplification of an imaginary-weak-value-like response. The amplification is induced in the difference signal of both detectors allowing robustness to different sources of technical noise, and offering in addition the advantages of balanced signals for precision metrology. All of the Fisher information about the parameter of interest is collected. A tunable phase controls the strength of the amplification response. We experimentally demonstrate the proposed technique by measuring polarization rotations in a linearly polarized laser pulse. We show that in the presence of technical noise the effective sensitivity and precision of a split detector is increased when compared to a conventional continuous-wave balanced detection technique.

Can Anomalous Amplification be Attained without Postselection?

NASA Astrophysics Data System (ADS)

Martínez-Rincón, Julián; Liu, Wei-Tao; Viza, Gerardo I.; Howell, John C.

2016-03-01

We present a parameter estimation technique based on performing joint measurements of a weak interaction away from the weak-value-amplification approximation. Two detectors are used to collect full statistics of the correlations between two weakly entangled degrees of freedom. Without discarding of data, the protocol resembles the anomalous amplification of an imaginary-weak-value-like response. The amplification is induced in the difference signal of both detectors allowing robustness to different sources of technical noise, and offering in addition the advantages of balanced signals for precision metrology. All of the Fisher information about the parameter of interest is collected. A tunable phase controls the strength of the amplification response. We experimentally demonstrate the proposed technique by measuring polarization rotations in a linearly polarized laser pulse. We show that in the presence of technical noise the effective sensitivity and precision of a split detector is increased when compared to a conventional continuous-wave balanced detection technique.

A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

PubMed Central

2010-01-01

Background The hepatitis C virus (HCV) genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM), at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP)/COBAS TaqMan (CTM) assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection. PMID:20529244

A Closed-tube Loop-Mediated Isothermal Amplification Assay for the Visual Endpoint Detection of Brucella spp. and Mycobacterium avium subsp. paratuberculosis.

PubMed

Trangoni, Marcos D; Gioffré, Andrea K; Cravero, Silvio L

2017-01-01

LAMP (loop-mediated isothermal amplification) is an isothermal nucleic acid amplification technique that is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity, and specificity, with the blueprint that it can be implemented in laboratories of low technological complexity. Despite the conceptual complexity underlying the mechanistic basis for the nucleic acid amplification, the technique is simple to use and the amplification and detection can be carried out in just one step. In this chapter, we present a protocol based on LAMP for the rapid identification of isolates of Brucella spp. and Mycobacterium avium subsp. paratuberculosis, two major bacterial pathogens in veterinary medicine.

Ultrasensitive electrochemical detection of DNA based on Zn²⁺ assistant DNA recycling followed with hybridization chain reaction dual amplification.

PubMed

Qian, Yong; Wang, Chunyan; Gao, Fenglei

2015-01-15

A new strategy to combine Zn(2+) assistant DNA recycling followed with hybridization chain reaction dual amplification was designed for highly sensitive electrochemical detection of target DNA. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of the target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB and catalyzed its cleavage in the presence of Zn(2+) cofactor and resulting in a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles triggering the cleavage of MB, thus forming numerous MB fragments. The MB fragments triggered the HCR and formed a long double-helix DNA structure. Because both H1 and H2 were labeled by biotin, a lot of SA-ALP was captured on the electrode surface, thus catalyzing a silver deposition process for electrochemical stripping analysis. This novel cascade signal amplification strategy can detect target DNA down to the attomolar level with a dynamic range spanning 6 orders of magnitude. This highly sensitive and specific assay has a great potential to become a promising DNA quantification method in biomedical research and clinical diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

A graphene-based biosensing platform based on the release of DNA probes and rolling circle amplification.

PubMed

Liu, Meng; Song, Jinping; Shuang, Shaomin; Dong, Chuan; Brennan, John D; Li, Yingfu

2014-06-24

We report a versatile biosensing platform capable of achieving ultrasensitive detection of both small-molecule and macromolecular targets. The system features three components: reduced graphene oxide for its ability to adsorb single-stranded DNA molecules nonspecifically, DNA aptamers for their ability to bind reduced graphene oxide but undergo target-induced conformational changes that facilitate their release from the reduced graphene oxide surface, and rolling circle amplification (RCA) for its ability to amplify a primer-template recognition event into repetitive sequence units that can be easily detected. The key to the design is the tagging of a short primer to an aptamer sequence, which results in a small DNA probe that allows for both effective probe adsorption onto the reduced graphene oxide surface to mask the primer domain in the absence of the target, as well as efficient probe release in the presence of the target to make the primer available for template binding and RCA. We also made an observation that the circular template, which on its own does not cause a detectable level of probe release from the reduced graphene oxide, augments target-induced probe release. The synergistic release of DNA probes is interpreted to be a contributing factor for the high detection sensitivity. The broad utility of the platform is illustrated though engineering three different sensors that are capable of achieving ultrasensitive detection of a protein target, a DNA sequence and a small-molecule analyte. We envision that the approach described herein will find useful applications in the biological, medical, and environmental fields.

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection.

PubMed

Lau, Han Yih; Botella, Jose R

2017-01-01

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail.

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

PubMed Central

Lau, Han Yih; Botella, Jose R.

2017-01-01

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail. PMID:29375588

Gravitational lensing statistics of amplified supernovae

NASA Technical Reports Server (NTRS)

Linder, Eric V.; Wagoner, Robert V.; Schneider, P.

1988-01-01

Amplification statistics of gravitationally lensed supernovae can provide a valuable probe of the lensing matter in the universe. A general probability distribution for amplification by compact objects is derived which allows calculation of the lensed fraction of supernovae at or greater than an amplification A and at or less than an apparent magnitude. Comparison of the computed fractions with future results from ongoing supernova searches can lead to determination of the mass density of compact dark matter components with masses greater than about 0.001 solar mass, while the time-dependent amplification (and polarization) of the expanding supernovae constrain the individual masses. Type II supernovae are found to give the largest fraction for deep surveys, and the optimum flux-limited search is found to be at approximately 23d magnitude, if evolution of the supernova rate is neglected.

Subtelomeric multiplex ligation-dependent probe amplification as a supplement for rapid prenatal detection of fetal chromosomal aberrations.

PubMed

Chen, Xiangnan; Li, Huanzheng; Mao, Yijian; Xu, Xueqin; Lv, Jiaojiao; Zhou, Lili; Lin, Xiaoling; Tang, Shaohua

2014-01-01

Pregnant women with high-risk indications are highly suspected of fetal chromosomal aberrations. To determine whether Multiplex Ligation-dependent Probe Amplification (MLPA) using subtelomeric probe mixes (P036-E2 and P070-B2) is a reliable method for rapid detection of fetal chromosomal aberrations. The subtelomeric MLPA probe mixes were used to evaluate 50 blood samples from healthy individuals. 168 amniocytes and 182 umbilical cord blood samples from high-risk fetuses were analyzed using the same subtelomeric MLPA probe sets. Karyotyping was also performed in all cases of high-risk pregnancies, and single nucleotide polymorphism array analysis was used to confirm submicroscopic and ambiguous results from MLPA/karyotyping. Subtelomeric MLPA analysis of normal samples showed normal result in all cases by use of P036-E2 probe mix, while P070-B2 probe mix gave normal results for all but one case. In one normal control case P070-B2 produced a duplicated signal of probe for 13q34. In the high-risk group, totally 44 chromosomal abnormalities were found by karyotyping and MLPA, including 23 aneuploidies and 21 rearrangements or mosaics. MLPA detected all 23 aneuploidies, 12 rearrangements and 1 mosaic. Importantly, MLPA revealed 4 chromosomal translocations, 2 small supernumerary marker chromosomes (sSMCs), and 3 subtelomeric imbalances that were not well characterized or not detectable by karyotyping. However, MLPA showed negetive results for the remaining 8 rearrangements or mosaics, including 3 low mosaic aneuploidies, 1 inherited sSMC, and 4 paracentric inversions. Results suggest that combined use of subtelomeric MLPA and karyotyping may be an alternative method for using karyotype analyses alone in rapid detection of aneuploidies, rearrangements, and sSMCs.

Diagnostic testing for Giardia infections.

PubMed

Heyworth, Martin F

2014-03-01

The traditional method for diagnosing Giardia infections involves microscopic examination of faecal specimens for Giardia cysts. This method is subjective and relies on observer experience. From the 1980s onwards, objective techniques have been developed for diagnosing Giardia infections, and are superseding diagnostic techniques reliant on microscopy. Detection of Giardia antigen(s) by immunoassay is the basis of commercially available diagnostic kits. Various nucleic acid amplification techniques (NAATs) can demonstrate DNA of Giardia intestinalis, and have the potential to become standard approaches for diagnosing Giardia infections. Of such techniques, methods involving either fluorescent microspheres (Luminex) or isothermal amplification of DNA (loop-mediated isothermal amplification; LAMP) are especially promising.

Biosensor-based microRNA detection: techniques, design, performance, and challenges.

PubMed

Johnson, Blake N; Mutharasan, Raj

2014-04-07

The current state of biosensor-based techniques for amplification-free microRNA (miRNA) detection is critically reviewed. Comparison with non-sensor and amplification-based molecular techniques (MTs), such as polymerase-based methods, is made in terms of transduction mechanism, associated protocol, and sensitivity. Challenges associated with miRNA hybridization thermodynamics which affect assay selectivity and amplification bias are briefly discussed. Electrochemical, electromechanical, and optical classes of miRNA biosensors are reviewed in terms of transduction mechanism, limit of detection (LOD), time-to-results (TTR), multiplexing potential, and measurement robustness. Current trends suggest that biosensor-based techniques (BTs) for miRNA assay will complement MTs due to the advantages of amplification-free detection, LOD being femtomolar (fM)-attomolar (aM), short TTR, multiplexing capability, and minimal sample preparation requirement. Areas of future importance in miRNA BT development are presented which include focus on achieving high measurement confidence and multiplexing capabilities.

Gonorrhea Test

MedlinePlus

... for Targeted Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker ... NAT Neisseria gonorrhoeae Nucleic Acid Amplification Test, Culture, Gram Stain, DNA Probe Formal Name Neisseria gonorrhoeae This article ...

Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplification assay.

PubMed

Yang, Yang; Qin, Xiaodong; Sun, Yingjun; Chen, Ting; Zhang, Zhidong

2016-12-01

A novel fluorescent probe-based real-time reverse transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed for rapid detection of highly pathogenic type 2 porcine reproductive and respiratory syndrome virus (HP-PRRSV). The sensitivity analysis showed that the detection limit of RPA was 70 copies of HP-PRRSV RNA/reaction. The real-time RT-RPA highly specific amplified HP-PRRSV with no cross-reaction with classic PRRSV, classic swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. Assessment with 125 clinical samples showed that the developed real-time RT-RPA assay was well correlated with real-time RT-qPCR assays for detection of HP-PRRSV. These results suggest that the developed real-time RT-RPA assay is suitable for rapid detection of HP-PRRSV.

Ultrasensitive electrochemical detection of avian influenza A (H7N9) virus DNA based on isothermal exponential amplification coupled with hybridization chain reaction of DNAzyme nanowires.

PubMed

Yu, Yanyan; Chen, Zuanguang; Jian, Wensi; Sun, Duanping; Zhang, Beibei; Li, Xinchun; Yao, Meicun

2015-02-15

In this work, a simple and label-free electrochemical biosensor with duel amplification strategy was developed for DNA detection based on isothermal exponential amplification (EXPAR) coupled with hybridization chain reaction (HCR) of DNAzymes nanowires. Through rational design, neither the primer nor the DNAzymes containing molecular beacons (MBs) could react with the duplex probe which were fixed on the electrode surface. Once challenged with target, the duplex probe cleaved and triggered the EXPAR mediated target recycle and regeneration circles as well as the HCR process. As a result, a greater amount of targets were generated to cleave the duplex probes. Subsequently, the nanowires consisting of the G-quadruplex units were self-assembled through hybridization with the strand fixed on the electrode surface. In the presence of hemin, the resulting catalytic G-quadruplex-hemin HRP-mimicking DNAzymes were formed. Electrochemical signals can be obtained by measuring the increase in reduction current of oxidized 3.3',5.5'-tetramethylbenzidine sulfate (TMB), which was generated by DNAzyme in the presence of H2O2. This method exhibited ultrahigh sensitivity towards avian influenza A (H7N9) virus DNA sequence with detection limits of 9.4 fM and a detection range of 4 orders of magnitude. The biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences and performed well in spiked cell lysates. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel selection methods for DNA-encoded chemical libraries.

PubMed

Chan, Alix I; McGregor, Lynn M; Liu, David R

2015-06-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Different approaches in the molecular analysis of the SHOX gene dysfunctions.

PubMed

Stuppia, L; Gatta, V; Antonucci, I; Giuliani, R; Palka, G

2010-06-01

Deficit of the short stature homeobox containing gene (SHOX) accounts for 2.15% of cases of idiopathic short stature (ISS) and 50-100% of cases of Leri-Weill dyschondrosteosis (LWD). It has been demonstrated that patients with SHOX deficit show a good response to treatment with GH. Thus, the early identification of SHOX alterations is a crucial point in order to choose the best treatment for ISS and LWD patients. In this study, we analyze the most commonly used molecular techniques for the detection of SHOX gene alterations. multiple ligation-dependent probe amplification analysis appears to represent the gold standard for the detection of deletion involving the SHOX gene or the enhancer region, being able to show both alterations in a single assay.

Identification of deletions in children with congenital hypothyroidism and thyroid dysgenesis with the use of multiplex ligation-dependent probe amplification.

PubMed

Kumorowicz-Czoch, Malgorzata; Madetko-Talowska, Anna; Tylek-Lemanska, Dorota; Pietrzyk, Jacek J; Starzyk, Jerzy

2015-01-01

Thyroid dysgenesis (TD) is the most common cause of congenital hypothyroidism (CH). Important genetic factors possibly contributing to TD etiologies include mutations of thyroid transcription factors and TSHR-encoding genes. Our objective was to determine multiplex ligation-dependent probe amplification (MLPA) utility in detecting the copy number changes in patients with CH and TD. The study included 45 children from southeastern Poland selected via already established neonatal screening for CH. Genomic DNA was extracted from peripheral blood samples and used in MLPA analysis. Genetic variations were analyzed within selected fragments of the PAX8, FOXE1, NKX2-1, thyroid stimulating hormone receptor (TSHR), and TPO genes. Three heterozygous deletion types in probe hybridization regions were identified for the following genes: PAX8 (exon 7), TSHR (exon 2), and FOXE1 (exon 1). Monoallelic deletions were identified in 5/45 TD subjects. MLPA is a useful tool for copy number changes detection and might both improve and expand genetic analysis for CH and TD.

An effective established biosensor of bifunctional probes-labeled AuNPs combined with LAMP for detection of fish pathogen Streptococcus iniae.

PubMed

Zhou, Ya; Xiao, Jingfan; Ma, Xin; Wang, Qiyao; Zhang, Yuanxing

2018-06-01

In purpose of valid Streptococcus iniae detection, we established a colorimetric biosensor using gold nanoparticles (AuNPs) labeled with dual functional probes and along with loop-mediated isothermal amplification (LAMP) assay (LAMP-AuNPs). Based on the characteristics of self-aggregation and bio-conjugation with ligands, AuNPs were chosen for observable color change in tandem with LAMP amplification method to reach high sensitivity and easy operation. Meanwhile, the improvement of dual probes that could fully utilize the LAMP product gave the biosensor a stable result exhibition. LAMP-AuNPs targeting gene ftsB, one of the ATP transporter-related genes, turned out favorable specificity in cross reaction among other fish pathogens. The detect limit of 10 2 CFU revealed a better sensitivity compared with polymerase chain reaction (PCR) method and AuNPs lateral flow test strip (LFTS). It was also proved to be effective by zebrafish infection model trials with less than 2-h time consumption and nearly no devices which make it a convenient biosensor for point-to-care S. iniae detection.

A mass spectrometry-based multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification.

PubMed

Park, Jung Hun; Jang, Hyowon; Jung, Yun Kyung; Jung, Ye Lim; Shin, Inkyung; Cho, Dae-Yeon; Park, Hyun Gyu

2017-05-15

We herein describe a new mass spectrometry-based method for multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification (SDA) reaction. In this method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site within the PCR-amplified target DNA. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific allele in the target DNA and the resulting ligated product serves as a template to promote the SDA reaction using a universal primer. This process isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. By varying the sizes of the mass markers, we successfully demonstrated the multiplex SNP genotyping capability of this method by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrochemical biosensor based on enzyme substrate as a linker: Application for aldolase activity with pectin-thionine complex as recognization element and signal amplification probe.

PubMed

Wang, Xiaonan; Wang, Meiwen; Zhang, Yuanyuan; Miao, Xiaocao; Huang, Yuanyuan; Zhang, Juan; Sun, Lizhou

2016-09-15

A new strategy to fabricate electrochemical biosensor is reported based on the linkage of enzyme substrate, thereby an electrochemical method to detect aldolase activity is established using pectin-thionine complex (PTC) as recognization element and signal probe. The linkage effect of fructose-1,6-bisphosphate (FBP), the substrate of aldolase, can be achieved via its strong binding to magnetic nanoparticles (MNPs)/aminophenylboronic acid (APBA) and the formation of phosphoramidate bond derived from its reaction with p-phenylenediamine (PDA) on the surface of electrode. Aldolase can reversibly catalyze the substrates into the products which have no binding capacity with MNPs/APBA, resulting in the exposure of the corresponding binding sites and its subsequent recognization on signal probe. Meanwhile, signal amplification can be accomplished by using the firstly prepared PTC which can bind with MNPs/APBA, and accuracy can be strengthened through magnetic separation. With good precision and accuracy, the established sensor may be extended to other proteins with reversible catalyzed ability. Copyright © 2016 Elsevier B.V. All rights reserved.

PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, S. N.

2013-08-08

Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

Detection of amplified or deleted chromosomal regions

DOEpatents

Stokke, Trond; Pinkel, Daniel; Gray, Joe W.

1995-01-01

The present invention relates to in situ hybridization methods for the identification of new chromosomal abnormalities associated with various diseases. In particular, it provides probes which are specific to a region of amplification in chromosome 20.

Detection Of Amplified Or Deleted Chromosomal Regions

DOEpatents

Stokke, Trond , Pinkel, Daniel , Gray, Joe W.

1997-05-27

The present invention relates to in situ hybridization methods for the identification of new chromosomal abnormalities associated with various diseases. In particular, it provides probes which are specific to a region of amplification in chromosome 20.

Detection of genetically modified organisms in foods by DNA amplification techniques.

PubMed

García-Cañas, Virginia; Cifuentes, Alejandro; González, Ramón

2004-01-01

In this article, the different DNA amplification techniques that are being used for detecting genetically modified organisms (GMOs) in foods are examined. This study intends to provide an updated overview (including works published till June 2002) on the principal applications of such techniques together with their main advantages and drawbacks in GMO detection in foods. Some relevant facts on sampling, DNA isolation, and DNA amplification methods are discussed. Moreover; these analytical protocols are discuissed from a quantitative point of view, including the newest investigations on multiplex detection of GMOs in foods and validation of methods.

[Synthesis of Circular DNA Templates with T4 RNA Ligase for Rolling Circle Amplification].

PubMed

Sakhabutdinova, A R; Maksimova, M A; Garafutdinov, R R

2017-01-01

Currently, isothermal methods of nucleic acid amplification have been well established; in particular, rolling circle amplification is of great interest. In this approach, circular ssDNA molecules have been used as a target that can be obtained by the intramolecular template-dependent ligation of an oligonucleotide C-probe. Here, a new method of synthesizing small circular DNA molecules via the cyclization of ssDNA based on T4 RNA ligase has been proposed. Circular ssDNA is further used as the template for the rolling circle amplification. The maximum yield of the cyclization products was observed in the presence of 5-10% polyethylene glycol 4000, and the optimum DNA length for the cyclization constituted 50 nucleotides. This highly sensitive method was shown to detect less than 10^(2) circular DNA molecules. The method reliability was proved based on artificially destroyed dsDNA, which suggests its implementation for analyzing any significantly fragmented dsDNA.

Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

PubMed Central

Schmidt, Thorsten L.; Beliveau, Brian J.; Uca, Yavuz O.; Theilmann, Mark; Da Cruz, Felipe; Wu, Chao-Ting; Shih, William M.

2015-01-01

Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes. PMID:26567534

Analysis of a Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for yellow fever diagnostic.

PubMed

Nunes, Marcio R T; Vianez, João Lídio; Nunes, Keley N B; da Silva, Sandro Patroca; Lima, Clayton P S; Guzman, Hilda; Martins, Lívia C; Carvalho, Valéria L; Tesh, Robert B; Vasconcelos, Pedro F C

2015-12-15

Yellow Fever virus (YFV) is an important human pathogen in tropical areas of Africa and South America. Although an efficient vaccine is available and has been used since the early 1940s, sylvatic YFV transmission still occurs in forested areas where anthropogenic actions are present, such as mineral extraction, rearing livestock and agriculture, and ecological tourism. In this context, two distinct techniques based on the RT-PCR derived method have been previously developed, however both methods are expensive due to the use of thermo cyclers and labeled probes. We developed isothermal genome amplification, which is a rapid, sensitive, specific and low cost molecular approach for YFV genome detection. This assay used a set of degenerate primers designed for the NS1 gene and was able to amplify, within 30 min in isothermal conditions, the YFV 17D vaccine strain derived from an African wild prototype strain (Asibi), as well as field strains from Brazil, other endemic countries from South and Central America, and the Caribbean. The generic RT-LAMP assay could be helpful for YFV surveillance in field and rapid response during outbreaks in endemic areas. Copyright © 2015 Elsevier B.V. All rights reserved.

Current molecular genetics strategies for the diagnosis of lysosomal storage disorders.

PubMed

Giugliani, Roberto; Brusius-Facchin, Ana-Carolina; Pasqualim, Gabriela; Leistner-Segal, Sandra; Riegel, Mariluce; Matte, Ursula

2016-01-01

Lysosomal storage disorders (LSDs) are a group of almost 50 monogenic diseases characterized by mutations causing deficiency of lysosomal enzymes or non-enzyme proteins involved in transport across the lysosomal membrane, protein maturation or lysosomal biogenesis. Usually, affected patients are normal at birth and have a progressive and severe disease with high morbidity and reduced life expectancy. The overall incidence of LSDs is usually estimated as 1:5000, but newborn screening studies are indicating that it could be much higher. Specific therapies were already developed for selected LSDs, making the timely and correct diagnosis very important for successful treatment and also for genetic counseling. In most LSD cases the biochemical techniques provide a reliable diagnosis. However, the identification of pathogenic mutations by genetic analysis is being increasingly recommended to provide additional information. In this paper we discuss the conventional methods for genetic analysis used in the LSDs [restriction fragment length polymorphism (RFLP), amplification-refractory mutation system (ARMS), single strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC), real-time polymerase chain reaction, high resolution melting (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing] and also the newer approaches [massive parallel sequencing, array comparative genomic hybridization (CGH)].

Electrochemical and photoelectrochemical nano-immunesensing using origami paper based method.

PubMed

Hasanzadeh, Mohammad; Shadjou, Nasrin

2016-04-01

Patterned paper has characteristics that lead to miniaturized assays that run by capillary action with small volumes of fluids. These methods suggest a path for the development of simple, inexpensive, and portable diagnostic assays that can be useful in remote settings, where simple immunoassays are becoming increasingly important for detecting disease and monitoring health. Incorporation of nanomaterials plays a major role in sensing probe immobilization and detection sensitivity of paper-based devices. Nanomaterial properties, such as increased surface area, have aided with signal amplification and lower detection limits. This review focuses on application of nanomaterials as signal amplification elements on origami paper-based electro-analytical devices for immune biomarkers detection with a brief introduction about various fabrication techniques and designs, biological and detection methods. In this review, we comprehensively summarize the selected latest research articles from 2013 to May 2015 on application of nanomaterials in various types of origami paper based electrochemical and photoelectrochemical immunosensors. The review breaks into two parts. The first part devotes to the development and applications of nanomaterials in electrochemical immunesensing. The second part provides an overview of recent origami paper based photoelectrochemical immunosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

Recent patents on self-quenching DNA probes.

PubMed

Knemeyer, Jens-Peter; Marmé, Nicole

2007-01-01

In this review, we report on patents concerning self-quenching DNA probes for assaying DNA during or after amplification as well as for direct assaying DNA or RNA, for example in living cells. Usually the probes consist of fluorescently labeled oligonucleotides whose fluorescence is quenched in the absence of the matching target DNA. Thereby the fluorescence quenching is based on fluorescence resonance energy transfer (FRET), photoinduced electron transfer (PET), or electronically interactions between dye and quencher. However, upon hybridization to the target or after the degradation during a PCR, the fluorescence of the dye is restored. Although the presented probes were originally developed for use in homogeneous assay formats, most of them are also appropriate to improve surface-based assay methods. In particular we describe patents for self-quenching primers, self-quenching probes for TaqMan assays, probes based on G-quartets, Molecular Beacons, Smart Probes, and Pleiades Probes.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

PubMed

Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

A catalytic and dual recycling amplification ATP sensor based on target-driven allosteric structure switching of aptamer beacons.

PubMed

Peng, Ying; Li, Daxiu; Yuan, Ruo; Xiang, Yun

2018-05-15

Abnormal concentrations of ATP are associated with many diseases and cancers, and quantitative detection of ATP is thus of great importance for disease diagnosis and prognosis. In the present work, we report a new dual recycling amplification sensor integrated with catalytic hairpin assembly (CHA) to achieve high sensitivity for fluorescent detection of ATP. The association of the target ATP with the aptamer beacons causes the allosteric structure switching of the aptamer beacons to expose the toehold regions, which hybridize with and unfold the fluorescently quenched hairpin signal probes (HP1) to recycle the target ATP and to trigger CHA between HP1 and the secondary hairpin probes (HP2) to form HP1/HP2 duplexes. Due to the recycling amplification, the presence of ATP leads to the formation of many HP1/HP2 duplexes, generating dramatically amplified fluorescent signals for sensitive detection of ATP. Under optimal experimental conditions, our sensor linearly responds to ATP in the range from 25 to 600nM with a calculated detection limit of 8.2nM. Furthermore, the sensor shows a high selectivity and can also be used to detect ATP in human serums to realize its application for real samples. With the distinct advantage of significant signal amplification without the involvement of any nanomaterial and enzyme, the developed sensor thus holds great potential for simple and sensitive detection of different small molecules and proteins. Copyright © 2018 Elsevier B.V. All rights reserved.

Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.

PubMed

Kivlehan, Francine; Mavré, François; Talini, Luc; Limoges, Benoît; Marchal, Damien

2011-09-21

We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.

Experimental Demonstration of Higher Precision Weak-Value-Based Metrology Using Power Recycling

NASA Astrophysics Data System (ADS)

Wang, Yi-Tao; Tang, Jian-Shun; Hu, Gang; Wang, Jian; Yu, Shang; Zhou, Zong-Quan; Cheng, Ze-Di; Xu, Jin-Shi; Fang, Sen-Zhi; Wu, Qing-Lin; Li, Chuan-Feng; Guo, Guang-Can

2016-12-01

The weak-value-based metrology is very promising and has attracted a lot of attention in recent years because of its remarkable ability in signal amplification. However, it is suggested that the upper limit of the precision of this metrology cannot exceed that of classical metrology because of the low sample size caused by the probe loss during postselection. Nevertheless, a recent proposal shows that this probe loss can be reduced by the power-recycling technique, and thus enhance the precision of weak-value-based metrology. Here we experimentally realize the power-recycled interferometric weak-value-based beam-deflection measurement and obtain the amplitude of the detected signal and white noise by discrete Fourier transform. Our results show that the detected signal can be strengthened by power recycling, and the power-recycled weak-value-based signal-to-noise ratio can surpass the upper limit of the classical scheme, corresponding to the shot-noise limit. This work sheds light on higher precision metrology and explores the real advantage of the weak-value-based metrology over classical metrology.

Detection of amplified or deleted chromosomal regions

DOEpatents

Stokke, T.; Pinkel, D.; Gray, J.W.

1995-12-05

The present invention relates to in situ hybridization methods for the identification of new chromosomal abnormalities associated with various diseases. In particular, it provides probes which are specific to a region of amplification in chromosome 20. 3 figs.

[Combined G-banded karyotyping and multiplex ligation-dependent probe amplification for the detection of chromosomal abnormalities in fetuses with congenital heart defects].

PubMed

Liu, Yang; Xie, Jiansheng; Geng, Qian; Xu, Zhiyong; Wu, Weiqin; Luo, Fuwei; Li, Suli; Wang, Qin; Chen, Wubin; Tan, Hongxi; Zhang, Hu

2017-02-10

To assess the value of G-banded karyotyping in combination with multiplex ligation-dependent probe amplification (MLPA) as a tool for the detection of chromosomal abnormalities in fetuses with congenital heart defects. The combined method was used to analyze 104 fetuses with heart malformations identified by ultrasonography. Abnormal findings were confirmed with chromosomal microarray analysis (CMA). Nineteen (18%) fetuses were found to harbor chromosomal aberrations by G-banded karyotyping and MLPA. For 93 cases, CMA has detected abnormalities in 14 cases including 10 pathogenic copy number variations (CNVs) and 4 CNVs of uncertain significance (VOUS). MLPA was able to detect all of the pathogenic CNVs and 1 VOUS CNV. Combined use of G-banded karyotyping and MLPA is a rapid, low-cost and effective method to detect chromosomal abnormalities in fetuses with various heart malformations.

Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

PubMed

Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

2018-01-01

The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Genetic diagnosis of Duchenne and Becker muscular dystrophy using multiplex ligation-dependent probe amplification in Rwandan patients.

PubMed

Uwineza, Annette; Hitayezu, Janvier; Murorunkwere, Seraphine; Ndinkabandi, Janvier; Kalala Malu, Celestin Kaputu; Caberg, Jean Hubert; Dideberg, Vinciane; Bours, Vincent; Mutesa, Leon

2014-04-01

Duchenne and Becker muscular dystrophies are the most common clinical forms of muscular dystrophies. They are genetically X-linked diseases caused by a mutation in the dystrophin (DMD) gene. A genetic diagnosis was carried out in six Rwandan patients presenting a phenotype of Duchenne and Becker muscular dystrophies and six asymptomatic female carrier relatives using multiplex ligation-dependent probe amplification (MLPA). Our results revealed deletion of the exons 48-51 in one patient, an inherited deletion of the exons 8-21 in two brothers and a de novo deletion of the exons 46-50 in the fourth patient. No copy number variation was found in two patients. Only one female carrier presented exon deletion in the DMD gene. This is the first cohort of genetic analysis in Rwandan patients affected by Duchenne and Becker muscular dystrophies. This report confirmed that MLPA assay can be easily implemented in low-income countries.

High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

PubMed Central

Kondrashova, Olga; Love, Clare J.; Lunke, Sebastian; Hsu, Arthur L.; Waring, Paul M.; Taylor, Graham R.

2015-01-01

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. PMID:26569395

Target-catalyzed hairpin assembly and metal-organic frameworks mediated nonenzymatic co-reaction for multiple signal amplification detection of miR-122 in human serum.

PubMed

Li, Yuliang; Yu, Chao; Yang, Bo; Liu, Zhirui; Xia, Peiyuan; Wang, Qian

2018-04-15

Herein, a new type of multifunctional iron based metal-organic frameworks (PdNPs@Fe-MOFs) has been synthesized by assembly palladium nanoparticles on the surface of Fe-MIL-88NH 2 MOFs microcrystals, and first applied in electrochemical biosensor for ultrasensitive detection of microRNA-122 (miR-122, a biomarker of drug-induced liver injury). The nanohybrids have not only been utilized as ideal nanocarriers for immobilization of signal probes, but also used as redox probes and electrocatalysts. In this biosensor, two hairpin probes were designed as capture probes and signal probes, respectively. The nanohybrids conjugated with streptavidin and biotinylated signal probes were used as the tracer labels, target miR-122 was sandwiched between the tracer labels and thiol-terminated capture probes inserted in MCH monolayer on the gold nanoparticles-functionalized nitrogen-doped graphene sheets (AuNPs@N-G) modified electrode. Based on target-catalyzed hairpin assembly, target miR-122 could trigger the hybridization of capture probes and signal probes to further be released to initiate the next reaction process resulted in numerous tracer indicators anchored onto the sensing interfaces. Thus, the detection signal could be dramatically enhanced towards the electrocatalytic oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of H 2 O 2 owing to the intrinsic and intriguing peroxidase-like activity of the nanohybrids. With the assist of target-catalyzed hairpin assembly and PdNPs@Fe-MOFs mimetic co-reaction for signal amplification, a wide detection range from 0.01fM to 10pM was achieved with a low detection limit of 0.003fM (S/N =3). Furthermore, the proposed biosensor exhibited excellent specificity and recovery in spiked serum samples, and was successfully used for detecting miR-122 in real biological samples, which provided a rapid and efficient method for detecting drug-induced liver injury at an early stage. Copyright © 2017. Published by Elsevier B.V.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

Brillouin Amplification--A Powerful New Scheme for Microwave Photonic Communications

NASA Technical Reports Server (NTRS)

Yao, S.; Maleki, L.

1997-01-01

We introduce the Brillouin selective sideband amplification technique and demonstrate many important applications of this technique in photonic microwave systems, including efficient phase modulation to amplitude modulation conversion, photonic frequency multiplication, photonic signal mixing with gain, and frequency multiplied signal up conversion.

Development of a cost-efficient novel method for rapid, concurrent genotyping of five common single nucleotide polymorphisms of the brain derived neurotrophic factor (BDNF) gene by tetra-primer amplification refractory mutation system.

PubMed

Wang, Cathy K; Xu, Michael S; Ross, Colin J; Lo, Ryan; Procyshyn, Ric M; Vila-Rodriguez, Fidel; White, Randall F; Honer, William G; Barr, Alasdair M

2015-09-01

Brain derived neurotrophic factor (BDNF) is a molecular trophic factor that plays a key role in neuronal survival and plasticity. Single nucleotide polymorphisms (SNPs) of the BDNF gene have been associated with specific phenotypic traits in a large number of neuropsychiatric disorders and the response to psychotherapeutic medications in patient populations. Nevertheless, due to study differences and occasionally contrasting findings, substantial further research is required to understand in better detail the association between specific BDNF SNPs and these psychiatric disorders. While considerable progress has been made recently in developing advanced genotyping platforms of SNPs, many high-throughput probe- or array-based detection methods currently available are limited by high costs, slow processing times or access to advanced instrumentation. The polymerase chain reaction (PCR)-based, tetra-primer amplification refractory mutation system (T-ARMS) method is a potential alternative technique for detecting SNP genotypes efficiently, quickly, easily, and cheaply. As a tool in psychopathology research, T-ARMS was shown to be capable of detecting five common SNPs in the BDNF gene (rs6265, rs988748, rs11030104, 11757G/C and rs7103411), which are all SNPs with previously demonstrated clinical relevance to schizophrenia and depression. The present technique therefore represents a suitable protocol for many research laboratories to study the genetic correlates of BDNF in psychiatric disorders. Copyright Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

c-MYC amplification and c-myc protein expression in pancreatic acinar cell carcinomas. New insights into the molecular signature of these rare cancers.

PubMed

La Rosa, Stefano; Bernasconi, Barbara; Vanoli, Alessandro; Sciarra, Amedeo; Notohara, Kenji; Albarello, Luca; Casnedi, Selenia; Billo, Paola; Zhang, Lizhi; Tibiletti, Maria Grazia; Sessa, Fausto

2018-05-02

The molecular alterations of pancreatic acinar cell carcinomas (ACCs) and mixed acinar-neuroendocrine carcinomas (MANECs) are not completely understood, and the possible role of c-MYC amplification in tumor development, progression, and prognosis is not known. We have investigated c-MYC gene amplification in a series of 35 ACCs and 4 MANECs to evaluate its frequency and a possible prognostic role. Gene amplification was investigated using interphasic fluorescence in situ hybridization analysis simultaneously hybridizing c-MYC and the centromere of chromosome 8 probes. Protein expression was immunohistochemically investigated using a specific monoclonal anti-c-myc antibody. Twenty cases had clones with different polysomies of chromosome 8 in absence of c-MYC amplification, and 5 cases had one amplified clone and other clones with chromosome 8 polysomy, while the remaining 14 cases were diploid for chromosome 8 and lacked c-MYC amplification. All MANECs showed c-MYC amplification and/or polysomy which were observed in 54% pure ACCs. Six cases (15.3%) showed nuclear immunoreactivity for c-myc, but only 4/39 cases showed simultaneous c-MYC amplification/polysomy and nuclear protein expression. c-myc immunoreactivity as well as c-MYC amplification and/or chromosome 8 polysomy was not statistically associated with prognosis. Our study demonstrates that a subset of ACCs shows c-MYC alterations including gene amplification and chromosome 8 polysomy. Although they are not associated with a different prognostic signature, the fact that these alterations are present in all MANECs suggests a role in the acinar-neuroendocrine differentiation possibly involved in the pathogenesis of MANECs.

Highly sensitive electrochemical nuclear factor kappa B aptasensor based on target-induced dual-signal ratiometric and polymerase-assisted protein recycling amplification strategy.

PubMed

Peng, Kanfu; Xie, Pan; Yang, Zhe-Han; Yuan, Ruo; Zhang, Keqin

2018-04-15

In this work, an amplified electrochemical ratiometric aptasensor for nuclear factor kappa B (NF-κB) assay based on target binding-triggered ratiometric signal readout and polymerase-assisted protein recycling amplification strategy is described. To demonstrate the effect of "signal-off" and "signal-on" change for the dual-signal electrochemical ratiometric readout, the thiol-hairpin DNA (SH-HD) hybridizes with methylene blue (MB)-modified protection DNA (MB-PD) to form capture probes, which is rationally introduced for the construction of the assay platform. On the interface, the probes can specifically bind to target NF-κB and expose a toehold region which subsequently hybridizes with the ferrocene (Fc)-modified DNA strand to take the Fc group to the electrode surface, accompanied by displacing MB-PD to release the MB group from the electrode surface, leading to the both "signal-on" of Fc (I Fc ) and "signal-off" of MB (I MB ). In order to improve the sensitivity of the electrochemical aptasensor, phi29-assisted target protein recycling amplification strategy was designed to achieve an amplified ratiometric signal. With the above advantages, the prepared aptasensor exhibits a wide linear range of 0.1pgmL -1 to 15ngmL -1 with a low detection limit of 0.03pgmL -1 . This strategy provides a simple and ingenious approach to construct ratiometric electrochemical aptasensor and shows promising potential applications in multiple disease marker detection by changing the recognition probe. Copyright © 2017. Published by Elsevier B.V.

Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification.

PubMed

Murinda, Shelton E; Ibekwe, A Mark; Zulkaffly, Syaizul; Cruz, Andrew; Park, Stanley; Razak, Nur; Paudzai, Farah Md; Ab Samad, Liana; Baquir, Khairul; Muthaiyah, Kokilah; Santiago, Brenna; Rusli, Amirul; Balkcom, Sean

2014-07-01

Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ∼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

PubMed

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing

PubMed Central

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing. PMID:14519201

Application of advanced cytometric and molecular technologies to minimal residual disease monitoring

NASA Astrophysics Data System (ADS)

Leary, James F.; He, Feng; Reece, Lisa M.

2000-04-01

Minimal residual disease monitoring presents a number of theoretical and practical challenges. Recently it has been possible to meet some of these challenges by combining a number of new advanced biotechnologies. To monitor the number of residual tumor cells requires complex cocktails of molecular probes that collectively provide sensitivities of detection on the order of one residual tumor cell per million total cells. Ultra-high-speed, multi parameter flow cytometry is capable of analyzing cells at rates in excess of 100,000 cells/sec. Residual tumor selection marker cocktails can be optimized by use of receiver operating characteristic analysis. New data minimizing techniques when combined with multi variate statistical or neural network classifications of tumor cells can more accurately predict residual tumor cell frequencies. The combination of these techniques can, under at least some circumstances, detect frequencies of tumor cells as low as one cell in a million with an accuracy of over 98 percent correct classification. Detection of mutations in tumor suppressor genes requires insolation of these rare tumor cells and single-cell DNA sequencing. Rare residual tumor cells can be isolated at single cell level by high-resolution single-cell cell sorting. Molecular characterization of tumor suppressor gene mutations can be accomplished using a combination of single- cell polymerase chain reaction amplification of specific gene sequences followed by TA cloning techniques and DNA sequencing. Mutations as small as a single base pair in a tumor suppressor gene of a single sorted tumor cell have been detected using these methods. Using new amplification procedures and DNA micro arrays it should be possible to extend the capabilities shown in this paper to screening of multiple DNA mutations in tumor suppressor and other genes on small numbers of sorted metastatic tumor cells.

Rapid isothermal detection of Phytophthora species on plant samples using recombinase polymerase amplification

USDA-ARS?s Scientific Manuscript database

Recently several isothermal amplification techniques have been developed that are extremely tolerant towards inhibitors present in many plant extracts. Recombinase polymerase amplification (RPA) assays for the genus Phytophthora have been developed which provide a simple and rapid method to macerate...

Detection of KIT Genotype in Pigs by TaqMan MGB Real-Time Quantitative Polymerase Chain Reaction.

PubMed

Li, Xiuxiu; Li, Xiaoning; Luo, Rongrong; Wang, Wenwen; Wang, Tao; Tang, Hui

2018-05-01

The dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene: a 450 kb duplication containing the entire KIT gene together with flanking sequences and one splice mutation with a G:A substitution in intron 17. The purpose of this study was to establish a simple, rapid method to determine KIT genotype in pigs. First, to detect KIT copy number variation (CNV), primers for exon 2 of the KIT gene, along with a TaqMan minor groove binder (MGB) probe, were designed. The single-copy gene, estrogen receptor (ESR), was used as an internal control. A real-time fluorescence-based quantitative PCR (FQ-PCR) protocol was developed to accurately detect KIT CNVs. Second, to detect the splice mutation ratio of the G:A substitution in intron 17, a 175 bp region, including the target mutation, was amplified from genomic DNA. Based on the sequence of the resulting amplified fragment, an MGB probe set was designed to detect the ratio of splice mutation to normal using FQ-PCR. A series of parallel amplification curves with the same internal distances were obtained using gradually diluted DNA as templates. The CT values among dilutions were significantly different (p < 0.001) and the coefficients of variation from each dilution were low (from 0.13% to 0.26%). The amplification efficiencies for KIT and ESR were approximately equal, indicating ESR was an appropriate control gene. Furthermore, use of the MGB probe set resulted in detection of the target mutation at a high resolution and stability; standard curves illustrated that the amplification efficiencies of KIT1 (G) and KIT2 (A) were approximately equal (98.8% and 97.2%). In conclusion, a simple, rapid method, with high specificity and stability, for the detection of the KIT genotype in pigs was established using TaqMan MGB probe real-time quantitative PCR.

ChIP-chip.

PubMed

Kim, Tae Hoon; Dekker, Job

2018-05-01

ChIP-chip can be used to analyze protein-DNA interactions in a region-wide and genome-wide manner. DNA microarrays contain PCR products or oligonucleotide probes that are designed to represent genomic sequences. Identification of genomic sites that interact with a specific protein is based on competitive hybridization of the ChIP-enriched DNA and the input DNA to DNA microarrays. The ChIP-chip protocol can be divided into two main sections: Amplification of ChIP DNA and hybridization of ChIP DNA to arrays. A large amount of DNA is required to hybridize to DNA arrays, and hybridization to a set of multiple commercial arrays that represent the entire human genome requires two rounds of PCR amplifications. The relative hybridization intensity of ChIP DNA and that of the input DNA is used to determine whether the probe sequence is a potential site of protein-DNA interaction. Resolution of actual genomic sites bound by the protein is dependent on the size of the chromatin and on the genomic distance between the probes on the array. As with expression profiling using gene chips, ChIP-chip experiments require multiple replicates for reliable statistical measure of protein-DNA interactions. © 2018 Cold Spring Harbor Laboratory Press.

Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses

USDA-ARS?s Scientific Manuscript database

Current technologies with next generation sequencing have revolutionized metagenomics analysis of clinical samples. To achieve the non-selective amplification and recovery of low abundance genetic sequences, a simplified Sequence-Independent, Single-Primer Amplification (SISPA) technique in combinat...

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

Sparse representation and Bayesian detection of genome copy number alterations from microarray data.

PubMed

Pique-Regi, Roger; Monso-Varona, Jordi; Ortega, Antonio; Seeger, Robert C; Triche, Timothy J; Asgharzadeh, Shahab

2008-02-01

Genomic instability in cancer leads to abnormal genome copy number alterations (CNA) that are associated with the development and behavior of tumors. Advances in microarray technology have allowed for greater resolution in detection of DNA copy number changes (amplifications or deletions) across the genome. However, the increase in number of measured signals and accompanying noise from the array probes present a challenge in accurate and fast identification of breakpoints that define CNA. This article proposes a novel detection technique that exploits the use of piece wise constant (PWC) vectors to represent genome copy number and sparse Bayesian learning (SBL) to detect CNA breakpoints. First, a compact linear algebra representation for the genome copy number is developed from normalized probe intensities. Second, SBL is applied and optimized to infer locations where copy number changes occur. Third, a backward elimination (BE) procedure is used to rank the inferred breakpoints; and a cut-off point can be efficiently adjusted in this procedure to control for the false discovery rate (FDR). The performance of our algorithm is evaluated using simulated and real genome datasets and compared to other existing techniques. Our approach achieves the highest accuracy and lowest FDR while improving computational speed by several orders of magnitude. The proposed algorithm has been developed into a free standing software application (GADA, Genome Alteration Detection Algorithm). http://biron.usc.edu/~piquereg/GADA

Long-stem shaped multifunctional molecular beacon for highly sensitive nucleic acids determination via intramolecular and intermolecular interactions based strand displacement amplification.

PubMed

Xu, Jianguo; Zheng, Tingting; Le, Jingqing; Jia, Lee

2017-11-20

Occurrence and application of oligonucleotide probes have promoted great progress in the biochemical analysis field due to their unique biological and chemical properties. In this work, a long-stem shaped multifunctional molecular beacon (LS-MMB) that is responsive to a cancer-related gene, p53, is well-prepared. By designing the probe with long-paired bases at its two ends and short-paired bases between the middle region and the 3' end, the LS-MMB is intelligently endowed with the ability to recognize the target analyte, serve as the polymerization primer/template, and signal the hybridization event synchronously, which is distinctly advantageous over the traditional molecular beacons (MBs). Moreover, it is excitingly found that the LS-MMB can be employed to exert intramolecular and intermolecular interactions for strand displacement amplification (SDA) without the involvement of any assistant probes; this therapy results in a really easy and rapid sensing system that provides an extremely low background noise and high target output signal. In this case, an excellent sensitivity and specificity to detect target gene down to picomolar level and resolution to even one nucleotide variation are achieved, respectively. In addition, the application potential for real genomic DNA analysis is realized. We envision that the probe of LS-MMB can act as a universal platform for biosensing and biomedical research.

Combined subtraction hybridization and polymerase chain reaction amplification procedure for isolation of strain-specific Rhizobium DNA sequences.

PubMed Central

Bjourson, A J; Stone, C E; Cooper, J E

1992-01-01

A novel subtraction hybridization procedure, incorporating a combination of four separation strategies, was developed to isolate unique DNA sequences from a strain of Rhizobium leguminosarum bv. trifolii. Sau3A-digested DNA from this strain, i.e., the probe strain, was ligated to a linker and hybridized in solution with an excess of pooled subtracter DNA from seven other strains of the same biovar which had been restricted, ligated to a different, biotinylated, subtracter-specific linker, and amplified by polymerase chain reaction to incorporate dUTP. Subtracter DNA and subtracter-probe hybrids were removed by phenol-chloroform extraction of a streptavidin-biotin-DNA complex. NENSORB chromatography of the sequences remaining in the aqueous layer captured biotinylated subtracter DNA which may have escaped removal by phenol-chloroform treatment. Any traces of contaminating subtracter DNA were removed by digestion with uracil DNA glycosylase. Finally, remaining sequences were amplified by polymerase chain reaction with a probe strain-specific primer, labelled with 32P, and tested for specificity in dot blot hybridizations against total genomic target DNA from each strain in the subtracter pool. Two rounds of subtraction-amplification were sufficient to remove cross-hybridizing sequences and to give a probe which hybridized only with homologous target DNA. The method is applicable to the isolation of DNA and RNA sequences from both procaryotic and eucaryotic cells. Images PMID:1637166

Superparamagnetic nanoparticles for enhanced magnetic resonance and multimodal imaging

NASA Astrophysics Data System (ADS)

Sikma, Elise Ann Schultz

Magnetic resonance imaging (MRI) is a powerful tool for noninvasive tomographic imaging of biological systems with high spatial and temporal resolution. Superparamagnetic (SPM) nanoparticles have emerged as highly effective MR contrast agents due to their biocompatibility, ease of surface modification and magnetic properties. Conventional nanoparticle contrast agents suffer from difficult synthetic reproducibility, polydisperse sizes and weak magnetism. Numerous synthetic techniques and nanoparticle formulations have been developed to overcome these barriers. However, there are still major limitations in the development of new nanoparticle-based probes for MR and multimodal imaging including low signal amplification and absence of biochemical reporters. To address these issues, a set of multimodal (T2/optical) and dual contrast (T1/T2) nanoparticle probes has been developed. Their unique magnetic properties and imaging capabilities were thoroughly explored. An enzyme-activatable contrast agent is currently being developed as an innovative means for early in vivo detection of cancer at the cellular level. Multimodal probes function by combining the strengths of multiple imaging techniques into a single agent. Co-registration of data obtained by multiple imaging modalities validates the data, enhancing its quality and reliability. A series of T2/optical probes were successfully synthesized by attachment of a fluorescent dye to the surface of different types of nanoparticles. The multimodal nanoparticles generated sufficient MR and fluorescence signal to image transplanted islets in vivo. Dual contrast T1/T2 imaging probes were designed to overcome disadvantages inherent in the individual T1 and T2 components. A class of T1/T2 agents was developed consisting of a gadolinium (III) complex (DTPA chelate or DO3A macrocycle) conjugated to a biocompatible silica-coated metal oxide nanoparticle through a disulfide linker. The disulfide linker has the ability to be reduced in vivo by glutathione, releasing large payloads of signal-enhancing T1 probes into the surrounding environment. Optimization of the agent occurred over three sequential generations, with each generation addressing a new challenge. The result was a T2 nanoparticle containing high levels of conjugated T1 complex demonstrating enhanced MR relaxation properties. The probes created here have the potential to play a key role in the advancement of nanoparticle-based agents in biomedical MRI applications.

DAGON: a 3D Maxwell-Bloch code

NASA Astrophysics Data System (ADS)

Oliva, Eduardo; Cotelo, Manuel; Escudero, Juan Carlos; González-Fernández, Agustín.; Sanchís, Alberto; Vera, Javier; Vicéns, Sergio; Velarde, Pedro

2017-05-01

The amplification of UV radiation and high order harmonics (HOH) in plasmas is a subject of raising interest due to its different potential applications in several fields like environment and security (detection at distance), biology, materials science and industry (3D imaging) and atomic and plasma physics (pump-probe experiments). In order to develop these sources, it is necessary to properly understand the amplification process. Being the plasma an inhomogeneous medium which changes with time, it is desirable to have a full time-dependent 3D description of the interaction of UV and XUV radiation with plasmas. For these reasons, at the Instituto de Fusíon Nuclear we have developed DAGON, a 3D Maxwell-Bloch code capable of studying the full spationtemporal structure of the amplification process abovementioned.

Attomolar detection of proteins via cascade strand-displacement amplification and polystyrene nanoparticle enhancement in fluorescence polarization aptasensors.

PubMed

Huang, Yong; Liu, Xiaoqian; Huang, Huakui; Qin, Jian; Zhang, Liangliang; Zhao, Shulin; Chen, Zhen-Feng; Liang, Hong

2015-08-18

Extremely sensitive and accurate measurements of protein markers for early detection and monitoring of diseases pose a formidable challenge. Herein, we develop a new type of amplified fluorescence polarization (FP) aptasensor based on allostery-triggered cascade strand-displacement amplification (CSDA) and polystyrene nanoparticle (PS NP) enhancement for ultrasensitive detection of proteins. The assay system consists of a fluorescent dye-labeled aptamer hairpin probe and a PS NP-modified DNA duplex (assistant DNA/trigger DNA duplex) probe with a single-stranded part and DNA polymerase. Two probes coexist stably in the absence of target, and the dye exhibits relatively low FP background. Upon recognition and binding with a target protein, the stem of the aptamer hairpin probe is opened, after which the opened hairpin probe hybridizes with the single-stranded part in the PS NP-modified DNA duplex probe and triggers the CSDA reaction through the polymerase-catalyzed recycling of both target protein and trigger DNA. Throughout this CSDA process, numerous massive dyes are assembled onto PS NPs, which results in a substantial FP increase that provides a readout signal for the amplified sensing process. Our newly proposed amplified FP aptasensor enables the quantitative measurement of proteins with the detection limit in attomolar range, which is about 6 orders of magnitude lower than that of traditional homogeneous aptasensors. Moreover, this sensing method also exhibits high specificity for target proteins and can be performed in homogeneous solutions. In addition, the suitability of this method for the quantification of target protein in biological samples has also been shown. Considering these distinct advantages, the proposed sensing method can be expected to provide an ultrasensitive platform for the analysis of various types of target molecules.

A homogeneous and "off-on" fluorescence aptamer-based assay for chloramphenicol using vesicle quantum dot-gold colloid composite probes.

PubMed

Miao, Yang-Bao; Ren, Hong-Xia; Gan, Ning; Zhou, You; Cao, Yuting; Li, Tianhua; Chen, Yinji

2016-07-27

In this work, a novel homogeneous and signal "off-on" aptamer based fluorescence assay was successfully developed to detect chloramphenicol (CAP) residues in food based on the fluorescence resonance energy transfer (FRET). The vesicle nanotracer was prepared through labeling single stranded DNA binding protein (SSB) on limposome-CdSe/ZnS quantum dot (SSB/L-QD) complexes. It was worth mentioning that the signal tracer (SSB/L-QD) with vesicle shape, which was fabricated being encapsulated with a number of quantum dots and SSB. The nanotracer has excellent signal amplification effects. The vesicle composite probe was formed by combining aptamer labeled nano-gold (Au-Apt) and SSB/L-QD. Which based on SSB's specific affinity towards aptamer. This probe can't emit fluoresce which is in "off" state because the signal from SSB/L-QD as donor can be quenched by the Au-aptas acceptor. When CAP was added in the composite probe solution, the aptamer on the Au-Apt can be preferentially bounded with CAP then release from the composite probe, which can turn the "off" signal of SSB/L-QD tracer into "on" state. The assay indicates excellent linear response to CAP from 0.001 nM to 10 nM and detection limit down to 0.3 pM. The vesicle probes with size of 88 nm have strong signal amplification. Because a larger number of QDs can be labeled inside the double phosphorus lipid membrane. Besides, it was employed to detect CAP residues in the milk samples with results being agreed well with those from ELISA, verifying its accuracy and reliability. Copyright © 2016 Elsevier B.V. All rights reserved.

PDGFRA amplification is common in pediatric and adult high-grade astrocytomas and identifies a poor prognostic group in IDH1 mutant glioblastoma

PubMed Central

Phillips, Joanna J.; Aranda, Derick; Ellison, David W.; Judkins, Alexander R.; Croul, Sidney E.; Brat, Daniel J.; Ligon, Keith L.; Horbinski, Craig; Venneti, Sriram; Zadeh, Gelareh; Santi, Mariarita; Zhou, Shengmei; Appin, Christina L.; Sioletic, Stefano; Sullivan, Lisa M.; Martinez-Lage, Maria; Robinson, Aaron E.; Yong, William H.; Cloughesy, Timothy; Lai, Albert; Phillips, Heidi S.; Marshall, Roxanne; Mueller, Sabine; Haas-Kogan, Daphne A.; Molinaro, Annette M.; Perry, Arie

2013-01-01

High-grade astrocytomas (HGAs), corresponding to WHO grades III (AA) and IV (GBM), are biologically aggressive and their molecular classification is increasingly relevant to clinical management. PDGFRA amplification is common in HGAs, although its prognostic significance remains unclear. Using fluorescence in situ hybridization (FISH), the most sensitive technique for detecting PDGFRA copy number gains, we determined PDGFRA amplification status in 123 pediatric and 263 adult HGAs. A range of PDGFRA FISH patterns were identified and cases were scored as non-amplified (normal and polysomy) or amplified (low-level and high-level). PDGFRA amplification was frequent in pediatric (29.3%) and adult (20.9%) tumors. Amplification was not prognostic in pediatric HGAs. In adult tumors diagnosed initially as GBM, the presence of combined PDGFRA amplification and IDH1R132H mutation was a significant independent prognostic factor (p=0.01). In HGAs, PDGFRA amplification is common and can manifest as high-level and focal or low-level amplifications. Our data indicate that the latter is more prevalent than previously reported with copy number averaging techniques. To our knowledge, this is the largest survey of PDGFRA status in adult and pediatric HGAs and suggests PDGFRA amplification increases with grade and is associated with a less favorable prognosis in IDH1 mutant de novo GBMs. PMID:23438035

Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rhee, Minsoung; Light, Yooli K.; Meagher, Robert J.

Here, multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently,more » the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.« less

Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples

DOE PAGES

Rhee, Minsoung; Light, Yooli K.; Meagher, Robert J.; ...

2016-05-04

Here, multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently,more » the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.« less

Development of one-step Loop-Mediated Isothermal Amplification (LAMP) for the detection of norovirus in oysters

USDA-ARS?s Scientific Manuscript database

The aim of this study was to develop a simple and rapid technique for detecting human norovirus (NoV). The loop-mediated isothermal amplification (LAMP) technique was evaluated and found to be sensitive, highly specific, and useful for routine oyster testing. Reverse transcription-LAMP (RT-LAMP) pri...

A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

NASA Astrophysics Data System (ADS)

Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

2015-06-01

Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.

Signal amplification of FISH for automated detection using image cytometry.

PubMed

Truong, K; Boenders, J; Maciorowski, Z; Vielh, P; Dutrillaux, B; Malfoy, B; Bourgeois, C A

1997-05-01

The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.

Sensitive SERS detection of DNA methyltransferase by target triggering primer generation-based multiple signal amplification strategy.

PubMed

Li, Ying; Yu, Chuanfeng; Han, Huixia; Zhao, Caisheng; Zhang, Xiaoru

2016-07-15

A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57×10(-4)UmL(-1). This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase. Copyright © 2016 Elsevier B.V. All rights reserved.

A NASBA on microgel-tethered molecular-beacon microarray for real-time microbial molecular diagnostics.

PubMed

Ma, Y; Dai, X; Hong, T; Munk, G B; Libera, M

2016-12-19

Despite their many advantages and successes, molecular beacon (MB) hybridization probes have not been extensively used in microarray formats because of the complicating probe-substrate interactions that increase the background intensity. We have previously shown that tethering to surface-patterned microgels is an effective means for localizing MB probes to specific surface locations in a microarray format while simultaneously maintaining them in as water-like an environment as possible and minimizing probe-surface interactions. Here we extend this approach to include both real-time detection together with integrated NASBA amplification. We fabricate small (∼250 μm × 250 μm) simplex, duplex, and five-plex assays with microarray spots of controllable size (∼20 μm diameter), position, and shape to detect bacteria and fungi in a bloodstream-infection model. The targets, primers, and microgel-tethered probes can be combined in a single isothermal reaction chamber with no post-amplification labelling. We extract total RNA from clinical blood samples and differentiate between Gram-positive and Gram-negative bloodstream infection in a duplex assay to detect RNA- amplicons. The sensitivity based on our current protocols in a simplex assay to detect specific ribosomal RNA sequences within total RNA extracted from S. aureus and E. coli cultures corresponds to tens of bacteria per ml. We furthermore show that the platform can detect RNA- amplicons from synthetic target DNA with 1 fM sensitivity in sample volumes that contain about 12 000 DNA molecules. These experiments demonstrate an alternative approach that can enable rapid and real-time microarray-based molecular diagnostics.

Absorption spectrum of a two-level atom in a bad cavity with injected squeezed vacuum

NASA Astrophysics Data System (ADS)

Zhou, Peng; Swain, S.

1996-02-01

We study the absorption spectrum of a coherently driven two-level atom interacting with a resonant cavity mode which is coupled to a broadband squeezed vacuum through its input-output mirror in the bad cavity limit. We study the modification of the two-photon correlation strength of the injected squeezed vacuum inside the cavity, and show that the equations describing probe absorption in the cavity environment are formally identical to these in free space, but with modified parameters describing the squeezed vacuum. The two photon correlations induced by the squeezed vacuum are always weaker than in free space. We pay particular attention to the spectral behaviour at line centre in the region of intermediate trength driving intensities, where anomalous spectral features such as hole-burning and dispersive profiles are displayed. These unusual spectral features are very sensitive to the squeezing phase and the Rabi frequency of the driving field. We also derive the threshold value of the Rabi frequency which gives rise to the transparency of the probe beam at the driving frequency. When the Rabi frequency is less than the threshold value, the probe beam is absorbed, whilst the probe beam is amplified (without population inversion under certain conditions) when the Rabi frequency is larger than this threshold. The anomalous spectral features all take place in the vicinity of the critical point dividing the different dynamical regimes, probe absorption and amplification, of the atomic radiation. The physical origin of the strong amplification without population inversion, and the feasibility of observing it, are discussed.

Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification.

PubMed

Agersborg, Sally; Mixon, Christopher; Nguyen, Thanh; Aithal, Sramila; Sudarsanam, Sucha; Blocker, Forrest; Weiss, Lawrence; Gasparini, Robert; Jiang, Shiping; Chen, Wayne; Hess, Gregory; Albitar, Maher

2018-03-22

While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases. Between 2014 and 2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of the patients tested by FISH, 505/3908 (12.9%) also had IHC data. Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. However, 50.5% of equivocal cases were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe: 78% of positive cases by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (p < 0.0001) among the equivocal cases by conventional FISH testing, 44% of IHC negative cases became positive while 36% of the positive IHC cases became negative by alternative FISH testing. Available data showed that 41% of patients were treated with palbociclib and were positive by alternative FISH. The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine the benefit of anti-HER2 therapy in these patients are urgently needed.

Methylation-Specific Multiplex Ligation-Dependent Probe Amplification and Identification of Deletion Genetic Subtypes in Prader-Willi Syndrome

PubMed Central

Henkhaus, Rebecca S.; Kim, Soo-Jeong; Kimonis, Virginia E.; Gold, June-Anne; Dykens, Elisabeth M.; Driscoll, Daniel J.

2012-01-01

Purpose: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are complex neurodevelopmental disorders caused by loss of expression of imprinted genes from the 15q11-q13 region depending on the parent of origin. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) kits from MRC-Holland (Amsterdam, The Netherlands) were used to detect PWS and AS deletion subtypes. We report our experience with two versions of the MS-MLPA-PWS/AS kit (original A1 and newer B1) in determining methylation status and deletion subtypes in individuals with PWS. Methods: MS-MLPA analysis was performed on DNA isolated from a large cohort of PWS subjects with the MS-MLPA-PWS/AS-A1 and -B1 probe sets. Results: Both MS-MLPA kits will identify deletions in the 15q11-q13 region but the original MS-MLPA-A1 kit has a higher density of probes at the telomeric end of the 15q11-q13 region, which is more useful for identifying individuals with atypical deletions. The newer B1 kit contains more probes in the imprinting center (IC) and adjoining small noncoding RNAs useful in identifying small microdeletions. Conclusion: The A1 kit identified the typical deletions and smaller atypical deletions, whereas the B1 kit was more informative for identifying microdeletions including the IC and SNORD116 regions. Both kits should be made available for accurate characterization of PWS/AS deletion subtypes as well as evaluating for IC and SNORD116 microdeletions. PMID:21977908

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strizhkov, B. N.; Drobyshev, A. L.; Mikhailovich, V. M.

PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency,more » the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.« less

Monochrome Multiplexing in Polymerase Chain Reaction by Photobleaching of Fluorogenic Hydrolysis Probes.

PubMed

Schuler, Friedrich; Trotter, Martin; Zengerle, Roland; von Stetten, Felix

2016-03-01

Multiplexing in polymerase chain reaction (PCR) is a technique widely used to save cost and sample material and to increase sensitivity compared to distributing a sample to several singleplex reactions. One of the most common methods to detect the different amplification products is the use of fluorogenic probes that emit at different wavelengths (colors). To reduce the number of detection channels, several methods for monochrome multiplexing have been suggested. However, they pose restrictions to the amplifiable target length, the sequence, or the melting temperature. To circumvent these limitations, we suggest a novel approach that uses different fluorophores with the same emission maximum. Discrimination is achieved by their different fluorescence stability during photobleaching. Atto488 (emitting at the same wavelength as 6-carboxyfluorescein, FAM) and Atto467N (emitting at the same wavelength as cyanine 5, Cy5) were found to bleach significantly less than FAM and Cy5; i.e., the final fluorescence of Atto dyes was more than tripled compared to FAM and Cy5. We successfully applied this method by performing a 4-plex PCR targeting antibiotic resistance genes in S. aureus using only 2 color channels. Confidence of discrimination between the targets was >99.9% at high copy initial copy numbers of 100 000 copies. Cases where both targets were present could be discriminated with equal confidence for Cy5 channel and reduced levels of confidence (>68%) for FAM channel. Moreover, a 2-plex digital PCR reaction in 1 color channel was shown. In the future, the degree of multiplexing may be increased by adding fluorogenic probe pairs with other emission wavelengths. The method may also be applied to other probe and assay formats, such as Förster resonance energy transfer (FRET) probes and immunoassays.

An "off-on" electrochemiluminescent biosensor based on DNAzyme-assisted target recycling and rolling circle amplifications for ultrasensitive detection of microRNA.

PubMed

Zhang, Pu; Wu, Xiaoyan; Yuan, Ruo; Chai, Yaqin

2015-03-17

In this study, an off-on switching of a dual amplified electrochemiluminescence (ECL) biosensor based on Pb(2+)-induced DNAzyme-assisted target recycling and rolling circle amplification (RCA) was constructed for microRNA (miRNA) detection. First, the primer probe with assistant probe and miRNA formed Y junction which was cleaved with the addition of Pb(2+) to release miRNA. Subsequently, the released miRNA could initiate the next recycling process, leading to the generation of numerous intermediate DNA sequences (S2). Afterward, bare glassy carbon electrode (GCE) was immersed into HAuCl4 solution to electrodeposit a Au nanoparticle layer (depAu), followed by the assembly of a hairpin probe (HP). Then, dopamine (DA)-modified DNA sequence (S1) was employed to hybridize with HP, which switching off the sensing system. This is the first work that employs DA to quench luminol ECL signal, possessing the biosensor ultralow background signal. Afterward, S2 produced by the target recycling process was loaded onto the prepared electrode to displace S1 and served as an initiator for RCA. With rational design, numerous repeated DNA sequences coupling with hemin to form hemin/G-quadruplex were generated, which could exhibit strongly catalytic toward H2O2, thus amplified the ECL signal and switched the ON state of the sensing system. The liner range for miRNA detection was from 1.0 fM to 100 pM with a low detection limit down to 0.3 fM. Moreover, with the high sensitivity and specificity induced by the dual signal amplification, the proposed miRNA biosensor holds great potential for analysis of other interesting tumor markers.

Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification

USDA-ARS?s Scientific Manuscript database

The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (or 6) primers targeting 6 (or 8) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an in...

Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

PubMed

Marras, Salvatore A E; Tyagi, Sanjay; Kramer, Fred Russell

2006-01-01

A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

Multicolor microRNA FISH effectively differentiates tumor types

PubMed Central

Renwick, Neil; Cekan, Pavol; Masry, Paul A.; McGeary, Sean E.; Miller, Jason B.; Hafner, Markus; Li, Zhen; Mihailovic, Aleksandra; Morozov, Pavel; Brown, Miguel; Gogakos, Tasos; Mobin, Mehrpouya B.; Snorrason, Einar L.; Feilotter, Harriet E.; Zhang, Xiao; Perlis, Clifford S.; Wu, Hong; Suárez-Fariñas, Mayte; Feng, Huichen; Shuda, Masahiro; Moore, Patrick S.; Tron, Victor A.; Chang, Yuan; Tuschl, Thomas

2013-01-01

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH. As a proof of concept, we used this method to differentiate two skin tumors, basal cell carcinoma (BCC) and Merkel cell carcinoma (MCC), with overlapping histologic features but distinct cellular origins. Using sequencing-based miRNA profiling and discriminant analysis, we identified the tumor-specific miRNAs miR-205 and miR-375 in BCC and MCC, respectively. We addressed three major shortcomings in miRNA FISH, identifying optimal conditions for miRNA fixation and ribosomal RNA (rRNA) retention using model compounds and high-pressure liquid chromatography (HPLC) analyses, enhancing signal amplification and detection by increasing probe-hapten linker lengths, and improving probe specificity using shortened probes with minimal rRNA sequence complementarity. We validated our method on 4 BCC and 12 MCC tumors. Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals. Tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis. Our study establishes a reliable miRNA FISH technique for parallel visualization of differentially expressed miRNAs in FFPE tumor tissues. PMID:23728175

Robust detection of EGFR copy number changes and EGFR variant III: technical aspects and relevance for glioma diagnostics.

PubMed

Jeuken, Judith; Sijben, Angelique; Alenda, Cristina; Rijntjes, Jos; Dekkers, Marieke; Boots-Sprenger, Sandra; McLendon, Roger; Wesseling, Pieter

2009-10-01

Epidermal growth factor receptor (EGFR) is commonly affected in cancer, generally in the form of an increase in DNA copy number and/or as mutation variants [e.g., EGFR variant III (EGFRvIII), an in-frame deletion of exons 2-7]. While detection of EGFR aberrations can be expected to be relevant for glioma patients, such analysis has not yet been implemented in a routine setting, also because feasible and robust assays were lacking. We evaluated multiplex ligation-dependent probe amplification (MLPA) for detection of EGFR amplification and EGFRvIII in DNA of a spectrum of 216 diffuse gliomas. EGFRvIII detection was verified at the protein level by immunohistochemistry and at the RNA level using the conventionally used endpoint RT-PCR as well as a newly developed quantitative RT-PCR. Compared to these techniques, the DNA-based MLPA assay for EGFR/EGFRvIII analysis tested showed 100% sensitivity and specificity. We conclude that MLPA is a robust assay for detection of EGFR/EGFRvIII aberrations. While the exact diagnostic, prognostic and predictive value of such EGFR testing remains to be seen, MLPA has great potential as it can reliably and relatively easily be performed on routinely processed (formalin-fixed, paraffin-embedded) tumor tissue in combination with testing for other relevant glioma markers.

Brillouin Selective Sideband Amplification of Microwave Photonic Signals

NASA Technical Reports Server (NTRS)

Yao, S.

1997-01-01

We introduce a powerful Brillouin selective sideband amplification technique and demonstrate its application for achieving gain in photonix signal up- and down- conversions in microwave photonic systems.

Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.

PubMed

Jørgensen, P L; Tangney, M; Pedersen, P E; Hastrup, S; Diderichsen, B; Jørgensen, S T

2000-02-01

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis.

PubMed

Zaghloul, Hosam; El-Shahat, Mahmoud

2014-12-27

Hepatitis C virus (HCV) infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20% of the total population are infected. Initial infection is usually asymptomatic and result in chronic hepatitis that give rise to complications including cirrhosis and hepatocellular carcinoma. The management of HCV infection should not only be focus on therapy, but also to screen carrier individuals in order to prevent transmission. In the present, molecular detection and quantification of HCV genome by real time polymerase chain reaction (PCR) represent the gold standard in HCV diagnosis and plays a crucial role in the management of therapeutic regimens. However, real time PCR is a complicated approach and of limited distribution. On the other hand, isothermal DNA amplification techniques have been developed and offer molecular diagnosis of infectious dieses at point-of-care. In this review we discuss recombinase polymerase amplification technique and illustrate its diagnostic value over both PCR and other isothermal amplification techniques.

Identification of a candidate oncogene SEI-1 within a minimal amplified region at 19q13.1 in ovarian cancer cell lines.

PubMed

Tang, Terence C-M; Sham, Jonathan S T; Xie, Dan; Fang, Yan; Huo, Ke-Ke; Wu, Qiu-Liang; Guan, Xin-Yuan

2002-12-15

High-level amplification of DNA sequence at 19q13.1 is one of the frequent genetic alterations in ovarian cancer. In an attempt to verify the minimal amplified region (MAR) at 19q13.1 and to identify the target oncogenes, 49 probes within a region from D19S425 to D19S907 ( approximately 19.5 cM) were used to survey the amplification status in four ovarian cancer cell lines that have been confirmed as containing amplification at 19q13.1. Two separated overlapping MARs, MAR1 (approximately 200 kb) and MAR2 (approximately 1.1 Mb), were identified at 19q13.1. Two candidate oncogenes, AKT2 and SEI-1, were identified in MAR2. Amplification and overexpression of these two genes in four ovarian cancer cell lines were confirmed by Southern and Northern blot analyses. The proliferation-related function of AKT2 and SEI-1 suggests that both genes are likely to be biological targets of an amplification event at 19q13.1 in ovarian cancer and to play important roles in ovarian tumorigenesis.

Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

PubMed Central

2010-01-01

Background Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level. Results A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins. Conclusions Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range. PMID:20569466

Multiplex ligation-dependant probe amplification study of children with idiopathic mental retardation in South India

PubMed Central

John, Neetha; Rajasekhar, Moka; Girisha, Katta Mohan; Sharma, Podila Satya Venkata Narasimha; Gopinath, Puthiya Mundyat

2013-01-01

BACKGROUND: Mental retardation (MR) is a heterogeneous dysfunction of the central nervous system exhibiting complex phenotypes and has an estimated prevalence of 1-3% in the general population. However, in about 50% of the children diagnosed with any form of intellectual disability or developmental delay the cause goes undetected contributing to idiopathic intellectual disability. MATERIALS AND METHODS: A total of 122 children with developmental delay/MR were studied to identify the microscopic and submicroscopic chromosome rearrangements by using the conventional cytogenetics and multiplex ligation dependent probe amplification (MLPA) analysis using SALSA MLPA kits from Microbiology Research Centre Holland [MRC] Holland. RESULTS: All the recruited children were selected for this study, after thorough clinical assessment and metaphases prepared were analyzed by using automated karyotyping system. None was found to have chromosomal abnormality; MLPA analysis was carried out in all subjects and identified in 11 (9%) patients. CONCLUSION: Karyotype analysis in combination with MLPA assays for submicroscopic micro-deletions may be recommended for children with idiopathic MR. PMID:24019617

An Electrochemical DNA Sensing System Using Modified Nanoparticle Probes for Detecting Methicillin-Resistant Staphylococcus aureus.

PubMed

Sakamoto, Hiroaki; Amano, Yoshihisa; Satomura, Takenori; Suye, Shin-Ichiro

2017-01-01

We have developed a novel, highly sensitive, biosensing system for detecting methicillin-resistant Staphylococcus aureus (MRSA). The system employs gold nanoparticles (AuNPs), magnetic nanoparticles (mNPs), and an electrochemical detection method. We have designed and synthesized ferrocene- and single-stranded DNA-conjugated nanoparticles that hybridize to MRSA DNA. Hybridized complexes are easily separated by taking advantage of mNPs. A current response could be obtained through the oxidation of ferrocene on the AuNP surface when a constant potential of +250 mV vs. Ag/AgCl is applied. The enzymatic reaction of L-proline dehydrogenase provides high signal amplification. This sensing system, using a nanoparticle-modified probe, has the ability to detect 10 pM of genomic DNA from MRSA without amplification by the polymerase chain reaction. Current responses are linearly related to the amount of genomic DNA in the range of 10-166 pM. Selectivity is confirmed by demonstrating that this sensing system could distinguish MRSA from Staphylococcus aureus (SA) DNA.

Rolling-circle amplification under topological constraints

PubMed Central

Kuhn, Heiko; Demidov, Vadim V.; Frank-Kamenetskii, Maxim D.

2002-01-01

We have performed rolling-circle amplification (RCA) reactions on three DNA templates that differ distinctly in their topology: an unlinked DNA circle, a linked DNA circle within a pseudorotaxane-type structure and a linked DNA circle within a catenane. In the linked templates, the single-stranded circle (dubbed earring probe) is threaded, with the aid of two peptide nucleic acid openers, between the two strands of double-stranded DNA (dsDNA). We have found that the RCA efficiency of amplification was essentially unaffected when the linked templates were employed. By showing that the DNA catenane remains intact after RCA reactions, we prove that certain DNA polymerases can carry out the replicative synthesis under topological constraints allowing detection of several hundred copies of a dsDNA marker without DNA denaturation. Our finding may have practical implications in the area of DNA diagnostics. PMID:11788721

Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio

PubMed Central

Clausson, Carl-Magnus; Arngården, Linda; Ishaq, Omer; Klaesson, Axel; Kühnemund, Malte; Grannas, Karin; Koos, Björn; Qian, Xiaoyan; Ranefall, Petter; Krzywkowski, Tomasz; Brismar, Hjalmar; Nilsson, Mats; Wählby, Carolina; Söderberg, Ola

2015-01-01

Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes. PMID:26202090

OCaPPI-Db: an oligonucleotide probe database for pathogen identification through hybridization capture.

PubMed

Gasc, Cyrielle; Constantin, Antony; Jaziri, Faouzi; Peyret, Pierre

2017-01-01

The detection and identification of bacterial pathogens involved in acts of bio- and agroterrorism are essential to avoid pathogen dispersal in the environment and propagation within the population. Conventional molecular methods, such as PCR amplification, DNA microarrays or shotgun sequencing, are subject to various limitations when assessing environmental samples, which can lead to inaccurate findings. We developed a hybridization capture strategy that uses a set of oligonucleotide probes to target and enrich biomarkers of interest in environmental samples. Here, we present Oligonucleotide Capture Probes for Pathogen Identification Database (OCaPPI-Db), an online capture probe database containing a set of 1,685 oligonucleotide probes allowing for the detection and identification of 30 biothreat agents up to the species level. This probe set can be used in its entirety as a comprehensive diagnostic tool or can be restricted to a set of probes targeting a specific pathogen or virulence factor according to the user's needs. : http://ocappidb.uca.works. © The Author(s) 2017. Published by Oxford University Press.

Detection of MET amplification in gastroesophageal tumor specimens using IQFISH.

PubMed

Jørgensen, Jan Trøst; Nielsen, Karsten Bork; Mollerup, Jens; Jepsen, Anna; Go, Ning

2017-12-01

The gene mesenchymal epithelial transition factor ( MET ) is a proto-oncogene that encodes a transmembrane receptor with intrinsic tyrosine kinase activity known as Met or cMet. MET is found to be amplified in several human cancers including gastroesophageal cancer. Here we report the MET amplification prevalence data from 159 consecutive tumor specimens from patients with gastric (G), gastroesophageal junction (GEJ) and esophageal (E) adenocarcinoma, using a novel fluorescence in situ hybridization (FISH) assay, MET /CEN-7 IQFISH Probe Mix [an investigational use only (IUO) assay]. MET amplification was defined as a MET /CEN-7 ratio ≥2.0. Furthermore, the link between the MET signal distribution and amplification status was investigated. The prevalence of MET amplification was found to be 6.9%. The FISH assay demonstrated a high inter-observer reproducibility. The inter-observer results showed a 100% overall agreement with respect to the MET status (amplified/non-amplified). The inter-observer CV was estimated to 11.8% (95% CI: 10.2-13.4). For the signal distribution, the inter-observer agreement was reported to be 98.7%. We also report an association of MET amplification and a unique signal distribution pattern in the G/GEJ/E tumor specimens. We found that the prevalence of MET amplification was markedly higher in tumors specimens with a heterogeneous (66.7%) versus homogeneous (2.0%) signal distribution. Furthermore, specimens with a heterogeneous signal distribution had a statically significantly higher median MET /CEN-7 ratio (2.35 versus 1.04; P<0.0001). The novel FISH assay showed a high inter-observer reproducibility both with respect to amplification status and signal distribution. Based on the finding in the study it is suggested that MET amplification mainly is associated with tumor cells that is represented by a heterogonous growth pattern.

Clinical significance of hTERC gene amplification detection by FISH in the screening of cervical lesions.

PubMed

Zhang, Yuan; Wang, Xiaobei; Ma, Ling; Wang, Zehua; Hu, Lihua

2009-06-01

This study evaluated the clinical significance of hTERC gene amplification detection by fluorescence in situ hybridization (FISH) in the screening of cervical lesions. Cervical specimens of 50 high risk patients were detected by thin liquid-based cytology. The patients whose cytological results were classified as ASCUS or above were subjected to the subsequent colposcopic biopsies. Slides prepared from these 50 cervical specimens were analyzed for hTERC gene amplification using interphase FISH with the two-color hTERC probe. The results of the cytological analysis and those of subsequent biopsies, when available, were compared with the FISH-detected hTERC abnormalities. It was found that the positive rates of hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 28.57%, 57.14%, 100%, and 100%, respectively. The positive rates of hTERC gene amplification in HSIL and SCC groups were significantly higher than those in NILM, ASCUS and LSIL groups (all P<0.05). The mean percentages of cells with hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 10.50%, 36.00%, 79.00%, and 96.50%, respectively. Patients with HSIL or SCC cytological diagnoses had significantly higher mean percentages of cells with hTERC gene amplification than did patients with NILM, ASCUS or LSIL cytological diagnoses (all P<0.05). It was concluded that two-color interphase FISH could detect hTERC gene amplification to accurately distinguish HSIL and ISIL of cervical cells. It may be an adjunct to cytology screening, especially high-risk patients.

Development and Evaluation of a Multiplex Real-Time Polymerase Chain Reaction Procedure to Clinically Type Prevalent Salmonella enterica Serovars

PubMed Central

Muñoz, Nélida; Diaz-Osorio, Miguel; Moreno, Jaime; Sánchez-Jiménez, Miryan; Cardona-Castro, Nora

2010-01-01

A multiplex real-time polymerase chain reaction procedure was developed to identify the most prevalent clinical isolates of Salmonella enterica subsp. enterica. Genes from the rfb, fliC, fljB, and viaB groups that encode the O, H, and Vi antigens were used to design 15 primer pairs and TaqMan probes specific for the genes rfbJ, wzx, fliC, fljB, wcdB, the sdf-l sequence, and invA, which was used as an internal amplification control. The primers and probes were variously combined into six sets. The first round of reactions used two of these sets to detect Salmonella O:4, O:9, O:7, O:8, and O:3,10 serogroups. Once the serogroups were identified, the results of a second round of reactions that used primers and probes for the flagellar antigen l genes, 1,2; e,h; g,m; d; e,n,x; and z10, and the Vi gene were used to identify individual serovars. The procedure was standardized using 18 Salmonella reference strains and other enterobacteria. The procedure's reliability and sensitivity was evaluated using 267 randomly chosen serotyped Salmonella clinical isolates. The procedure had a sensitivity of 95.5% and was 100% specific. Thus, our technique is a quick, sensitive, reliable, and specific means of identifying S. enterica serovars and can be used in conjunction with traditional serotyping. Other primer and probe combinations could be used to increase the number of identifiable serovars. PMID:20110454

Combined free-stream disturbance measurements and receptivity studies in hypersonic wind tunnels by means of a slender wedge probe and direct numerical simulation

NASA Astrophysics Data System (ADS)

Wagner, Alexander; Schülein, Erich; Petervari, René; Hannemann, Klaus; Ali, Syed R. C.; Cerminara, Adriano; Sandham, Neil D.

2018-05-01

Combined free-stream disturbance measurements and receptivity studies in hypersonic wind tunnels were conducted by means of a slender wedge probe and direct numerical simulation. The study comprises comparative tunnel noise measurements at Mach 3, 6 and 7.4 in two Ludwieg tube facilities and a shock tunnel. Surface pressure fluctuations were measured over a wide range of frequencies and test conditions including harsh test environments not accessible to measurement techniques such as pitot probes and hot-wire anemometry. Quantitative results of the tunnel noise are provided in frequency ranges relevant for hypersonic boundary layer transition. In combination with the experimental studies, direct numerical simulations of the leading-edge receptivity to fast and slow acoustic waves were performed for the slender wedge probe at conditions corresponding to the experimental free-stream conditions. The receptivity to fast acoustic waves was found to be characterized by an early amplification of the induced fast mode. For slow acoustic waves an initial decay was found close to the leading edge. At all Mach numbers, and for all considered frequencies, the leading-edge receptivity to fast acoustic waves was found to be higher than the receptivity to slow acoustic waves. Further, the effect of inclination angles of the acoustic wave with respect to the flow direction was investigated. The combined numerical and experimental approach in the present study confirmed the previous suggestion that the slow acoustic wave is the dominant acoustic mode in noisy hypersonic wind tunnels.

Implementing and diagnosing magnetic flux compression on the Z pulsed power accelerator

DOE Office of Scientific and Technical Information (OSTI.GOV)

McBride, Ryan D.; Bliss, David E.; Gomez, Matthew R.

2015-11-01

We report on the progress made to date for a Laboratory Directed Research and Development (LDRD) project aimed at diagnosing magnetic flux compression on the Z pulsed-power accelerator (0-20 MA in 100 ns). Each experiment consisted of an initially solid Be or Al liner (cylindrical tube), which was imploded using the Z accelerator's drive current (0-20 MA in 100 ns). The imploding liner compresses a 10-T axial seed field, B z ( 0 ) , supplied by an independently driven Helmholtz coil pair. Assuming perfect flux conservation, the axial field amplification should be well described by B z ( tmore » ) = B z ( 0 ) x [ R ( 0 ) / R ( t )] 2 , where R is the liner's inner surface radius. With perfect flux conservation, B z ( t ) and dB z / dt values exceeding 10 4 T and 10 12 T/s, respectively, are expected. These large values, the diminishing liner volume, and the harsh environment on Z, make it particularly challenging to measure these fields. We report on our latest efforts to do so using three primary techniques: (1) micro B-dot probes to measure the fringe fields associated with flux compression, (2) streaked visible Zeeman absorption spectroscopy, and (3) fiber-based Faraday rotation. We also mention two new techniques that make use of the neutron diagnostics suite on Z. These techniques were not developed under this LDRD, but they could influence how we prioritize our efforts to diagnose magnetic flux compression on Z in the future. The first technique is based on the yield ratio of secondary DT to primary DD reactions. The second technique makes use of the secondary DT neutron time-of-flight energy spectra. Both of these techniques have been used successfully to infer the degree of magnetization at stagnation in fully integrated Magnetized Liner Inertial Fusion (MagLIF) experiments on Z [P. F. Schmit et al. , Phys. Rev. Lett. 113 , 155004 (2014); P. F. Knapp et al. , Phys. Plasmas, 22 , 056312 (2015)]. Finally, we present some recent developments for designing and fabricating novel micro B-dot probes to measure B z ( t ) inside of an imploding liner. In one approach, the micro B-dot loops were fabricated on a printed circuit board (PCB). The PCB was then soldered to off-the-shelf 0.020- inch-diameter semi-rigid coaxial cables, which were terminated with standard SMA connectors. These probes were recently tested using the COBRA pulsed power generator (0-1 MA in 100 ns) at Cornell University. In another approach, we are planning to use new multi-material 3D printing capabilities to fabricate novel micro B-dot packages. In the near future, we plan to 3D print these probes and then test them on the COBRA generator. With successful operation demonstrated at 1-MA, we will then make plans to use these probes on a 20-MA Z experiment.« less

Frequency-domain nonlinear optics in two-dimensionally patterned quasi-phase-matching media.

PubMed

Phillips, C R; Mayer, B W; Gallmann, L; Keller, U

2016-07-11

Advances in the amplification and manipulation of ultrashort laser pulses have led to revolutions in several areas. Examples include chirped pulse amplification for generating high peak-power lasers, power-scalable amplification techniques, pulse shaping via modulation of spatially-dispersed laser pulses, and efficient frequency-mixing in quasi-phase-matched nonlinear crystals to access new spectral regions. In this work, we introduce and demonstrate a new platform for nonlinear optics which has the potential to combine these separate functionalities (pulse amplification, frequency transfer, and pulse shaping) into a single monolithic device that is bandwidth- and power-scalable. The approach is based on two-dimensional (2D) patterning of quasi-phase-matching (QPM) gratings combined with optical parametric interactions involving spatially dispersed laser pulses. Our proof of principle experiment demonstrates this technique via mid-infrared optical parametric chirped pulse amplification of few-cycle pulses. Additionally, we present a detailed theoretical and numerical analysis of such 2D-QPM devices and how they can be designed.

Christmas-tree Derived Amplification Immuno-strategy for Sensitive Visual Detection of Vibrio parahaemolyticus Based on Gold Label Silver Stain Technology.

PubMed

Song, Xinxin; Wu, Yanjie; Wu, Lin; Hu, Yufang; Li, Wenrou; Guo, Zhiyong; Su, Xiurong; Jiang, Xiaohua

2017-01-01

A developed Christmas-tree derived immunosensor based on a gold label silver stain (GLSS) technique was fabricated for a highly sensitive analysis of Vibrio parahaemolyticu (VP). In this strategy, captured VP antibody (cAb) was immobilized on a solid substrate; then, the VPs were sequentially tagged with a signal probe by incubating the assay with a detection VP antibody (dAb) conjugated gold nanoparticles (AuNPs)-labeled graphite-like carbon nitride (g-C 3 N 4 ). Finally, the attached signal probe could harvest a visible signal by the silver meal deposition, and then followed by homebrew Matlab 6.0 as a grey value acquisition. In addition, the overall design of the biosensor was established in abundant AuNPs and g-C 3 N 4 with a two-dimensional structure, affording a bulb-decorated Christmas-tree model. Moreover, with the optimized conditions, the detection limit of the as-proposed biosensor is as low as 10 2 CFU (Colony-Forming Units) mL -1 , exhibiting an increase of two orders of magnitude compared with the traditional immune-gold method. Additionally, the developed visible immunosensor was also successfully applied to the analysis of complicated samples.

Digital detection of multiple minority mutants and expression levels of multiple colorectal cancer-related genes using digital-PCR coupled with bead-array.

PubMed

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed "multiplex ligation-dependent probe amplification-digital amplification coupled with hydrogel bead-array" (MLPA-DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA-DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA-DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.

Practical and regenerable electrochemical aptasensor based on nanoporous gold and thymine-Hg2+-thymine base pairs for Hg2+ detection.

PubMed

Zeng, Guangming; Zhang, Chen; Huang, Danlian; Lai, Cui; Tang, Lin; Zhou, Yaoyu; Xu, Piao; Wang, Hou; Qin, Lei; Cheng, Min

2017-04-15

A simple, practical and reusable electrochemical aptasensor, based on thymine-Hg 2+ -thymine (T-Hg 2+ -T) coordination chemistry and nanoporous gold (NPG) for signal amplification, was designed for sensitive and selective detection of mercury ions (Hg 2+ ). The thiol modified T-rich hairpin capture probe was self-assembled onto the surface of the NPG modified electrode for hybridizing with ferrocene-labeled T-rich probe in the presence of Hg 2+ via T-Hg 2+ -T coordination chemistry. As a result, the hairpin capture probe was opened, and the ferrocene tags were close to the NPG modified electrode. Taking advantage of the amplification effect of NPG electrode for increasing the reaction sites of thiol modified capture probe, the proposed electrochemical aptasensor could detect Hg 2+ quantitatively in the range of 0.01-5000nM, with a detection limit as low as 0.0036nM which is much lower than the maximum contamination level for Hg 2+ in drinking water defined by the U.S. Environmental Protection Agency. Moreover, the proposed electrochemical aptasensor can be regenerated by adding cysteine and Mg 2+ . The aptasensor was also used to detect Hg 2+ from real water samples, and the results showed excellent agreement with the values determined by atomic fluorescence spectrometer. This aptasensor showed a promising potential for on-site detecting Hg 2+ in drinking water. Copyright © 2016 Elsevier B.V. All rights reserved.

Sensitive detection of microRNAs based on the conversion of colorimetric assay into electrochemical analysis with duplex-specific nuclease-assisted signal amplification

PubMed Central

Xia, Ning; Liu, Ke; Zhou, Yingying; Li, Yuanyuan; Yi, Xinyao

2017-01-01

miRNAs have emerged as new biomarkers for the detection of a wide variety of cancers. By employing duplex-specific nuclease for signal amplification and gold nanoparticles (AuNPs) as the carriers of detection probes, a novel electrochemical assay of miRNAs was performed. The method is based on conversion of the well-known colorimetric assay into electrochemical analysis with enhanced sensitivity. DNA capture probes immobilized on the electrode surface and ferrocene (Fc)-labeled DNA detection probes (denoted “Fc-DNA-Fc”) presented in the solution induced the assembly of positively charged AuNPs on the electrode surface through the electrostatic interaction. As a result, a large number of Fc-DNA-Fc molecules were attached on the electrode surface, thus amplifying the electrochemical signal. When duplex-specific nuclease was added to recycle the process of miRNA-initiated digestion of the immobilized DNA probes, Fc-DNA-Fc-induced assembly of AuNPs on the electrode surface could not occur. This resulted in a significant fall in the oxidation current of Fc. The current was found to be inversely proportional to the concentration of miRNAs in the range of 0–25 fM, and a detection limit of 0.1 fM was achieved. Moreover, this work presents a new method for converting colorimetric assays into sensitive electrochemical analyses, and thus would be valuable for design of novel chemical/biosensors. PMID:28761341

MYC gene amplification is a rare event in atypical fibroxanthoma and pleomorphic dermal sarcoma

PubMed Central

Bach, Marisa; Kind, Peter; Helbig, Doris; Quaas, Alexander; Utikal, Jochen; Marx, Alexander; Gaiser, Maria Rita

2018-01-01

Atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS) are rare malignancies typically occurring in elderly patients and predominantly located in skin regions exposed to UV-light. Thus, a role of UV-radiation-induced damage for AFX and PDS tumorigenesis has been postulated. MYC gene amplification has been demonstrated as a distinctive feature of radiation-induced angiosarcoma. In order to investigate whether chronic exposure to UV-light might also lead to MYC copy number changes, 51 AFX and 24 PDS samples were retrospectively analyzed for MYC amplification by fluorescence in situ hybridization using a MYC and a CEP8 gene probe. Of the 44 analyzable AFX samples, one case showed MYC amplification (defined as a MYC/CEP8 ratio ≥2.0), whereas 13 cases demonstrated low level copy number gains (defined as MYC/CEP8 ratio ≥ 1.2−< 2.0). MYC amplification was seen in an AFX sample of extraordinary tumor thickness of 17.5 mm (vs. median 3.25 mm for all samples). Of the 24 PDS cases, five specimen demonstrated MYC low level copy number gains. Immunohistochemically, neither the AFX nor the PDS cases showed MYC protein expression. In summary, these findings rule out that MYC amplification is a major genetic driver in the process of AFX or PDS tumorigenesis. However, MYC amplification may occur as a late event during AFX development and hence might only be detectable in advanced, thick lesions. PMID:29765529

MYC gene amplification is a rare event in atypical fibroxanthoma and pleomorphic dermal sarcoma.

PubMed

Gaiser, Timo; Hirsch, Daniela; Orouji, Azadeh; Bach, Marisa; Kind, Peter; Helbig, Doris; Quaas, Alexander; Utikal, Jochen; Marx, Alexander; Gaiser, Maria Rita

2018-04-20

Atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS) are rare malignancies typically occurring in elderly patients and predominantly located in skin regions exposed to UV-light. Thus, a role of UV-radiation-induced damage for AFX and PDS tumorigenesis has been postulated. MYC gene amplification has been demonstrated as a distinctive feature of radiation-induced angiosarcoma. In order to investigate whether chronic exposure to UV-light might also lead to MYC copy number changes, 51 AFX and 24 PDS samples were retrospectively analyzed for MYC amplification by fluorescence in situ hybridization using a MYC and a CEP8 gene probe. Of the 44 analyzable AFX samples, one case showed MYC amplification (defined as a MYC /CEP8 ratio ≥2.0), whereas 13 cases demonstrated low level copy number gains (defined as MYC /CEP8 ratio ≥ 1.2-< 2.0). MYC amplification was seen in an AFX sample of extraordinary tumor thickness of 17.5 mm (vs. median 3.25 mm for all samples). Of the 24 PDS cases, five specimen demonstrated MYC low level copy number gains. Immunohistochemically, neither the AFX nor the PDS cases showed MYC protein expression. In summary, these findings rule out that MYC amplification is a major genetic driver in the process of AFX or PDS tumorigenesis. However, MYC amplification may occur as a late event during AFX development and hence might only be detectable in advanced, thick lesions.

Diagnostic application of polymerase chain reaction for detection of Ehrlichia risticii in equine monocytic ehrlichiosis (Potomac horse fever).

PubMed

Biswas, B; Mukherjee, D; Mattingly-Napier, B L; Dutta, S K

1991-10-01

Genomic amplification by the polymerase chain reaction (PCR) was used to identify a unique genomic sequence of Ehrlichia risticii directly in DNA isolated from peripheral-blood buffy coat cells of E. risticii-infected horses (Potomac horse fever) and from infected cell cultures. A specific primer pair, selected from a cloned, species-specific, 1-kb DNA fragment of the E. risticii genome as a template, was used for the amplification of the target DNA of 247 bp. The optimal number of 40 PCR cycles, determined by analyzing an amplification profile obtained with a constant Taq polymerase concentration, was used to achieve maximum amplification of the E. risticii DNA segment. Efficient amplification of target DNA was achieved with specimens processed by either the phenol extraction or rapid lysis method. The specificity of the amplified DNA product was confirmed by the proper size (247 bp) and appropriate restriction enzyme cleavage pattern of the amplified target DNA, as well as by the specific hybridization signal obtained by using a PCR-amplified 185-bp internal DNA probe. A 10(5)- to 10(6)-fold amplification of target DNA, which allowed detection of E. risticii from as few as two to three infected cells in culture and from a very small volume of buffy coat cells from infected horses, was achieved. This PCR amplification procedure was found to be highly specific and sensitive for the detection of E. risticii for the study of Potomac horse fever.

De novo amplification within a silent human cholinesterase gene in a family subjected to prolonged exposure to organophosphorus insecticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prody, C.A.; Dreyfus, P.; Soreq, H.

1989-01-01

A 100-fold DNA amplification in the CHE gene, coding for serum butyrylcholinesterase (BtChoEase), was found in a farmer expressing silent CHE phenotype. Individuals homozygous for this gene display a defective serum BtChoEase and are particularly vulnerable to poisoning by agricultural organophosphorus insecticides, to which all members of this family had long been exposed. DNA blot hybridization with regional BtChoEase cDNA probes suggested that the amplification was most intense in regions encoding central sequences within BtChoEase cDNA, whereas distal sequences were amplified to a much lower extent. This is in agreement with the onion skin model, based on amplification of genesmore » in cultured cells and primary tumors. The amplification was absent in the grandparents but present at the same extent in one of their sons and in a grandson, with similar DNA blot hybridization patterns. In situ hybridization experiments localized the amplified sequences to the long arm of chromosome 3, close to the site where the authors previously mapped the CHE gene. Altogether, these observations suggest that the initial amplification event occurred early in embryogenesis, spermatogenesis, or oogenesis, where the CHE gene is intensely active and where cholinergic functioning was indicated to be physiologically necessary. These findings demonstrate a de novo amplification in apparently healthy individuals within an autosomal gene producing a target protein to an inhibitor.« less

Research Advances

ERIC Educational Resources Information Center

King, Angela G.

2004-01-01

Nanotechnology are employed by researchers at Northwestern University to develop a method of labeling disease markers present in blood with unique DNA tags they have dubbed "bio-bar-codes". The preparation of nanoparticle and magnetic microparticle probes and a nanoparticle-based PSR-less DNA amplification scheme are involved by the DNA-BCA assay.

Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase.

PubMed

Wang, Li-Juan; Ren, Ming; Zhang, Qianyi; Tang, Bo; Zhang, Chun-Yang

2017-04-18

Uracil-DNA glycosylase (UDG) is an important base excision repair (BER) enzyme responsible for the repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using excision repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-excision repair, (2) excision repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA repair enzyme-related biomedical research and clinical diagnosis.

Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.

PubMed

Ponchel, Frederique; Toomes, Carmel; Bransfield, Kieran; Leong, Fong T; Douglas, Susan H; Field, Sarah L; Bell, Sandra M; Combaret, Valerie; Puisieux, Alain; Mighell, Alan J; Robinson, Philip A; Inglehearn, Chris F; Isaacs, John D; Markham, Alex F

2003-10-13

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Double-probe signal enhancing strategy for toxin aptasensing based on rolling circle amplification.

PubMed

Tong, Ping; Zhao, Wei-Wei; Zhang, Lan; Xu, Jing-Juan; Chen, Hong-Yuan

2012-03-15

On the basis of aptamer-based rolling circle amplification (RCA) and magnetic beads (MBs), a highly sensitive electrochemical method was developed for the determination of Ochratoxin A (OTA). Initially, an amino-modified capture DNA was immobilized onto MBs for the following hybridization with an OTA aptamer and a phosphate labeled padlock DNA. In the presence of OTA, the aptamer would dissociate from the bioconjugate, and the padlock DNA would subsequently hybridize with the capture DNA to form a circular template with the aid of the T4 ligase. Next, capture DNA would act as primer to initiate a linear RCA reaction and hence generate a long tandem repeated sequences by phi29 DNA polymerase and dNTPs. Then, two quantum dots (QDs) labeled DNA probes were tagged on the resulted RCA product to indicate the OTA recognition event by electrochemical readout. This strategy, based on the novel design of OTA-mediated DNA circularization, the combination of RCA and double signal probes introduction, could detect OTA down to the level of 0.2 pg mL(-1) with a dynamic range spanning more than 4 orders of magnitude. The proposed approach is tested to determine OTA in red wines and shows good application potential in real samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Non-crosslinking gold nanoprobe-LAMP for simple, colorimetric, and specific detection of Salmonella typhi

NASA Astrophysics Data System (ADS)

Bozorgmehr, Ali; Yazdanparast, Razieh; Mollasalehi, Hamidreza

2016-12-01

In this study, we developed a non-crosslinking gold nanoprobe loop-mediated isothermal amplification (LAMP) method for nanodiagnosis of bacterial typhoid fever source, Salmonella typhi. Therefore, a unique region in the S. typhi genomic DNA was targeted for LAMP amplification using a specific set of four precisely designed primers. Also, for specific colorimetric visualization of the amplicons, a thiolated oligonucleotide probe, complementary to the single-stranded loop region of the amplicons between F2 and F1C segments, was designed. The probe was bound to the surface of gold nanoparticles via covalent bonds. Increasing the salt concentration in the detection reaction medium led to aggregation of nanoprobes in the blank and the negative vessels in a time-dependent form. That was followed by a change in the surface plasmon resonance (SPR) leading to blue/black color that was observable by the naked eyes after about 5 min. Meanwhile, the original pink/red color was retained in the positive sample due to the large interparticle spaces and the stability against the ionic strength elevation which persisted for about 30 min. The whole process of DNA extraction, amplification, and detection took less than 2 h with a sensitivity of 20 CFU/ml. The developed gold nanoprobe-LAMP could serve as a simple, rapid, and cost-effective method for nanodiagnosis of S. typhi in point-of-need applications.

Dual-cyclical nucleic acid strand-displacement polymerization based signal amplification system for highly sensitive determination of p53 gene.

PubMed

Xu, Jianguo; Wu, Zai-Sheng; Li, Hongling; Wang, Zhenmeng; Le, Jingqing; Zheng, Tingting; Jia, Lee

2016-12-15

In the present study, we proposed a novel dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) based signal amplification system for highly sensitive determination of tumor suppressor genes. The system primarily consisted of a signaling hairpin probe (SHP), a label-free hairpin probe (LHP) and an initiating primer (IP). The presence of target DNA was able to induce one CNDP through continuous process of ligation, polymerization and nicking, leading to extensively accumulation of two nicked triggers (NT1 and NT2). Intriguingly, the NT1 could directly hybridize SHP, while the NT2 could act as the target analog to induce another CNDP. The resulting dual-CNDP contributed the striking signal amplification, and only a very weak blank noise existed since the ligation template of target was not involved. In this case, the target could be detected in a wide linear range (5 orders of magnitude), and a low detection limit (78 fM) was obtained, which is superior to most of the existing fluorescent methods. Moreover, the dual-CNDP sensing system provided a high selectivity towards target DNA against mismatched target and was successfully applied to analysis of target gene extracted from cancer cells or in human serum-contained samples, indicating its great potential for practical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of HER2/neu gene amplification by competitive pcr using fluorescent melting curve analysis.

PubMed

Lyon, E; Millson, A; Lowery, M C; Woods, R; Wittwer, C T

2001-05-01

Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick.

PubMed

Wang, H; Sun, M; Xu, D; Podok, P; Xie, J; Jiang, Ys; Lu, Lq

2018-05-28

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV-2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV-2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV-2. The highly conserved ORF72 of CyHV-2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA-LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross-reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA-LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV-2 when resources are limited. © 2018 John Wiley & Sons Ltd.

The significance of gtf genes in caries expression: a rapid identification of Streptococcus mutans from dental plaque of child patients.

PubMed

Mishra, Apurva; Pandey, Ramesh K; Manickam, Natesan

2015-01-01

Rapid phylogenetic and functional gene (gtfB) identification of S. mutans from the dental plaque derived from children. Dental plaque collected from fifteen patients of age group 7-12 underwent centrifugation followed by genomic DNA extraction for S. mutans. Genomic DNA was processed with S. mutans specific primers in suitable PCR condtions for phylogenetic and functional gene (gtfB) identification. The yield and results were confirmed by agarose gel electrophoresis. 1% agarose gel electrophoresis depicts the positive PCR amplification at 1,485 bp when compared with standard 1 kbp indicating the presence of S. mutans in the test sample. Another PCR reaction was set using gtfB primers specific for S. mutans for functional gene identification. 1.2% agarose gel electrophoresis was done and a positive amplication was observed at 192 bp when compared to 100 bp standards. With the advancement in molecular biology techniques, PCR based identification and quantification of the bacterial load can be done within hours using species-specific primers and DNA probes. Thus, this technique may reduce the laboratory time spend in conventional culture methods, reduces the possibility of colony identification errors and is more sensitive to culture techniques.

Dual-primer self-generation SERS signal amplification assay for PDGF-BB using label-free aptamer.

PubMed

Ye, SuJuan; Zhai, XiaoMo; Wu, YanYing; Kuang, ShaoPing

2016-05-15

Highly sensitive detection of proteins, especially those associated with cancers, is essential to biomedical research as well as clinical diagnosis. In this work, a simple and novel one-two-three signal amplification surface-enhanced Raman scattering (SERS) method for the detection of protein is fabricated by using label-free aptamer and dual-primer self-generation. Platelet-derived growth factor B-chain (PDGF-BB) is selected as the model protein. The one-two-three cascade DNA amplification means one target-aptamer binding event, two hairpin DNA switches and three DNA amplification reactions. This strategy possesses some remarkable features compared to conventional signal amplification methods: (i) A smart probe including a label-free aptamer is fabricated, for suitable hybridization without hindering the affinity of the aptamer toward its target. (ii) Using the unique structure switch of the aptamer and cooperator, a one-two-three working mode is developed to amplify the SERS signal. The amplification efficiency is enhanced. Given the unique and attractive characteristics, a simple and universal strategy is designed to accomplish ultrasensitive detection of proteins. The detection limit of PDGF-BB via SERS detection is 0.42 pM, with the linear range from 1.0×10(-12)M to 10(-8)M. It is potentially universal because the aptamer can be easily designed for biomolecules whose aptamers undergo similar conformational changes. Copyright © 2015 Elsevier B.V. All rights reserved.

Unknown sequence amplification: Application to in vitro genome walking in Chlamydia trachomatis L2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Copley, C.G.; Boot, C.; Bundell, K.

1991-01-01

A recently described technique, Chemical Genetics' unknown sequence amplification method, which requires only one specific oligonucleotide, has broadened the applicability of the polymerase chain reaction to DNA of unknown sequence. The authors have adapted this technique to the study of the genome of Chlamydia trachomatis, an obligate intracellular bacterium, and describe modifications that significantly improve the utility of this approach. These techniques allow for rapid genomic analysis entirely in vitro, using DNA of limited quantity of purity.

Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa.

PubMed

Manajit, Orapan; Longyant, Siwaporn; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

2018-04-01

Pseudomonas aeruginosa (P. aeruginosa) is an important opportunistic pathogen that causes serious infections in humans, including keratitis in contact lens wearers. Therefore, establishing a rapid, specific and sensitive method for the identification of P. aeruginosa is imperative. In the present study, the uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification combined with nanogold labeled hybridization probe (UDG-LAMP-AuNP) was developed for the detection of P. aeruginosa. UDG-LAMP was performed to prevent carry over contamination and the LAMP reactions can be readily observed using the nanogold probe. A set of 4 primers and a hybridization probe were designed based on the ecfX gene. The UDG-LAMP reactions were performed at 65˚C for 60 min using the ratio of 40% deoxyuridine triphosphate to 60% deoxythymidine triphosphate. The detection of UDG-LAMP products using the nanogold labeled hybridization probe, which appeared as a red-purple color, was examined at 65˚C for 5 min with 40 mM MgSO4. The UDG-LAMP-AuNP demonstrated specificity to all tested isolates of P. aeruginosa without cross reaction to other bacteria. The sensitivity for the detection of pure culture was 1.6x103 colony-forming units (CFU) ml-1 or equivalent to 3 CFU per reaction while that of polymerase chain reaction was 30 CFU per reaction. The detection limit of spiked contact lenses was 1.1x103 CFU ml-1 or equivalent to 2 CFU per reaction. In conclusion, the UDG-LAMP-AuNP assay was rapid, simple, specific and was effective for the identification of P. aeruginosa in contaminated samples.

Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

PubMed Central

Manajit, Orapan; Longyant, Siwaporn; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

2018-01-01

Pseudomonas aeruginosa (P. aeruginosa) is an important opportunistic pathogen that causes serious infections in humans, including keratitis in contact lens wearers. Therefore, establishing a rapid, specific and sensitive method for the identification of P. aeruginosa is imperative. In the present study, the uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification combined with nanogold labeled hybridization probe (UDG-LAMP-AuNP) was developed for the detection of P. aeruginosa. UDG-LAMP was performed to prevent carry over contamination and the LAMP reactions can be readily observed using the nanogold probe. A set of 4 primers and a hybridization probe were designed based on the ecfX gene. The UDG-LAMP reactions were performed at 65°C for 60 min using the ratio of 40% deoxyuridine triphosphate to 60% deoxythymidine triphosphate. The detection of UDG-LAMP products using the nanogold labeled hybridization probe, which appeared as a red-purple color, was examined at 65°C for 5 min with 40 mM MgSO4. The UDG-LAMP-AuNP demonstrated specificity to all tested isolates of P. aeruginosa without cross reaction to other bacteria. The sensitivity for the detection of pure culture was 1.6×103 colony-forming units (CFU) ml−1 or equivalent to 3 CFU per reaction while that of polymerase chain reaction was 30 CFU per reaction. The detection limit of spiked contact lenses was 1.1×103 CFU ml−1 or equivalent to 2 CFU per reaction. In conclusion, the UDG-LAMP-AuNP assay was rapid, simple, specific and was effective for the identification of P. aeruginosa in contaminated samples. PMID:29436623

High-sensitive electrochemical detection of point mutation based on polymerization-induced enzymatic amplification.

PubMed

Feng, Kejun; Zhao, Jingjin; Wu, Zai-Sheng; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

2011-03-15

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection. Copyright © 2010 Elsevier B.V. All rights reserved.

An advanced dual labeled gold nanoparticles probe to detect Cryptosporidium parvum using rapid immuno-dot blot assay.

PubMed

Thiruppathiraja, Chinnasamy; Kamatchiammal, Senthilkumar; Adaikkappan, Periyakaruppan; Alagar, Muthukaruppan

2011-07-15

The zoonotic protozoan parasite Cryptosporidium parvum poses a significant risk to public health. Due to the low infectious dose of C. parvum, remarkably sensitive detection methods are required for water and food industries analysis. However PCR affirmed sensing method of the causative nucleic acid has numerous advantages, still criterion demands for simple techniques and expertise understanding to extinguish its routine use. In contrast, protein based immuno detecting techniques are simpler to perform by a commoner, but lack of sensitivity due to inadequate signal amplification. In this paper, we focused on the development of a mere sensitive immuno detection method by coupling anti-cyst antibody and alkaline phosphatase on gold nanoparticle for C. parvum is described. Outcome of the sensitivity in an immuno-dot blot assay detection is enhanced by 500 fold (using conventional method) and visually be able to detect up to 10 oocysts/mL with minimal processing period. Techniques reported in this paper substantiate the convenience of immuno-dot blot assay for the routine screening of C. parvum in water/environmental examines and most importantly, demonstrates the potential of a prototype development of simple and inexpensive diagnostic technique. Copyright © 2011 Elsevier B.V. All rights reserved.

Rolling circle amplification of metazoan mitochondrialgenomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simison, W. Brian; Lindberg, D.R.; Boore, J.L.

2005-07-31

Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.

[Recombinase Polymerase Amplification and its Applications in Parasite Detection].

PubMed

ZHENG, Wen-bin; WU, Yao-dong; MA, Jian-gang; ZHU, Xing-quan; ZHOU, Dong-hui

2015-10-01

Recombinase polymerase amplification (RPA) is a recently -developed isothermal nucleic-acid-amplification technology that is based on the nucleic acid replication mechanism in T4 bacteriophage. With this technique, nucleic-acid templates can be amplified to measurable levels within 20 min at 37-42 °C. The. RPA process has high sensitivity and specificity, and is simple to operate, thus nucleic acids can be detected rapidly in non-laboratory conditions. Since its development in 2006, the RPA technique has been applied in agriculture, food safety, medicine, transgene detection, etc. In this review, we will give an overview on the research progress of RPA and its application in parasite detection.

Precise and absolute measurements of complex third-order optical susceptibility

NASA Astrophysics Data System (ADS)

Santran, Stephane; Canioni, Lionel; Cardinal, Thierry; Fargin, Evelyne; Le Flem, Gilles; Rouyer, Claude; Sarger, Laurent

2000-11-01

We present precise and absolute measurements of full complex third order optical susceptibility on different fused silica and original glasses composed of tellurium, titanium, niobium erbium. These materials are designed to be the key point for applications ranging form high power laser systems to optoelectronics, their nonlinear index of refraction is a major property and thus must be accurately known. Due to the accuracy and sensitivity of our technique, we have been able to find a large dispersion (more than 30%) of the non linear index of fused silica glasses as a function of their processing mode. On the other hand, measurements on tellurium glasses have shown very strong nonlinearities (40 times higher than fused silica), to be linked to the configurations of their cations and anions. Although the titanium and niobium glasses are less nonlinear, they can be promising matrices for addition of luminescent entities like erbium leading to very interesting laser amplification materials. The experimental set-up is a collinear pump-probe (orthogonally polarized) experiment using transient absorption technique. It is built with around a 100 femtosecond laser oscillator. A fast oscillating delay between the pump and the probe allows us to measure the electronic nonlinearity in quasi real-time. This experiment has the following specifications: an absolute measurement accuracy below 10% mainly due to the laser parameters characterization, a relative measurement accuracy of 1% and a resolution less than 5.10-24m2/V2(50 times less than fused silica).

Chemiluminescence resonance energy transfer imaging on magnetic particles for single-nucleotide polymorphism detection based on ligation chain reaction.

PubMed

Bi, Sai; Zhang, Zhipeng; Dong, Ying; Wang, Zonghua

2015-03-15

A novel ligation chain reaction (LCR) methodology for single-nucleotide polymorphism (SNP) detection was developed based on luminol-H2O2-horseradish peroxidase (HRP)-mimicking DNAzyme-fluorescein chemiluminescence resonance energy transfer (CRET) imaging on magnetic particles. For LCR, four unique target-complement probes (X and X(⁎), YG and Y(⁎)) for the amplification of K-ras (G12C) were designed by modifying G-quadruplex sequence at 3'-end of YG and fluorescein at 5'-end of Y(⁎). After the LCR, the resulting products of XYG/X(⁎)Y(⁎) with biotin-labeled X(⁎) were captured onto streptavidin-coated magnetic particles (SA-MPs) via specific biotin-SA interaction, which stimulated the CRET reaction from hemin/G-quadruplex-catalyzed luminol-H2O2 CL system to fluorescein. By collecting signals by a cooled low-light CCD, a CRET imaging method was proposed for visual detection and quantitative analysis of SNP. As low as 0.86fM mutant DNA was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10,000:1. This high sensitivity and specificity could be attributed to not only the exponential amplification and excellent discrimination of LCR but also the employment of SA-MPs. SA-MPs ensured the feasibility of the proposed strategy, which also simplified the operations through magnetic separation and separated the reaction and detection procedures to improve sensitivity. The proposed LCR-CRET imaging strategy extends the application of signal amplification techniques to SNP detection, providing a promising platform for effective and high-throughput genetic diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

PubMed

Wang, Jianchang; Wang, Jinfeng; Li, Ruiwen; Liu, Libing; Yuan, Wanzhe

2017-08-15

Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R 2 value of the positive results was 0.947. The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

A comparative study of electrical probe techniques for plasma diagnostics

NASA Technical Reports Server (NTRS)

Szuszczewicz, E. P.

1972-01-01

Techniques for using electrical probes for plasma diagnostics are reviewed. Specific consideration is given to the simple Langmuir probe, the symmetric double probe of Johnson and Malter, the variable-area probe of Fetz and Oeschsner, and a floating probe technique. The advantages and disadvantages of each technique are discussed.

Cascade signal amplification for electrochemical immunosensing by integrating biobarcode probes, surface-initiated enzymatic polymerization and silver nanoparticle deposition.

PubMed

Lin, Dajie; Mei, Chengyang; Liu, Aili; Jin, Huile; Wang, Shun; Wang, Jichang

2015-04-15

A cascade signal amplification strategy through combining surface-initiated enzymatic polymerization (SIEP) and the subsequent deposition of strepavidin functionalized silver nanoparticles (AgNPs) was proposed. The first step of constructing the electrochemical immunosensor involves covalently immobilizing capture antibody on a chitosan modified glass carbon electrode, which then catalyzes DNA addition of deoxynucleotides (dNTP) at the 3'-OH group by terminal deoxynucleotidyl transferase (TdT), leading to the formation of long single-stranded DNAs labeled with numerous biotins. Following the deposition of numerous strepavidin functionalized AgNPs on those long DNA chains, electrochemical stripping signal of silver was used to monitor the immunoreaction in KCl solution. Using α-fetoprotein as a model analyte, this amplification strategy could detect fetoprotein down to 0.046pg/mL with a wide linear range from 0.1pg/mL to 1.0ng/mL. The achieved high sensitivity and good reproducibility suggest that this cascade signal amplification strategy has great potential for detecting biological samples and possibly clinical application. Copyright © 2014 Elsevier B.V. All rights reserved.

Soliton-induced relativistic-scattering and amplification.

PubMed

Rubino, E; Lotti, A; Belgiorno, F; Cacciatori, S L; Couairon, A; Leonhardt, U; Faccio, D

2012-01-01

Solitons are of fundamental importance in photonics due to applications in optical data transmission and also as a tool for investigating novel phenomena ranging from light generation at new frequencies and wave-trapping to rogue waves. Solitons are also moving scatterers: they generate refractive index perturbations moving at the speed of light. Here we found that such perturbations scatter light in an unusual way: they amplify light by the mixing of positive and negative frequencies, as we describe using a first Born approximation and numerical simulations. The simplest scenario in which these effects may be observed is within the initial stages of optical soliton propagation: a steep shock front develops that may efficiently scatter a second, weaker probe pulse into relatively intense positive and negative frequency modes with amplification at the expense of the soliton. Our results show a novel all-optical amplification scheme that relies on soliton induced scattering.

Biochip for Real-Time Monitoring of Hepatitis B Virus (HBV) by Combined Loop-Mediated Isothermal Amplification and Solution-Phase Electrochemical Detection

NASA Astrophysics Data System (ADS)

Tien, Bui Quang; Ngoc, Nguyen Thy; Loc, Nguyen Thai; Thu, Vu Thi; Lam, Tran Dai

2017-06-01

Accurate in situ diagnostic tests play a key role in patient management and control of most infectious diseases. To achieve this, use of handheld biochips that implement sample handling, sample analysis, and result readout together is an ideal approach. We present herein a fluid-handling biochip for real-time electrochemical monitoring of nucleic acid amplification based on loop-mediated isothermal amplification and real-time electrochemical detection on a microfluidic platform. Intercalation between amplifying DNA and free redox probe in solution phase was used to monitor the number of DNA copies. The whole diagnostic process is completed within 70 min. Our platform offers a fast and easy tool for quantification of viral pathogens in shorter time and with limited risk of all potential forms of cross-contamination. Such diagnostic tools have potential to make a huge difference to the lives of millions of people worldwide.

Aptamer-aided target capturing with biocatalytic metal deposition: an electrochemical platform for sensitive detection of cancer cells.

PubMed

Yi, Zi; Li, Xiao-Yan; Gao, Qing; Tang, Li-Juan; Chu, Xia

2013-04-07

A novel aptamer biosensor for cancer cell assay has been reported on the basis of ultrasensitive electrochemical detection. Cancer cell capturing is first accomplished via aptamer-aided recognition, and the cell-aptamer binding events then mediate an alkaline phosphatase-catalyzed silver deposition reaction which can be probed by electrochemical detection. Following biocatalytic silver deposition, an efficient amplification approach for sensitive electrochemical measurements is demonstrated, for cell detection with high sensitivity. Ramos cell are used as a model case, a typical biomarker of the acute blood cell cancer, Burkitt's lymphoma. The results reveal that the developed technique displays desirable selectivity in Ramos cell discrimination, and linear response range from 10 to 10(6) cells with a detection limit as low as 10 cells. Due to the simple procedures, label-free and electrochemistry based detection format, this technique is simple and cost-effective, and exhibits excellent compatibility with miniaturization technologies. The electrochemical cell detection strategy may create an intrinsically specific and sensitive platform for cancer cell assay and associated studies.

Simplified Microarray Technique for Identifying mRNA in Rare Samples

NASA Technical Reports Server (NTRS)

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

Two simplified methods of identifying messenger ribonucleic acid (mRNA), and compact, low-power apparatuses to implement the methods, are at the proof-of-concept stage of development. These methods are related to traditional methods based on hybridization of nucleic acid, but whereas the traditional methods must be practiced in laboratory settings, these methods could be practiced in field settings. Hybridization of nucleic acid is a powerful technique for detection of specific complementary nucleic acid sequences, and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes. A traditional microarray study entails at least the following six steps: 1. Purification of cellular RNA, 2. Amplification of complementary deoxyribonucleic acid [cDNA] by polymerase chain reaction (PCR), 3. Labeling of cDNA with fluorophores of Cy3 (a green cyanine dye) and Cy5 (a red cyanine dye), 4. Hybridization to a microarray chip, 5. Fluorescence scanning the array(s) with dual excitation wavelengths, and 6. Analysis of the resulting images. This six-step procedure must be performed in a laboratory because it requires bulky equipment.

NASBA: A detection and amplification system uniquely suited for RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sooknanan, R.; Malek, L.T.

1995-06-01

The invention of PCR (polymerase chain reaction) has revolutionized our ability to amplify and manipulate a nucleic acid sequence in vitro. The commercial rewards of this revolution have driven the development of other nuclei acid amplification and detection methodologies. This has created an alphabet soup of technologies that use different amplification methods, including NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), SDA (strand displacement amplification), QBR (Q-beta replicase), CPR (cycling probe reaction), and bDNA (branched DNA). Despite the differences in their processes, these amplification systems can be separated into two broad categories based on how they achieve their goal:more » sequence-based amplification systems, such as PCR, NASBA, and SDA, amplify a target nucleic acid sequence. Signal-based amplification systems, such as LCR, QBR, CPR and bDNA, amplify or alter a signal from a detection reaction that is target-dependent. While the various methods have relative strengths and weaknesses, only NASBA offers the unique ability to homogeneously amplify an RNA analyte in the presence of homologous genomic DNA under isothermal conditions. Since the detection of RNA sequences almost invariably measures biological activity, it is an excellent prognostic indicator of activities as diverse as virus production, gene expression, and cell viability. The isothermal nature of the reaction makes NASBA especially suitable for large-scale manual screening. These features extend NASBA`s application range from research to commercial diagnostic applications. Field test kits are presently under development for human diagnostics as well as the burgeoning fields of food and environmental diagnostic testing. These developments suggest future integration of NASBA into robotic workstations for high-throughput screening as well. 17 refs., 1 tab.« less

Amplification and Attenuation Across USArray Using Ambient Noise Wavefront Tracking

NASA Astrophysics Data System (ADS)

Bowden, Daniel C.; Tsai, Victor C.; Lin, Fan-Chi

2017-12-01

As seismic traveltime tomography continues to be refined using data from the vast USArray data set, it is advantageous to also exploit the amplitude information carried by seismic waves. We use ambient noise cross correlation to make observations of surface wave amplification and attenuation at shorter periods (8-32 s) than can be observed with only traditional teleseismic earthquake sources. We show that the wavefront tracking approach can be successfully applied to ambient noise correlations, yielding results quite similar to those from earthquake observations at periods of overlap. This consistency indicates that the wavefront tracking approach is viable for use with ambient noise correlations, despite concerns of the inhomogeneous and unknown distribution of noise sources. The resulting amplification and attenuation maps correlate well with known tectonic and crustal structure; at the shortest periods, our amplification and attenuation maps correlate well with surface geology and known sedimentary basins, while our longest period amplitudes are controlled by crustal thickness and begin to probe upper mantle materials. These amplification and attenuation observations are sensitive to crustal materials in different ways than traveltime observations and may be used to better constrain temperature or density variations. We also value them as an independent means of describing the lateral variability of observed Rayleigh wave amplitudes without the need for 3-D tomographic inversions.

Whole Genome Amplification of Labeled Viable Single Cells Suited for Array-Comparative Genomic Hybridization.

PubMed

Kroneis, Thomas; El-Heliebi, Amin

2015-01-01

Understanding details of a complex biological system makes it necessary to dismantle it down to its components. Immunostaining techniques allow identification of several distinct cell types thereby giving an inside view of intercellular heterogeneity. Often staining reveals that the most remarkable cells are the rarest. To further characterize the target cells on a molecular level, single cell techniques are necessary. Here, we describe the immunostaining, micromanipulation, and whole genome amplification of single cells for the purpose of genomic characterization. First, we exemplify the preparation of cell suspensions from cultured cells as well as the isolation of peripheral mononucleated cells from blood. The target cell population is then subjected to immunostaining. After cytocentrifugation target cells are isolated by micromanipulation and forwarded to whole genome amplification. For whole genome amplification, we use GenomePlex(®) technology allowing downstream genomic analysis such as array-comparative genomic hybridization.

A new class of homogeneous nucleic acid probes based on specific displacement hybridization

PubMed Central

Li, Qingge; Luan, Guoyan; Guo, Qiuping; Liang, Jixuan

2002-01-01

We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but they become fluorescent upon displacement hybridization with the target. These probes display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. A comparison of double-stranded probes with corresponding linear probes confirms that the presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant. PMID:11788731

ProbeDesigner: for the design of probesets for branched DNA (bDNA) signal amplification assays.

PubMed

Bushnell, S; Budde, J; Catino, T; Cole, J; Derti, A; Kelso, R; Collins, M L; Molino, G; Sheridan, P; Monahan, J; Urdea, M

1999-05-01

The sensitivity and specificity of branched DNA (bDNA) assays are derived in part through the judicious design of the capture and label extender probes. To minimize non-specific hybridization (NSH) events, which elevate assay background, candidate probes must be computer screened for complementarity with generic sequences present in the assay. We present a software application which allows for rapid and flexible design of bDNA probesets for novel targets. It includes an algorithm for estimating the magnitude of NSH contribution to background, a mechanism for removing probes with elevated contributions, a methodology for the simultaneous design of probesets for multiple targets, and a graphical user interface which guides the user through the design steps. The program is available as a commercial package through the Pharmaceutical Drug Discovery program at Chiron Diagnostics.

Diagnostic devices for isothermal nucleic acid amplification.

PubMed

Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

2012-01-01

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Diagnostic Devices for Isothermal Nucleic Acid Amplification

PubMed Central

Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

2012-01-01

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development. PMID:22969402

Applications of Flow Cytometry to Clinical Microbiology†

PubMed Central

Álvarez-Barrientos, Alberto; Arroyo, Javier; Cantón, Rafael; Nombela, César; Sánchez-Pérez, Miguel

2000-01-01

Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory. PMID:10755996

Specific 16S ribosomal RNA targeted oligonucleotide probe against Clavibacter michiganensis subsp. sepedonicus.

PubMed

Mirza, M S; Rademaker, J L; Janse, J D; Akkermans, A D

1993-11-01

In this article we report on the polymerase chain reaction amplification of a partial 16S rRNA gene from the plant pathogenic bacterium Clavibacter michiganensis subsp. sepedonicus. A partial sequence (about 400 base pairs) of the gene was determined that covered two variable regions important for oligonucleotide probe development. A specific 24mer oligonucleotide probe targeted against the V6 region of 16S rRNA was designed. Specificity of the probe was determined using dot blot hybridization. Under stringent conditions (60 degrees C), the probe hybridized with all 16 Cl. michiganensis subsp. sepedonicus strains tested. Hybridization did not occur with 32 plant pathogenic and saprophytic bacteria used as controls under the same conditions. Under less stringent conditions (55 degrees C) the related Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. nebraskensis, and Clavibacter michiganensis subsp. tesselarius also showed hybridization. At even lower stringency (40 degrees C), all Cl. michiganensis subspecies tested including Clavibacter michiganensis subsp. michiganensis showed hybridization signal, suggesting that under these conditions the probe may be used as a species-specific probe for Cl. michiganensis.

High sensitive and direct fluorescence detection of single viral DNA sequences by integration of double strand probes onto microgels particles.

PubMed

Aliberti, A; Cusano, A M; Battista, E; Causa, F; Netti, P A

2016-02-21

A novel class of probes for fluorescence detection was developed and combined to microgel particles for a high sensitive fluorescence detection of nucleic acids. A double strand probe with an optimized fluorescent-quencher couple was designed for the detection of different lengths of nucleic acids (39 nt and 100 nt). Such probe proved efficient in target detection in different contests and specific even in presence of serum proteins. The conjugation of double strand probes onto polymeric microgels allows for a sensitive detection of DNA sequences from HIV, HCV and SARS corona viruses with a LOD of 1.4 fM, 3.7 fM and 1.4 fM, respectively, and with a dynamic range of 10(-9)-10(-15) M. Such combination enhances the sensitivity of the detection of almost five orders of magnitude when compared to the only probe. The proposed platform based on the integration of innovative double strand probe into microgels particles represents an attractive alternative to conventional sensitive DNA detection technologies that rely on amplifications methods.

How could you use the ergonomics 'knowhow' transfer management to enhance human working for sustainable improvements in industrially developing countries?

PubMed

Helali, Faramarz

2012-01-01

This paper describes the different strategic understanding from getting ergonomics intervention programmes' conversations to 'Tip', including minimizing strategies; tipping point strategies; and maximizing strategies from building ergonomics intervention techniques. Those have indicated to different recognitions: 1) when amplification of the 'problem' is necessary; 2) when amplification of the 'tipping point' is necessary, and 3) when amplification of the 'success' is necessary. The practical applications and implications of the ergonomics intervention techniques are drawn from the findings of framing positive questions: 1) what is successful ergonomics intervention technique right now (Appreciative)? 2) What do we need to change for a better future (Imagine)? 3) How do we do this (Design)? 4) Who takes action and with what consequences (Act)? This requires re-framing of the ergonomics intervention techniques in an appreciative way, because of, the future action needs to be inspired by those things that participants feel are worth valuing, worth celebrating and sustaining.

Exonuclease III-Assisted Upconversion Resonance Energy Transfer in a Wash-Free Suspension DNA Assay.

PubMed

Chen, Yinghui; Duong, Hien T T; Wen, Shihui; Mi, Chao; Zhou, Yingzhu; Shimoni, Olga; Valenzuela, Stella M; Jin, Dayong

2018-01-02

Sensitivity is the key in optical detection of low-abundant analytes, such as circulating RNA or DNA. The enzyme Exonuclease III (Exo III) is a useful tool in this regard; its ability to recycle target DNA molecules results in markedly improved detection sensitivity. Lower limits of detection may be further achieved if the detection background of autofluorescence can be removed. Here we report an ultrasensitive and specific method to quantify trace amounts of DNA analytes in a wash-free suspension assay. In the presence of target DNA, the Exo III recycles the target DNA by selectively digesting the dye-tagged sequence-matched probe DNA strand only, so that the amount of free dye removed from the probe DNA is proportional to the number of target DNAs. Remaining intact probe DNAs are then bound onto upconversion nanoparticles (energy donor), which allows for upconversion luminescence resonance energy transfer (LRET) that can be used to quantify the difference between the free dye and tagged dye (energy acceptor). This scheme simply avoids both autofluorescence under infrared excitation and many tedious washing steps, as the free dye molecules are physically located away from the nanoparticle surface, and as such they remain "dark" in suspension. Compared to alternative approaches requiring enzyme-assisted amplification on the nanoparticle surface, introduction of probe DNAs onto nanoparticles only after DNA hybridization and signal amplification steps effectively avoids steric hindrance. Via this approach, we have achieved a detection limit of 15 pM in LRET assays of human immunodeficiency viral DNA.

Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Arakawa, C.K.; Deering, R.E.; Higman, K.H.; Oshima, K.H.; O'Hara, P.J.; Winton, J.R.

1990-01-01

The polymerase chain reaction [PCR) was used to amplify a portion of the nucleoprotein [NI gene of infectious hematopoietic necrosis virus (IHNV). Using a published sequence for the Round Butte isolate of IHNV, a pair of PCR pnmers was synthesized that spanned a 252 nucleotide region of the N gene from residue 319 to residue 570 of the open reading frame. This region included a 30 nucleotide target sequence for a synthetic oligonucleotide probe developed for detection of IHNV N gene messenger RNA. After 25 cycles of amplification of either messenger or genomic RNA, the PCR product (DNA) of the expected size was easily visible on agarose gels stained with ethidium bromide. The specificity of the amplified DNA was confirmed by Southern and dot-blot analysis using the biotinylated oligonucleotide probe. The PCR was able to amplify the N gene sequence of purified genomic RNA from isolates of IHNV representing 5 different electropherotypes. Using the IHNV primer set, no PCR product was obtained from viral hemorrhagic septicemia virus RNA, but 2 higher molecular weight products were synthesized from hirame rhabdovirus RNA that did not hybridize with the biotinylated probe. The PCR could be efficiently performed with all IHNV genomic RNA template concentrations tested (1 ng to 1 pg). The lowest level of sensitivity was not determined. The PCR was used to amplify RNA extracted from infected cell cultures and selected tissues of Infected rainbow trout. The combination of PCR and nucleic acid probe promises to provide a detection method for IHNV that is rapid, h~ghly specific, and sensitive.

Optical pulse synthesis using brillouin selective sideband amplification

NASA Technical Reports Server (NTRS)

Yao, X. Steve (Inventor)

2002-01-01

Techniques for producing optical pulses based on Brillouin selective sideband amplification by using a common modulation control signal to modulate both a signal beam to produce multiple sideband signals and a single pump beam to produce multiple pump beams.

Multiple displacement amplification on single cell and possible PGD applications.

PubMed

Hellani, Ali; Coskun, Serdar; Benkhalifa, Moncef; Tbakhi, Abelghani; Sakati, Nadia; Al-Odaib, Ali; Ozand, Pinar

2004-11-01

Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. We used MDA to amplify the whole genome directly from a single cell. The most common techniques used in PGD are PCR and fluorescent in-situ hybridization (FISH). There are many limitations to these techniques including, the number of chromosomes diagnosed for FISH or the quality of DNA issued from a single cell PCR. This report shows, for the first time, use of MDA for single cell whole genome amplification. A total of 16 short tandem repeats (STRs) were amplified successfully with a similar pattern to the genomic DNA. Furthermore, allelic drop out (ADO) derived from MDA was assessed in 40 single cells by analysing (i) heterozygosity for a known beta globin mutation (IVSI-5 C-G) and by studying (ii) the heterozygous loci present in the STRs. ADO turned out to be 10.25% for the beta globin gene sequencing and 5% for the fluorescent PCR analysis of STRs. Moreover, the amplification accuracy of MDA permitted the detection of trisomy 21 on a single cell using comparative genome hybridization-array. Altogether, these data suggest that MDA can be used for single cell molecular karyotyping and the diagnosis of any single gene disorder in PGD.

Energy transfer of nucleic acid products

NASA Astrophysics Data System (ADS)

Jung, Paul M.; Hu, Hsiang-Yun; Khalil, Omar S.

1995-04-01

Fluorescence energy transfer was investigated as a homogeneous detection method for the gapped ligase chain reaction (G-LCR). Oligonucleotides of a Chlamydia trachomatic G-LCR probe set were labeled with fluorescein as the donor and Texas Red as the acceptor fluorophore. Amplification and detection of 10 molecules of synthetic target was demonstrated in spiked urine samples.

RNA splicing process analysis for identifying antisense oligonucleotide inhibitors with padlock probe-based isothermal amplification.

PubMed

Ren, Xiaojun; Deng, Ruijie; Wang, Lida; Zhang, Kaixiang; Li, Jinghong

2017-08-01

RNA splicing, which mainly involves two transesterification steps, is a fundamental process of gene expression and its abnormal regulation contributes to serious genetic diseases. Antisense oligonucleotides (ASOs) are genetic control tools that can be used to specifically control genes through alteration of the RNA splicing pathway. Despite intensive research, how ASOs or various other factors influence the multiple processes of RNA splicing still remains obscure. This is largely due to an inability to analyze the splicing efficiency of each step in the RNA splicing process with high sensitivity. We addressed this limitation by introducing a padlock probe-based isothermal amplification assay to achieve quantification of the specific products in different splicing steps. With this amplified assay, the roles that ASOs play in RNA splicing inhibition in the first and second steps could be distinguished. We identified that 5'-ASO could block RNA splicing by inhibiting the first step, while 3'-ASO could block RNA splicing by inhibiting the second step. This method provides a versatile tool for assisting efficient ASO design and discovering new splicing modulators and therapeutic drugs.

SPR-DNA array for detection of methicillin-resistant Staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification.

PubMed

Nawattanapaiboon, Kawin; Kiatpathomchai, Wansika; Santanirand, Pitak; Vongsakulyanon, Apirom; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Srikhirin, Toemsak

2015-12-15

In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies/µl and LAMP-SPR sensing system showed a good selectivity toward the MRSA. Copyright © 2015 Elsevier B.V. All rights reserved.

High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.

PubMed

Andeer, Peter; Strand, Stuart E; Stahl, David A

2012-01-01

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of Orf virus.

PubMed

Yang, Yang; Qin, Xiaodong; Wang, Guangxiang; Zhang, Yuen; Shang, Youjun; Zhang, Zhidong

2015-12-02

Orf virus (ORFV) is the causative agent of Orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. The rapid detection of ORFV is of great importance in disease control and highly needed. A isothermal molecular diagnostic approach, termed recombinase polymerase amplification (RPA), is considered as an novel and rapid alternative techonology to PCR assay. In the present study, a novel fluorescent probe based on RPA assay (ORFV exo RPA assay) was developed. The developed ORFV exo RPA assay was capable of as low as 100 copies of ORFV DNA /reaction and was highly specific, with no cross-reaction with closely related viruses (capripox virus, foot-and-mouth disease virus or peste des petits ruminants virus). Further assessment with clinical samples showed that the developed ORFV exo RPA assay has good correlation with qPCR assays for detection of ORFV. These results suggest that the developed ORFV exo RPA assay is suitable for rapid detection of ORFV.

Use of Multiple Displacement Amplification as Pre-polymerase Chain Reaction (Pre-PCR) to amplify genomic DNA of siphonapterids preserved for long periods in scientific collections.

PubMed

Avelar, Daniel M; Linardi, Pedro M

2010-09-15

The recently developed Multiple Displacement Amplification technique (MDA) allows for the production of a large quantity of high quality genomic DNA from low amounts of the original DNA. The goal of this study was to evaluate the performance of the MDA technique to amplify genomic DNA of siphonapterids that have been stored for long periods in 70% ethanol at room temperature. We subjected each DNA sample to two different methodologies: (1) amplification of mitochondrial 16S sequences without MDA; (2) amplification of 16S after MDA. All the samples obtained from these procedures were then sequenced. Only 4 samples (15.4%) subjected to method 1 showed amplification. In contrast, the application of MDA (method 2) improved the performance substantially, with 24 samples (92.3%) showing amplification, with significant difference. Interestingly, one of the samples successfully amplified with this method was originally collected in 1909. All of the sequenced samples displayed satisfactory results in quality evaluations (Phred ≥ 20) and good similarities, as identified with the BLASTn tool. Our results demonstrate that the use of MDA may be an effective tool in molecular studies involving specimens of fleas that have traditionally been considered inadequately preserved for such purposes.

Use of Multiple Displacement Amplification as Pre-polymerase Chain Reaction (Pre-PCR) to amplify genomic DNA of siphonapterids preserved for long periods in scientific collections

PubMed Central

2010-01-01

The recently developed Multiple Displacement Amplification technique (MDA) allows for the production of a large quantity of high quality genomic DNA from low amounts of the original DNA. The goal of this study was to evaluate the performance of the MDA technique to amplify genomic DNA of siphonapterids that have been stored for long periods in 70% ethanol at room temperature. We subjected each DNA sample to two different methodologies: (1) amplification of mitochondrial 16S sequences without MDA; (2) amplification of 16S after MDA. All the samples obtained from these procedures were then sequenced. Only 4 samples (15.4%) subjected to method 1 showed amplification. In contrast, the application of MDA (method 2) improved the performance substantially, with 24 samples (92.3%) showing amplification, with significant difference. Interestingly, one of the samples successfully amplified with this method was originally collected in 1909. All of the sequenced samples displayed satisfactory results in quality evaluations (Phred ≥ 20) and good similarities, as identified with the BLASTn tool. Our results demonstrate that the use of MDA may be an effective tool in molecular studies involving specimens of fleas that have traditionally been considered inadequately preserved for such purposes. PMID:20840790

Pyrosequencing®-Based Identification of Low-Frequency Mutations Enriched Through Enhanced-ice-COLD-PCR.

PubMed

How-Kit, Alexandre; Tost, Jörg

2015-01-01

A number of molecular diagnostic assays have been developed in the last years for mutation detection. Although these methods have become increasingly sensitive, most of them are incompatible with a sequencing-based readout and require prior knowledge of the mutation present in the sample. Consequently, coamplification at low denaturation (COLD)-PCR-based methods have been developed and combine a high analytical sensitivity due to mutation enrichment in the sample with the identification of known or unknown mutations by downstream sequencing experiments. Among these methods, the recently developed Enhanced-ice-COLD-PCR appeared as the most powerful method as it outperformed the other COLD-PCR-based methods in terms of the mutation enrichment and due to the simplicity of the experimental setup of the assay. Indeed, E-ice-COLD-PCR is very versatile as it can be used on all types of PCR platforms and is applicable to different types of samples including fresh frozen, FFPE, and plasma samples. The technique relies on the incorporation of an LNA containing blocker probe in the PCR reaction followed by selective heteroduplex denaturation enabling amplification of the mutant allele while amplification of the wild-type allele is prevented. Combined with Pyrosequencing(®), which is a very quantitative high-resolution sequencing technology, E-ice-COLD-PCR can detect and identify mutations with a limit of detection down to 0.01 %.

Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick

PubMed Central

Zhao, Guimin; Wang, Hongmei; Hou, Peili; He, Chengqiang

2018-01-01

Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39℃ with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings. PMID:29284204

Trypanosomatidae: Phytomonas detection in plants and phytophagous insects by PCR amplification of a genus-specific sequence of the spliced leader gene.

PubMed

Serrano, M G; Nunes, L R; Campaner, M; Buck, G A; Camargo, E P; Teixeira, M M

1999-03-01

In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes. Copyright 1999 Academic Press.

Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick.

PubMed

Zhao, Guimin; Wang, Hongmei; Hou, Peili; He, Chengqiang; He, Hongbin

2018-03-31

Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis . First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39°C with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis . Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.

Prevalence and spectrum of large deletions or duplications in the major long QT syndrome-susceptibility genes and implications for long QT syndrome genetic testing.

PubMed

Tester, David J; Benton, Amber J; Train, Laura; Deal, Barbara; Baudhuin, Linnea M; Ackerman, Michael J

2010-10-15

Long QT syndrome (LQTS) is a cardiac channelopathy associated with syncope, seizures, and sudden death. Approximately 75% of LQTS is due to mutations in genes encoding for 3 cardiac ion channel α-subunits (LQT1 to LQT3). However, traditional mutational analyses have limited detection capabilities for atypical mutations such as large gene rearrangements. We set out to determine the prevalence and spectrum of large deletions/duplications in the major LQTS-susceptibility genes in unrelated patients who were mutation negative after point mutation analysis of LQT1- to LQT12-susceptibility genes. Forty-two unrelated, clinically strong LQTS patients were analyzed using multiplex ligation-dependent probe amplification, a quantitative fluorescent technique for detecting multiple exon deletions and duplications. The SALSA multiplex ligation-dependent probe amplification LQTS kit from MRC-Holland was used to analyze the 3 major LQTS-associated genes, KCNQ1, KCNH2, and SCN5A, and the 2 minor genes, KCNE1 and KCNE2. Overall, 2 gene rearrangements were found in 2 of 42 unrelated patients (4.8%, confidence interval 1.7 to 11). A deletion of KCNQ1 exon 3 was identified in a 10-year-old Caucasian boy with a corrected QT duration of 660 ms, a personal history of exercise-induced syncope, and a family history of syncope. A deletion of KCNQ1 exon 7 was identified in a 17-year-old Caucasian girl with a corrected QT duration of 480 ms, a personal history of exercise-induced syncope, and a family history of sudden cardiac death. In conclusion, because nearly 5% of patients with genetically elusive LQTS had large genomic rearrangements involving the canonical LQTS-susceptibility genes, reflex genetic testing to investigate genomic rearrangements may be of clinical value. Copyright © 2010 Elsevier Inc. All rights reserved.

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

PubMed Central

Zhou, Xiaoming; Xing, Da; Tang, Yonghong; Chen, Wei R.

2009-01-01

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids. PMID:19956680

Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

PubMed Central

Rodríguez-Lázaro, David; D'Agostino, Martin; Pla, Maria; Cook, Nigel

2004-01-01

An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5′ end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of Mycobacterium avium subsp. paratuberculosis is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results. PMID:15583319

A three-phase amplification of the cosmic magnetic field in galaxies

NASA Astrophysics Data System (ADS)

Martin-Alvarez, Sergio; Devriendt, Julien; Slyz, Adrianne; Teyssier, Romain

2018-06-01

Arguably the main challenge of galactic magnetism studies is to explain how the interstellar medium of galaxies reaches energetic equipartition despite the extremely weak cosmic primordial magnetic fields that are originally predicted to thread the inter-galactic medium. Previous numerical studies of isolated galaxies suggest that a fast dynamo amplification might suffice to bridge the gap spanning many orders of magnitude in strength between the weak early Universe magnetic fields and the ones observed in high redshift galaxies. To better understand their evolution in the cosmological context of hierarchical galaxy growth, we probe the amplification process undergone by the cosmic magnetic field within a spiral galaxy to unprecedented accuracy by means of a suite of constrained transport magnetohydrodynamical adaptive mesh refinement cosmological zoom simulations with different stellar feedback prescriptions. A galactic turbulent dynamo is found to be naturally excited in this cosmological environment, being responsible for most of the amplification of the magnetic energy. Indeed, we find that the magnetic energy spectra of simulated galaxies display telltale inverse cascades. Overall, the amplification process can be divided in three main phases, which are related to different physical mechanisms driving galaxy evolution: an initial collapse phase, an accretion-driven phase, and a feedback-driven phase. While different feedback models affect the magnetic field amplification differently, all tested models prove to be subdominant at early epochs, before the feedback-driven phase is reached. Thus the three-phase evolution paradigm is found to be quite robust vis-a-vis feedback prescriptions.

Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification.

PubMed

Sabaté Del Río, Jonathan; Steylaerts, Tim; Henry, Olivier Y F; Bienstman, Peter; Stakenborg, Tim; Van Roy, Wim; O'Sullivan, Ciara K

2015-11-15

In this work we present the use of a silicon-on-insulator (SOI) chip featuring an array of 64 optical ring resonators used as refractive index sensors for real-time and label-free DNA detection. Single ring functionalisation was achieved using a click reaction after precise nanolitre spotting of specific hexynyl-terminated DNA capture probes to link to an azido-silanised chip surface. To demonstrate detectability using the ring resonators and to optimise conditions for solid-phase amplification, hybridisation between short 25-mer single stranded DNA (ssDNA) fragments and a complementary capture probe immobilised on the surface of the ring resonators was carried out and detected through the shift in the resonant wavelength. Using the optimised conditions demonstrated via the solid-phase hybridisation, a 144-bp double stranded DNA (dsDNA) was then detected directly using recombinase and polymerase proteins through on-chip target amplification and solid-phase elongation of immobilised forward primers on specific rings, at a constant temperature of 37°C and in less than 60min, achieving a limit of detection of 7.8·10(-13)M (6·10(5) copies in 50µL). The use of an automatic liquid handler injection instrument connected to an integrated resealable chip interface (RCI) allowed programmable multiple injection protocols. Air plugs between different solutions were introduced to prevent intermixing and a proportional-integral-derivative (PID) temperature controller minimised temperature based drifts. Published by Elsevier B.V.

Terahertz spectroscopy for the isothermal detection of bacterial DNA by magnetic bead-based rolling circle amplification.

PubMed

Yang, Xiang; Yang, Ke; Zhao, Xiang; Lin, Zhongquan; Liu, Zhiyong; Luo, Sha; Zhang, Yang; Wang, Yunxia; Fu, Weiling

2017-12-04

The demand for rapid and sensitive bacterial detection is continuously increasing due to the significant requirements of various applications. In this study, a terahertz (THz) biosensor based on rolling circle amplification (RCA) was developed for the isothermal detection of bacterial DNA. The synthetic bacterium-specific sequence of 16S rDNA hybridized with a padlock probe (PLP) that contains a sequence fully complementary to the target sequence at the 5' and 3' ends. The linear PLP was circularized by ligation to form a circular PLP upon recognition of the target sequence; then the capture probe (CP) immobilized on magnetic beads (MBs) acted as a primer to initialize RCA. As DNA molecules are much less absorptive than water molecules in the THz range, the RCA products on the surface of the MBs cause a significant decrease in THz absorption, which can be sensitively probed by THz spectroscopy. Our results showed that 0.12 fmol of synthetic bacterial DNA and 0.05 ng μL -1 of genomic DNA could be effectively detected using this assay. In addition, the specificity of this strategy was demonstrated by its low signal response to interfering bacteria. The proposed strategy not only represents a new method for the isothermal detection of the target bacterial DNA but also provides a general methodology for sensitive and specific DNA biosensing using THz spectroscopy.

[Multiplex Ligation - dependent Probe Amplification (MLPA) as a screening test in children with developmental defects and intellectual disability of unknown etiology].

PubMed

Laczmańska, Izabela; Jakubiak, Aleksandra; Slęzak, Ryszard; Pesz, Karolina; Stembalska, Agnieszka; Laczmański, Lukasz; Sąsiadek, Maria M; Smigiel, Robert

2011-01-01

Developmental delay and intellectual disability are significant medical and social problems which concern 1-3% of population. The etiology remains unknown in over half of the cases. To evaluate the efficiency of MLPA (Multiplex Ligation-dependent Probe Amplification) as a screening test in diagnosis of patients with developmental delay and/or intellectual disability. 313 MLPA tests were performed in 256 patients with developmental delay and/ or intellectual disability with unknown etiology. MLPA test was made after exclusion of genetic disorders possible to diagnose by dysmorphological examination or using specifi c genetic tests. Positive results were confirmed by FISH analysis with appropriate probes. Chromosomal microaberrations were identifi ed in 15 patients (4,8%): deletions of 1p36 in 4 cases, in one case deletion of 22q11.21, 22q13.33, SNRPN1, 4ptel, 6qtel, 7q11.23, 16ptel, 18qtel as well as one ca se of deletion 3ptel/duplication 15qtel; deletion 18qtel/duplication Xqtel, and also duplication 7q11.23. Detail clinical analysis was performed in patients with diagnosed microaberrations in MLPA test. The molecular MLPA test, screening for chromosomal microaberration syndromes, should be performed in each patient with developmental delay and/or intellectual disability of unknown etiology and normal cytogenetic analysis, even if congenital defects and positive familial history do not exist.

Identification of salivary Lactobacillus rhamnosus species by DNA profiling and a specific probe.

PubMed

Richard, B; Groisillier, A; Badet, C; Dorignac, G; Lonvaud-Funel, A

2001-03-01

The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.

Establishment of a universal and rational gene detection strategy through three-way junction-based remote transduction.

PubMed

Tang, Yidan; Lu, Baiyang; Zhu, Zhentong; Li, Bingling

2018-01-21

The polymerase chain reaction and many isothermal amplifications are able to achieve super gene amplification. Unfortunately, most commonly-used transduction methods, such as dye staining and Taqman-like probing, still suffer from shortcomings including false signals or difficult probe design, or are incompatible with multi-analysis. Here a universal and rational gene detection strategy has been established by translating isothermal amplicons to enzyme-free strand displacement circuits via three-way junction-based remote transduction. An assistant transduction probe was imported to form a partial hybrid with the target single-stranded nucleic acid. After systematic optimization the hybrid could serve as an associative trigger to activate a downstream circuit detector via a strand displacement reaction across the three-way junction. By doing so, the detection selectivity can be double-guaranteed through both amplicon-transducer recognition and the amplicon-circuit reaction. A well-optimized circuit can be immediately applied to a new target detection through simply displacing only 10-12 nt on only one component, according to the target. More importantly, this property for the first time enables multi-analysis and logic-analysis in a single reaction, sharing a single fluorescence reporter. In an applicable model, trace amounts of Cronobacter and Enterobacteria genes have been clearly distinguished from samples with no bacteria or one bacterium, with ultra-high sensitivity and selectivity.

Broad-Spectrum Molecular Detection of Fungal Nucleic Acids by PCR-Based Amplification Techniques.

PubMed

Czurda, Stefan; Lion, Thomas

2017-01-01

Over the past decade, the incidence of life-threatening invasive fungal infections has dramatically increased. Infections caused by hitherto rare and emerging fungal pathogens are associated with significant morbidity and mortality among immunocompromised patients. These observations render the coverage of a broad range of clinically relevant fungal pathogens highly important. The so-called panfungal or, perhaps more correctly, broad-range nucleic acid amplification techniques do not only facilitate sensitive detection of all clinically relevant fungal species but are also rapid and can be applied to analyses of any patient specimens. They have therefore become valuable diagnostic tools for sensitive screening of patients at risk of invasive fungal infections. This chapter summarizes the currently available molecular technologies employed in testing of a wide range of fungal pathogens, and provides a detailed workflow for patient screening by broad-spectrum nucleic acid amplification techniques.

Detection of MET amplification in gastroesophageal tumor specimens using IQFISH

PubMed Central

Nielsen, Karsten Bork; Mollerup, Jens; Jepsen, Anna; Go, Ning

2017-01-01

Background The gene mesenchymal epithelial transition factor (MET) is a proto-oncogene that encodes a transmembrane receptor with intrinsic tyrosine kinase activity known as Met or cMet. MET is found to be amplified in several human cancers including gastroesophageal cancer. Methods Here we report the MET amplification prevalence data from 159 consecutive tumor specimens from patients with gastric (G), gastroesophageal junction (GEJ) and esophageal (E) adenocarcinoma, using a novel fluorescence in situ hybridization (FISH) assay, MET/CEN-7 IQFISH Probe Mix [an investigational use only (IUO) assay]. MET amplification was defined as a MET/CEN-7 ratio ≥2.0. Furthermore, the link between the MET signal distribution and amplification status was investigated. Results The prevalence of MET amplification was found to be 6.9%. The FISH assay demonstrated a high inter-observer reproducibility. The inter-observer results showed a 100% overall agreement with respect to the MET status (amplified/non-amplified). The inter-observer CV was estimated to 11.8% (95% CI: 10.2–13.4). For the signal distribution, the inter-observer agreement was reported to be 98.7%. We also report an association of MET amplification and a unique signal distribution pattern in the G/GEJ/E tumor specimens. We found that the prevalence of MET amplification was markedly higher in tumors specimens with a heterogeneous (66.7%) versus homogeneous (2.0%) signal distribution. Furthermore, specimens with a heterogeneous signal distribution had a statically significantly higher median MET/CEN-7 ratio (2.35 versus 1.04; P<0.0001). Conclusions The novel FISH assay showed a high inter-observer reproducibility both with respect to amplification status and signal distribution. Based on the finding in the study it is suggested that MET amplification mainly is associated with tumor cells that is represented by a heterogonous growth pattern. PMID:29285491

Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

PubMed Central

Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

2016-01-01

Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non-AHPND bacteria commonly found in shrimp ponds (including other Vibrio species). The new method significantly reduced the time, difficulty and cost for molecular detection of VPAHPND in shrimp hatchery and farm settings. PMID:27003504

New Signal Amplification Strategy Using Semicarbazide as Co-reaction Accelerator for Highly Sensitive Electrochemiluminescent Aptasensor Construction.

PubMed

Ma, Meng-Nan; Zhuo, Ying; Yuan, Ruo; Chai, Ya-Qin

2015-11-17

A highly sensitive electrochemiluminescent (ECL) aptasensor was constructed using semicarbazide (Sem) as co-reaction accelerator to promote the ECL reaction rate of CdTe quantum dots (CdTe QDs) and the co-reactant of peroxydisulfate (S2O8(2-)) for boosting signal amplification. The co-reaction accelerator is a species that when it is introduced into the ECL system containing luminophore and co-reactant, it can interact with co-reactant rather than luminophore to promote the ECL reaction rate of luminophore and co-reactant; thus the ECL signal is significantly amplified in comparison with that in which only luminophore and co-reactant are present. In this work, the ECL signal probes were first fabricated by alternately assembling the Sem and Au nanoparticles (AuNPs) onto the surfaces of hollow Au nanocages (AuNCs) via Au-N bond to obtain the multilayered nanomaterials of (AuNPs-Sem)n-AuNCs for immobilizing amino-terminated detection aptamer of thrombin (TBA2). Notably, the Sem with two -NH2 terminal groups could not only serve as cross-linking reagent to assemble AuNPs and AuNCs but also act as co-reaction accelerator to enhance the ECL reaction rate of CdTe QDs and S2O8(2-) for signal amplification. With the sandwich-type format, TBA2 signal probes could be trapped on the CdTe QD-based sensing interface in the presence of thrombin (TB) to achieve a considerably enhanced ECL signal in S2O8(2-) solution. As a result, the Sem in the TBA2 signal probes could accelerate the reduction of S2O8(2-) to produce the more oxidant mediators of SO4(•-), which further boosted the production of excited states of CdTe QDs to emit light. With the employment of the novel co-reaction accelerator Sem, the proposed ECL biosensor exhibited ultrahigh sensitivity to quantify the concentration of TB from 1 × 10(-7) to 1 nM with a detection limit of 0.03 fM, which demonstrated that the co-reaction accelerator could provide a simple, efficient, and low-cost approach for signal amplification and hold great potential for other ECL biosensors construction.

NanoCluster Beacons as reporter probes in rolling circle enhanced enzyme activity detection

NASA Astrophysics Data System (ADS)

Juul, Sissel; Obliosca, Judy M.; Liu, Cong; Liu, Yen-Liang; Chen, Yu-An; Imphean, Darren M.; Knudsen, Birgitta R.; Ho, Yi-Ping; Leong, Kam W.; Yeh, Hsin-Chih

2015-04-01

As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics.As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics. Electronic supplementary information (ESI) available: The detailed steps of NCB preparation, REEAD assay and STEM imaging. The sequences of the sNCB and the REEAD substrate. See DOI: 10.1039/c5nr01705j

Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification.

PubMed

Wang, Ye; Gan, Ning; Zhou, You; Li, Tianhua; Hu, Futao; Cao, Yuting; Chen, Yinji

2017-11-15

Novel label-free and multiplex aptasensors have been developed for simultaneous detection of several antibiotics based on a microchip electrophoresis (MCE) platform and target catalyzed hairpin assembly (CHA) for signal amplification. Kanamycin (Kana) and oxytetracycline (OTC) were employed as models for testing the system. These aptasensors contained six DNA strands termed as Kana aptamer-catalysis strand (Kana apt-C), Kana inhibit strand (Kana inh), OTC aptamer-catalysis strand (OTC apt-C), OTC inhibit strand (OTC inh), hairpin structures H1 and H2 which were partially complementary. Upon the addition of Kana or OTC, the binding event of aptamer and target triggered the self-assembly between H1 and H2, resulting in the formation of many H1-H2 complexes. They could show strong signals which represented the concentration of Kana or OTC respectively in the MCE system. With the help of the well-designed and high-quality CHA amplification, the assay could yield 300-fold amplified signal comparing that from non-amplified system. Under optimal conditions, this assay exhibited a linear correlation in the ranges from 0.001ngmL -1 to 10ngmL -1 , with the detection limits of 0.7pgmL -1 and 0.9pgmL -1 (S/N=3) toward Kana and OTC, respectively. The platform has the following advantages: firstly, the aptamer probes can be fabricated easily without labeling signal tags for MCE detection; Secondly, the targets can just react with probes and produce the amplified signal in one-pot. Finally, the targets can be simultaneously detected within 10min in different channels, thus high-throughput measurement can be achieved. Based on this work, it is estimated that this detection platform will be universally served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel Immunohistochemical Techniques Using Discrete Signal Amplification Systems for Human Cutaneous Peripheral Nerve Fiber Imaging

PubMed Central

Wang, Ningshan; Gibbons, Christopher H.; Freeman, Roy

2011-01-01

Confocal imaging uses immunohistochemical binding of specific antibodies to visualize tissues, but technical obstacles limit more widespread use of this technique in the imaging of peripheral nerve tissue. These obstacles include same-species antibody cross-reactivity and weak fluorescent signals of individual and co-localized antigens. The aims of this study were to develop new immunohistochemical techniques for imaging of peripheral nerve fibers. Three-millimeter punch skin biopsies of healthy individuals were fixed, frozen, and cut into 50-µm sections. Tissues were stained with a variety of antibody combinations with two signal amplification systems, streptavidin-biotin-fluorochrome (sABC) and tyramide-horseradish peroxidase-fluorochrome (TSA), used simultaneously to augment immunohistochemical signals. The combination of the TSA and sABC amplification systems provided the first successful co-localization of sympathetic adrenergic and sympathetic cholinergic nerve fibers in cutaneous human sweat glands and vasomotor and pilomotor systems. Primary antibodies from the same species were amplified individually without cross-reactivity or elevated background interference. The confocal fluorescent signal-to-noise ratio increased, and image clarity improved. These modifications to signal amplification systems have the potential for widespread use in the study of human neural tissues. PMID:21411809

High-throughput amplification of mature microRNAs in uncharacterized animal models using polyadenylated RNA and stem-loop reverse transcription polymerase chain reaction.

PubMed

Biggar, Kyle K; Wu, Cheng-Wei; Storey, Kenneth B

2014-10-01

This study makes a significant advancement on a microRNA amplification technique previously used for expression analysis and sequencing in animal models without annotated mature microRNA sequences. As research progresses into the post-genomic era of microRNA prediction and analysis, the need for a rapid and cost-effective method for microRNA amplification is critical to facilitate wide-scale analysis of microRNA expression. To facilitate this requirement, we have reoptimized the design of amplification primers and introduced a polyadenylation step to allow amplification of all mature microRNAs from a single RNA sample. Importantly, this method retains the ability to sequence reverse transcription polymerase chain reaction (RT-PCR) products, validating microRNA-specific amplification. Copyright © 2014 Elsevier Inc. All rights reserved.

Fast capacitive probe for electromagnetic pulse diagnostic.

PubMed

Lorusso, A; Nassisi, V; Siciliano, M V

2008-06-01

In this work, we report the study and the development of a capacitive probe which is suitable for getting fast and high voltage/current measurements. Due to the fact that fast pulses propagate generally in coaxial structures, the probe realized in this work was a capacitive divider with the divider electrode properly designed to assure the same characteristic impedance of the coaxial structure and the recombination time of the split signals during the propagation. It was a folded cylindrical ring of 1.4 cm long and 0.8 cm thick, which introduce a theoretical delay time of about 100 ps. Analyzing the behavior of the probe closed on 520 Omega, the voltage amplification resulted to be of (3.6+/-0.1) x 10(-4) and, as a consequence, the current attenuation factor of 56+/-1 AV. The response rise time was less than 320 ps, which was limited by oscilloscope bandwave. The capacitor probe can operate voltage measurements of the order of 100 kV.

Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

DOEpatents

Weier, H.U.G.; Gray, J.W.

1995-06-27

A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers and probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity. 18 figs.

Detection of proteins using a colorimetric bio-barcode assay.

PubMed

Nam, Jwa-Min; Jang, Kyung-Jin; Groves, Jay T

2007-01-01

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take approximately 3 h (whole protocol including probe preparations takes approximately 3 days).

Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

DOEpatents

Weier, Heinz-Ulrich G.; Gray, Joe W.

1995-01-01

A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers ard probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity.

Measurement of locus copy number by hybridisation with amplifiable probes

PubMed Central

Armour, John A. L.; Sismani, Carolina; Patsalis, Philippos C.; Cross, Gareth

2000-01-01

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicroscopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader–Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications. PMID:10606661

Measurement of locus copy number by hybridisation with amplifiable probes.

PubMed

Armour, J A; Sismani, C; Patsalis, P C; Cross, G

2000-01-15

Despite its fundamental importance in genome analysis, it is only recently that systematic approaches have been developed to assess copy number at specific genetic loci, or to examine genomic DNA for submicro-scopic deletions of unknown location. In this report we show that short probes can be recovered and amplified quantitatively following hybridisation to genomic DNA. This simple observation forms the basis of a new approach to determining locus copy number in complex genomes. The power and specificity of multiplex amplifiable probe hybridisation is demonstrated by the simultaneous assessment of copy number at a set of 40 human loci, including detection of deletions causing Duchenne muscular dystrophy and Prader-Willi/Angelman syndromes. Assembly of other probe sets will allow novel, technically simple approaches to a wide variety of genetic analyses, including the potential for extension to high resolution genome-wide screens for deletions and amplifications.

Nanoparticle based bio-bar code technology for trace analysis of aflatoxin B1 in Chinese herbs.

PubMed

Yu, Yu-Yan; Chen, Yuan-Yuan; Gao, Xuan; Liu, Yuan-Yuan; Zhang, Hong-Yan; Wang, Tong-Ying

2018-04-01

A novel and sensitive assay for aflatoxin B1 (AFB1) detection has been developed by using bio-bar code assay (BCA). The method that relies on polyclonal antibodies encoded with DNA modified gold nanoparticle (NP) and monoclonal antibodies modified magnetic microparticle (MMP), and subsequent detection of amplified target in the form of bio-bar code using a fluorescent quantitative polymerase chain reaction (FQ-PCR) detection method. First, NP probes encoded with DNA that was unique to AFB1, MMP probes with monoclonal antibodies that bind AFB1 specifically were prepared. Then, the MMP-AFB1-NP sandwich compounds were acquired, dehybridization of the oligonucleotides on the nanoparticle surface allows the determination of the presence of AFB1 by identifying the oligonucleotide sequence released from the NP through FQ-PCR detection. The bio-bar code techniques system for detecting AFB1 was established, and the sensitivity limit was about 10 -8  ng/mL, comparable ELISA assays for detecting the same target, it showed that we can detect AFB1 at low attomolar levels with the bio-bar-code amplification approach. This is also the first demonstration of a bio-bar code type assay for the detection of AFB1 in Chinese herbs. Copyright © 2017. Published by Elsevier B.V.

Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider).

PubMed

Amarger, V; Mercier, L

1995-01-01

We have applied the recently developed technique of random amplified polymorphic DNA (RAPD) for the discrimination between two jojoba clones at the genomic level. Among a set of 30 primers tested, a simple reproducible pattern with three distinct fragments for clone D and two distinct fragments for clone E was obtained with primer OPB08. Since RAPD products are the results of arbitrarily priming events and because a given primer can amplify a number of non-homologous sequences, we wondered whether or not RAPD bands, even those of similar size, were derived from different loci in the two clones. To answer this question, two complementary approaches were used: i) cloning and sequencing of the amplification products from clone E; and ii) complementary Southern analysis of RAPD gels using cloned or amplified fragments (directly recovered from agarose gels) as RFLP probes. The data reported here show that the RAPD reaction generates multiple amplified fragments. Some fragments, although resolved as a single band on agarose gels, contain different DNA species of the same size. Furthermore, it appears that the cloned RAPD products of known sequence that do not target repetitive DNA can be used as hybridization probes in RFLP to detect a polymorphism among individuals.

Digital Assays Part II: Digital Protein and Cell Assays.

PubMed

Basu, Amar S

2017-08-01

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

PubMed

Mohammadi-Yeganeh, S; Paryan, M; Mirab Samiee, S; Kia, V; Rezvan, H

2012-06-01

Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was <1000 copies/ml for HIV-1 and <500 copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

New Perspectives on Assessing Amplification Effects

PubMed Central

Souza, Pamela E.; Tremblay, Kelly L.

2006-01-01

Clinicians have long been aware of the range of performance variability with hearing aids. Despite improvements in technology, there remain many instances of well-selected and appropriately fitted hearing aids whereby the user reports minimal improvement in speech understanding. This review presents a multistage framework for understanding how a hearing aid affects performance. Six stages are considered: (1) acoustic content of the signal, (2) modification of the signal by the hearing aid, (3) interaction between sound at the output of the hearing aid and the listener's ear, (4) integrity of the auditory system, (5) coding of available acoustic cues by the listener's auditory system, and (6) correct identification of the speech sound. Within this framework, this review describes methodology and research on 2 new assessment techniques: acoustic analysis of speech measured at the output of the hearing aid and auditory evoked potentials recorded while the listener wears hearing aids. Acoustic analysis topics include the relationship between conventional probe microphone tests and probe microphone measurements using speech, appropriate procedures for such tests, and assessment of signal-processing effects on speech acoustics and recognition. Auditory evoked potential topics include an overview of physiologic measures of speech processing and the effect of hearing loss and hearing aids on cortical auditory evoked potential measurements in response to speech. Finally, the clinical utility of these procedures is discussed. PMID:16959734

Optimal energy growth in a stably stratified shear flow

NASA Astrophysics Data System (ADS)

Jose, Sharath; Roy, Anubhab; Bale, Rahul; Iyer, Krithika; Govindarajan, Rama

2018-02-01

Transient growth of perturbations by a linear non-modal evolution is studied here in a stably stratified bounded Couette flow. The density stratification is linear. Classical inviscid stability theory states that a parallel shear flow is stable to exponentially growing disturbances if the Richardson number (Ri) is greater than 1/4 everywhere in the flow. Experiments and numerical simulations at higher Ri show however that algebraically growing disturbances can lead to transient amplification. The complexity of a stably stratified shear flow stems from its ability to combine this transient amplification with propagating internal gravity waves (IGWs). The optimal perturbations associated with maximum energy amplification are numerically obtained at intermediate Reynolds numbers. It is shown that in this wall-bounded flow, the three-dimensional optimal perturbations are oblique, unlike in unstratified flow. A partitioning of energy into kinetic and potential helps in understanding the exchange of energies and how it modifies the transient growth. We show that the apportionment between potential and kinetic energy depends, in an interesting manner, on the Richardson number, and on time, as the transient growth proceeds from an optimal perturbation. The oft-quoted stabilizing role of stratification is also probed in the non-diffusive limit in the context of disturbance energy amplification.

A surface-confined DNA assembly amplification strategy on DNA nanostructural scaffold for electrochemiluminescence biosensing.

PubMed

Feng, Qiu-Mei; Guo, Yue-Hua; Xu, Jing-Juan; Chen, Hong-Yuan

2018-02-15

A critical challenge in surface-based DNA assembly amplification is the reduced accessibility of DNA strands arranged on a heterogeneous surface compared to that in homogeneous solution. Here, a novel in situ surface-confined DNA assembly amplification electrochemiluminescence (ECL) biosensor based on DNA nanostructural scaffold was presented. In this design, a stem-loop structural DNA segment (Hairpin 1) was constructed on the vertex of DNA nanostructural scaffold as recognition probe. In the present of target DNA, the hairpin structure changed to rod-like through complementary hybridization with target DNA, resulting in the formation of Hairpin 1:target DNA. When the obtained Hairpin 1:target DNA met Hairpin 2 labeled with glucose oxidase (GOD), the DNA cyclic amplification was activated, releasing target DNA into homogeneous solution for the next recycling. Thus, the ECL signal of Ru(bpy) 3 2+ -TPrA system was quenched by H 2 O 2 , the product of GOD catalyzing glucose. As a result, this proposed method achieved a linear range response from 50 aM to 10 pM with lower detection limit of 20 aM. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

PubMed Central

Bhadra, Sanchita; Jiang, Yu Sherry; Kumar, Mia R.; Johnson, Reed F.; Hensley, Lisa E.; Ellington, Andrew D.

2015-01-01

The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens. PMID:25856093

Correlation of MET gene amplification and TP53 mutation with PD-L1 expression in non-small cell lung cancer

PubMed Central

Albitar, Maher; Sudarsanam, Sucha; Ma, Wanlong; Jiang, Shiping; Chen, Wayne; Funari, Vincent; Blocker, Forrest; Agersborg, Sally

2018-01-01

Background The role of MET amplification in lung cancer, particularly in relation to checkpoint inhibition and EGFR WT, has not been fully explored. In this study, we correlated PD-L1 expression with MET amplification and EGFR, KRAS, or TP53 mutation in primary lung cancer. Methods In this retrospective study, tissue collected from 471 various tumors, including 397 lung cancers, was tested for MET amplification by FISH with a MET/centromere probe. PD-L1 expression was evaluated using clone SP142 and standard immunohistochemistry, and TP53, KRAS, and EGFR mutations were tested using next generation sequencing. Results Our results revealed that PD-L1 expression in non-small cell lung cancer is inversely correlated with EGFR mutation (P=0.0003), and positively correlated with TP53 mutation (P=0.0001) and MET amplification (P=0.004). Patients with TP53 mutations had significantly higher MET amplification (P=0.007), and were more likely (P=0.0002) to be EGFR wild type. There was no correlation between KRAS mutation and overall PD-L1 expression, but significant positive correlation between PD-L1 expression and KRAS with TP53 co-mutation (P=0.0002). A cut-off for the ratio of MET: centromere signal was determined as 1.5%, and 4% of lung cancer patients were identified as MET amplified. Conclusions This data suggests that in lung cancer both MET and TP53 play direct roles in regulating PD-L1 opposing EGFR. Moreover, KRAS and TP53 co-mutation may cooperate to drive PD-L1 expression in lung cancer. Adding MET or TP53 inhibitors to checkpoint inhibitors may be an attractive combination therapy in patients with lung cancer and MET amplification. PMID:29568386

High-resolution tyramide-FISH mapping of markers tightly linked to the male-fertility restoration (Ms) locus of onion

USDA-ARS?s Scientific Manuscript database

Fluorescence in situ hybridization (FISH) has not been readily exploited for physical mapping of molecular markers in plants due to the technical challenge to visualize small single-copy probes. Signal amplification using tyramide (tyr) FISH can increase sensitivity up to 100 fold. We used tyr-FISH ...

The successes and future prospects of the linear antisense RNA amplification methodology.

PubMed

Li, Jifen; Eberwine, James

2018-05-01

It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.

Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

PubMed

Ball, Cameron S; Light, Yooli K; Koh, Chung-Yan; Wheeler, Sarah S; Coffey, Lark L; Meagher, Robert J

2016-04-05

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation.

PubMed

Mauger, Florence; Kernaleguen, Magali; Lallemand, Céline; Kristensen, Vessela N; Deleuze, Jean-François; Tost, Jörg

2018-05-01

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

Design of a Sensitive and Selective Electrochemical Aptasensor for the Determination of the Complementary cDNA of miRNA-145 Based on the Intercalation and Electrochemical Reduction of Doxorubicin.

PubMed

Mohamadi, Maryam; Mostafavi, Ali; Torkzadeh-Mahani, Masoud

2017-11-01

The aim of this research was the determination of a microRNA (miRNA) using a DNA electrochemical aptasensor. In this biosensor, the complementary complementary DNA (cDNA) of miRNA-145 (a sense RNA transcript) was the target strand and the cDNA of miRNA-145 was the probe strand. Both cDNAs can be the product of the reverse transcriptase-polymerase chain reaction of miRNA. The proposed aptasensor's function was based on the hybridization of target strands with probes immobilized on the surface of a working electrode and the subsequent intercalation of doxorubicin (DOX) molecules functioning as the electroactive indicators of any double strands that formed. Electrochemical transduction was performed by measuring the cathodic current resulting from the electrochemical reduction of the intercalated molecules at the electrode surface. In the experiment, because many DOX molecules accumulated on each target strand on the electrode surface, amplification was inherently easy, without a need for enzymatic or complicated amplification strategies. The proposed aptasensor also had the excellent ability to regenerate as a result of the melting of the DNA duplex. Moreover, the use of DNA probe strands obviated the challenges of working with an RNA probe, such as sensitivity to RNase enzyme. In addition to the linear relationship between the electrochemical signal and the concentration of the target strands that ranged from 2.0 to 80.0 nM with an LOD of 0.27 nM, the proposed biosensor was clearly capable of distinguishing between complementary (target strand) and noncomplementary sequences. The presented biosensor was successfully applied for the quantification of DNA strands corresponding to miRNA-145 in human serum samples.

Graphene oxide@gold nanorods-based multiple-assisted electrochemiluminescence signal amplification strategy for sensitive detection of prostate specific antigen.

PubMed

Cao, Jun-Tao; Yang, Jiu-Jun; Zhao, Li-Zhen; Wang, Yu-Ling; Wang, Hui; Liu, Yan-Ming; Ma, Shu-Hui

2018-01-15

A novel and competitive electrochemiluminescence (ECL) aptasensor for prostate specific antigen (PSA) assay was constructed using gold nanorods functionalized graphene oxide (GO@AuNRs) multilabeled with glucose oxidase (GOD) and streptavidin (SA) toward luminol-based ECL system. A strong initial ECL signal was achieved by electrodeposited gold (DpAu) on the electrode because of gold nanoparticles (AuNPs) motivating the luminol ECL signal. The signal probes prepared by loading GOD and SA-biotin-DNA on GO@AuNRs were used for achieving multiple signal amplification. In the absence of PSA, the signal probes can be attached on the electrode by hybridization reaction between PSA aptamer and biotin-DNA. In this state, the GOD loaded on the probe could catalyze glucose to in situ produce H 2 O 2 and then AuNRs catalyze H 2 O 2 to generate abundant reactive oxygen species (ROSs) in luminol ECL reaction. Both the high-content GOD and AuNRs in the signal probe amplified the ECL signal in the ECL system. Moreover, the combination of SA with biotin-DNA further expands ECL intensity. The integration of such amplifying effects in this protocol endows the aptasensor with high sensitivity and good selectivity for PSA detection. This aptasensor exhibits a linear relation in the range of 0.5pgmL -1 to 5.0ngmL -1 with the detection limit of 0.17pgmL -1 (S/N = 3). Besides, the strategy was successfully applied in determination of human serum samples with recovery of 81.4-116.0%. Copyright © 2017 Elsevier B.V. All rights reserved.

Models and methods to characterize site amplification from a pair of records

USGS Publications Warehouse

Safak, E.

1997-01-01

The paper presents a tutorial review of the models and methods that are used to characterize site amplification from the pairs of rock- and soil-site records, and introduces some new techniques with better theoretical foundations. The models and methods discussed include spectral and cross-spectral ratios, spectral ratios for downhole records, response spectral ratios, constant amplification factors, parametric models, physical models, and time-varying filters. An extensive analytical and numerical error analysis of spectral and cross-spectral ratios shows that probabilistically cross-spectral ratios give more reliable estimates of site amplification. Spectral ratios should not be used to determine site amplification from downhole-surface recording pairs because of the feedback in the downhole sensor. Response spectral ratios are appropriate for low frequencies, but overestimate the amplification at high frequencies. The best method to be used depends on how much precision is required in the estimates.

Amplification in Technical Manuals: Theory and Practice.

ERIC Educational Resources Information Center

Killingsworth, M. Jimmie; And Others

1989-01-01

Examines how amplification (rhetorical techniques by which discourse is extended to enhance its appeal and information value) tends to increase and improve the coverage, rationale, warnings, behavioral alternatives, examples, previews, and general emphasis of technical manuals. Shows how classical and modern rhetorical theories can be applied to…

GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cronn, M.T.; Miyada, C.G.; Fucini, R.V.

1994-09-01

GeneChip{sup {trademark}} assays are based on high density, carefully designed arrays of short oligonucleotide probes (13-16 bases) built directly on derivatized silica substrates. DNA target sequence analysis is achieved by hybridizing fluorescently labeled amplification products to these arrays. Fluorescent hybridization signals located within the probe array are translated into target sequence information using the known probe sequence at each array feature. The mutation screening assay for cystic fibrosis includes sets of oligonucleotide probes designed to detect numerous different mutations that have been described in 14 exons and one intron of the CFTR gene. Each mutation site is addressed by amore » sub-array of at least 40 probe sequences, half designed to detect the wild type gene sequence and half designed to detect the reported mutant sequence. Hybridization with homozygous mutant, homozygous wild type or heterozygous targets results in distinctive hybridization patterns within a sub-array, permitting specific discrimination of each mutation. The GeneChip probe arrays are very small (approximately 1 cm{sup 2}). There miniature size coupled with their high information content make GeneChip probe arrays a useful and practical means for providing CF mutation analysis in a clinical setting.« less

The sandwich-type electrochemiluminescence immunosensor for α-fetoprotein based on enrichment by Fe3O4-Au magnetic nano probes and signal amplification by CdS-Au composite nanoparticles labeled anti-AFP.

PubMed

Zhou, Hankun; Gan, Ning; Li, Tianhua; Cao, Yuting; Zeng, Saolin; Zheng, Lei; Guo, Zhiyong

2012-10-09

A novel and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor was fabricated on a glassy carbon electrode (GCE) for ultra trace levels of α-fetoprotein (AFP) based on sandwich immunoreaction strategy by enrichment using magnetic capture probes and quantum dots coated with Au shell (CdS-Au) as the signal tag. The capture probe was prepared by immobilizing the primary antibody of AFP (Ab1) on the core/shell Fe(3)O(4)-Au nanoparticles, which was first employed to capture AFP antigens to form Fe(3)O(4)-Au/Ab1/AFP complex from the serum after incubation. The product can be separated from the background solution through the magnetic separation. Then the CdS-Au labeled secondary antibody (Ab2) as signal tag (CdS-Au/Ab2) was conjugated successfully with Fe(3)O(4)-Au/Ab1/AFP complex to form a sandwich-type immunocomplex (Fe(3)O(4)-Au/Ab1/AFP/Ab2/CdS-Au), which can be further separated by an external magnetic field and produce ECL signals at a fixed voltage. The signal was proportional to a certain concentration range of AFP for quantification. Thus, an easy-to-use immunosensor with magnetic probes and a quantum dots signal tag was obtained. The immunosensor performed at a level of high sensitivity and a broad concentration range for AFP between 0.0005 and 5.0 ng mL(-1) with a detection limit of 0.2 pg mL(-1). The use of magnetic probes was combined with pre-concentration and separation for trace levels of tumor markers in the serum. Due to the amplification of the signal tag, the immunosensor is highly sensitive, which can offer great promise for rapid, simple, selective and cost-effective detection of effective biomonitoring for clinical application. Copyright © 2012 Elsevier B.V. All rights reserved.

Inefficiency of Signal Amplification by Post-selection

NASA Astrophysics Data System (ADS)

Tanaka, Saki; Yamamoto, Naoki

Basing the two-state vector formalism, Aharonov, Albert and Vaidman found a measurement way such that spin 1/2 particle can turn out 100 [1]. The measurement result is called weak value and this value depends on pre-and post- selected states. The weak value becomes infinitely large when the post- selected state is orthogonal to pre-selected state. By using this feature, the weak measurement has been applied to amplification technique. However, the success of the post-selection depends on luck and this technique does not always work. We take into account of loss by post-selection, and evaluate this amplification by quantum estimation theory. As a result, we get an inequality which means that post-selection does not improve estate accuracy when the number of states is limited.

Aptamer/Au nanoparticles/cobalt sulfide nanosheets biosensor for 17β-estradiol detection using a guanine-rich complementary DNA sequence for signal amplification.

PubMed

Huang, Ke-Jing; Liu, Yu-Jie; Zhang, Ji-Zong; Cao, Jun-Tao; Liu, Yan-Ming

2015-05-15

We have developed a sensitive sensing platform for 17β-estradiol by combining the aptamer probe and hybridization reaction. In this assay, 2-dimensional cobalt sulfide nanosheet (CoS) was synthesized by a simple hydrothermal method with L-cysteine as sulfur donor. An electrochemical aptamer biosensor was constructed by assembling a thiol group tagged 17β-estradiol aptamer on CoS and gold nanoparticles (AuNPs) modified electrode. Methylene blue was applied as a tracer and a guanine-rich complementary DNA sequence was designed to bind with the unbound 17β-estradiol aptamer for signal amplification. The binding of guanine-rich DNA to the aptamer was inhibited when the aptamer captured 17β-estradiol. Using guanine-rich DNA in the assay greatly amplified the redox signal of methylene blue bound to the detection probe. The CoS/AuNPs film formed on the biosensor surface appeared to be a good conductor for accelerating the electron transfer. The method demonstrated a high sensitivity of detection with the dynamic concentration range spanning from 1.0×10(-9) to 1.0×10(-12) M and a detection limit of 7.0×10(-13) M. Besides, the fabricated biosensor exhibited good selectivity toward 17β-estradiol even when interferents were presented at 100-fold concentrations. Our attempt will extend the application of the CoS nanosheet and this signal amplification assay to biosensing areas. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of Fluorescent Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) using Quenching Probes for the Detection of the Middle East Respiratory Syndrome Coronavirus.

PubMed

Shirato, Kazuya; Semba, Shohei; El-Kafrawy, Sherif A; Hassan, Ahmed M; Tolah, Ahmed M; Takayama, Ikuyo; Kageyama, Tsutomu; Notomi, Tsugunori; Kamitani, Wataru; Matsuyama, Shutoku; Azhar, Esam Ibraheem

2018-05-12

Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Highly-sensitive microRNA detection based on bio-bar-code assay and catalytic hairpin assembly two-stage amplification.

PubMed

Tang, Songsong; Gu, Yuan; Lu, Huiting; Dong, Haifeng; Zhang, Kai; Dai, Wenhao; Meng, Xiangdan; Yang, Fan; Zhang, Xueji

2018-04-03

Herein, a highly-sensitive microRNA (miRNA) detection strategy was developed by combining bio-bar-code assay (BBA) with catalytic hairpin assembly (CHA). In the proposed system, two nanoprobes of magnetic nanoparticles functionalized with DNA probes (MNPs-DNA) and gold nanoparticles with numerous barcode DNA (AuNPs-DNA) were designed. In the presence of target miRNA, the MNP-DNA and AuNP-DNA hybridized with target miRNA to form a "sandwich" structure. After "sandwich" structures were separated from the solution by the magnetic field and dehybridized by high temperature, the barcode DNA sequences were released by dissolving AuNPs. The released barcode DNA sequences triggered the toehold strand displacement assembly of two hairpin probes, leading to recycle of barcode DNA sequences and producing numerous fluorescent CHA products for miRNA detection. Under the optimal experimental conditions, the proposed two-stage amplification system could sensitively detect target miRNA ranging from 10 pM to 10 aM with a limit of detection (LOD) down to 97.9 zM. It displayed good capability to discriminate single base and three bases mismatch due to the unique sandwich structure. Notably, it presented good feasibility for selective multiplexed detection of various combinations of synthetic miRNA sequences and miRNAs extracted from different cell lysates, which were in agreement with the traditional polymerase chain reaction analysis. The two-stage amplification strategy may be significant implication in the biological detection and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

PubMed

Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

2012-04-20

In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min. Published by Elsevier Inc.

Optically Tunable Gratings Based on Coherent Population Oscillation.

PubMed

Zhang, Xiao-Jun; Wang, Hai-Hua; Wang, Lei; Wu, Jin-Hui

2018-05-01

We theoretically study the optically tunable gratings based on a L-type atomic medium using coherent population oscillations from the angle of reflection and transmission of the probe field. Adopting a standing-wave driving field, the refractive index of the medium as well as the absorption are periodically modified. Consequently, the Bragg scattering causes the effective reflection. We show that different intensities of the control field lead to three types of reflection profile which actually correspond to different absorption/amplification features of the medium. We present a detailed analyses about the influence of amplification on the reflection profile as well. The coherent population oscillation is robust to the dephasing effect, and such induced gratings could have promising applications in nonlinear optics and all-optical information processing.

Rapid Identification of Pseudallescheria and Scedosporium Strains by Using Rolling Circle Amplification

PubMed Central

Lackner, Michaela; Najafzadeh, Mohammad Javad; Sun, Jiufeng; Lu, Qiaoyun

2012-01-01

The Pseudallescheria boydii complex, comprising environmental pathogens with Scedosporium anamorphs, has recently been subdivided into five main species: Scedosporium dehoogii, S. aurantiacum, Pseudallescheria minutispora, P. apiosperma, and P. boydii, while the validity of some other taxa is being debated. Several Pseudallescheria and Scedosporium species are indicator organisms of pollution in soil and water. Scedosporium dehoogii in particular is enriched in soils contaminated by aliphatic hydrocarbons. In addition, the fungi may cause life-threatening infections involving the central nervous system in severely impaired patients. For screening purposes, rapid and economic tools for species recognition are needed. Our aim is to establish rolling circle amplification (RCA) as a screening tool for species-specific identification of Pseudallescheria and Scedosporium. With this aim, a set of padlock probes was designed on the basis of the internal transcribed spacer (ITS) region, differing by up to 13 fixed mutations. Padlock probes were unique as judged from sequence comparison by BLAST search in GenBank and in dedicated research databases at CBS (Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre). RCA was applied as an in vitro tool, tested with pure DNA amplified from cultures. The species-specific padlock probes designed in this study yielded 100% specificity. The method presented here was found to be an attractive alternative to identification by restriction fragment length polymorphism (RFLP) or sequencing. The rapidity (<1 day), specificity, and low costs make RCA a promising screening tool for environmentally and clinically relevant fungi. PMID:22057865

Rapid identification of Pseudallescheria and Scedosporium strains by using rolling circle amplification.

PubMed

Lackner, Michaela; Najafzadeh, Mohammad Javad; Sun, Jiufeng; Lu, Qiaoyun; Hoog, G Sybren de

2012-01-01

The Pseudallescheria boydii complex, comprising environmental pathogens with Scedosporium anamorphs, has recently been subdivided into five main species: Scedosporium dehoogii, S. aurantiacum, Pseudallescheria minutispora, P. apiosperma, and P. boydii, while the validity of some other taxa is being debated. Several Pseudallescheria and Scedosporium species are indicator organisms of pollution in soil and water. Scedosporium dehoogii in particular is enriched in soils contaminated by aliphatic hydrocarbons. In addition, the fungi may cause life-threatening infections involving the central nervous system in severely impaired patients. For screening purposes, rapid and economic tools for species recognition are needed. Our aim is to establish rolling circle amplification (RCA) as a screening tool for species-specific identification of Pseudallescheria and Scedosporium. With this aim, a set of padlock probes was designed on the basis of the internal transcribed spacer (ITS) region, differing by up to 13 fixed mutations. Padlock probes were unique as judged from sequence comparison by BLAST search in GenBank and in dedicated research databases at CBS (Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre). RCA was applied as an in vitro tool, tested with pure DNA amplified from cultures. The species-specific padlock probes designed in this study yielded 100% specificity. The method presented here was found to be an attractive alternative to identification by restriction fragment length polymorphism (RFLP) or sequencing. The rapidity (<1 day), specificity, and low costs make RCA a promising screening tool for environmentally and clinically relevant fungi.

Expanding the mutational spectrum in Johanson-Blizzard syndrome: identification of whole exon deletions and duplications in the UBR1 gene by multiplex ligation-dependent probe amplification analysis.

PubMed

Sukalo, Maja; Schäflein, Eva; Schanze, Ina; Everman, David B; Rezaei, Nima; Argente, Jesús; Lorda-Sanchez, Isabel; Deshpande, Charu; Takahashi, Tsutomu; Kleger, Alexander; Zenker, Martin

2017-11-01

Johanson-Blizzard syndrome (JBS, MIM #243800) is a very rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, nasal wing hypoplasia, hypodontia, and other abnormalities. JBS is caused by mutations of the UBR1 gene (MIM *605981), encoding a ubiquitin ligase of the N-end rule pathway. Molecular findings in a total of 65 unrelated patients with a clinical diagnosis of JBS who were previously screened for UBR1 mutations by Sanger sequencing were reviewed and cases lacking a disease-causing UBR1 mutation on either one or both alleles were included in this study. In order to discover mutations that are not detectable by Sanger sequencing, we designed a probe set for multiplex ligation-dependent probe amplification (MLPA) analysis of the UBR1 gene and analyzed the copy number status of all 47 UBR1 exons. Our previous studies using Sanger sequencing could detect mutations in 93.1% of 130 disease-associated UBR1 alleles. Six patients with a highly suggestive clinical diagnosis of JBS and unsolved genotype were included in this study. MLPA analysis detected six alleles harboring exon deletions/duplications, thereby raising the mutation detection rate in the entire cohort to 97.7% (127/130 alleles). We conclude that single or multi-exon deletions or duplications account for a substantial proportion of JBS-associated UBR1 mutations. © 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

PubMed Central

SUN, Yu-Ling; YEN, Chon-Ho; TU, Ching-Fu

2013-01-01

ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. The optimum time and temperature for LAMP were 60 min and 65°C, respectively. The specific capture oligonucleotide probes, biotin-labeled CPV probe for LAMP–ELISA and fluorescein isothiocyanate-labeled CPV probe for LAMP–LFD were also designed for hybridization with LAMP amplicons on streptavidin-coated wells and LFD strips, respectively. For the comparison of detection sensitivity, conventional PCR and LAMP for CPV detection were also performed. The CPV detection limits by PCR, PCR–ELISA, LAMP, LAMP–ELISA and LAMP–LFD were 102, 102, 10−1, 10−1 and 10−1 TCID50/ml, respectively. In tests using artificially contaminated dog fecal samples, the samples with CPV inoculation levels of ≥1 TCID50/ml gave positive results by both LAMP–ELISA and LAMP–LFD. Our data indicated that both LAMP–ELISA and LAMP–LFD are promising as rapid, sensitive and specific methods for an efficient diagnosis of CPV infection. PMID:24334855

Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria.

PubMed

Salzman, Nita H; de Jong, Hendrik; Paterson, Yvonne; Harmsen, Hermie J M; Welling, Gjalt W; Bos, Nicolaas A

2002-11-01

Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers. Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences. Of these sequences, 25% (10/40) corresponded to described intestinal organisms of the mouse, including Lactobacillus spp., Helicobacter spp., segmented filamentous bacteria and members of the altered Schaedler flora (ASF360, ASF361, ASF502 and ASF519); 75% (30/40) represented novel sequences. A large number (11/40) of the novel sequences revealed a new operational taxonomic unit (OTU) belonging to the Cytophaga-Flavobacter-Bacteroides phylum, which the authors named 'mouse intestinal bacteria'. 16S rRNA probes were developed for this new OTU. Upon analysis of the novel sequences, eight were found to cluster within the Eubacterium rectale-Clostridium coccoides group and three clustered within the Bacteroides group. One of the novel sequences was distantly related to Verrucomicrobium spinosum and one was distantly related to Bacillus mycoides. Oligonucleotide probes specific for the 16S rRNA of these novel clones were generated. Using a combination of four previously described and four newly designed probes, approximately 80% of bacteria recovered from the murine large intestine and 71% of bacteria recovered from the murine caecum could be identified by fluorescence in situ hybridization (FISH).

Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization.

PubMed

Mills, D; Russell, B W; Hanus, J W

1997-08-01

ABSTRACT Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.

An Electrochemical Genosensing Assay Based on Magnetic Beads and Gold Nanoparticle-Loaded Latex Microspheres for Vibrio cholerae Detection.

PubMed

Low, Kim-Fatt; Rijiravanich, Patsamon; Singh, Kirnpal Kaur Banga; Surareungchai, Werasak; Yean, Chan Yean

2015-04-01

An ultrasensitive electrochemical genosensing assay was developed for the sequence-specific detection of Vibrio cholerae DNA using magnetic beads as the biorecognition surface and gold nanoparticle-loaded latex microspheres (latex-AuNPs) as a signal-amplified hybridization tag. This biorecognition surface was prepared by immobilizing specific biotinylated capturing probes onto the streptavidin-coupled magnetic beads. Fabricating a hybridization tag capable of amplifying the electrochemical signal involved loading multiple AuNPs onto polyelectrolyte multilayer film-coated poly(styrene-co-acrylic acid) latex microspheres as carrier particles. The detection targets, single-stranded 224-bp asymmetric PCR amplicons of the V. cholerae lolB gene, were sandwich-hybridized to magnetic bead-functionalized capturing probes and fluorescein-labeled detection probes and tagged with latex-AuNPs. The subsequent electrochemical stripping analysis of chemically dissolved AuNPs loaded onto the latex microspheres allowed for the quantification of the target amplicons. The high-loading capacity of the AuNPs on the latex microspheres for sandwich-type dual-hybridization genosensing provided eminent signal amplification. The genosensing variables were optimized, and the assay specificity was demonstrated. The clinical applicability of the assay was evaluated using spiked stool specimens. The current signal responded linearly to the different V. cholerae concentrations spiked into stool specimens with a detection limit of 2 colony-forming units (CFU)/ml. The proposed latex-AuNP-based magnetogenosensing platform is promising, exhibits an effective amplification performance, and offers new opportunities for the ultrasensitive detection of other microbial pathogens.

Weak value amplification considered harmful

NASA Astrophysics Data System (ADS)

Ferrie, Christopher; Combes, Joshua

2014-03-01

We show using statistically rigorous arguments that the technique of weak value amplification does not perform better than standard statistical techniques for the tasks of parameter estimation and signal detection. We show that using all data and considering the joint distribution of all measurement outcomes yields the optimal estimator. Moreover, we show estimation using the maximum likelihood technique with weak values as small as possible produces better performance for quantum metrology. In doing so, we identify the optimal experimental arrangement to be the one which reveals the maximal eigenvalue of the square of system observables. We also show these conclusions do not change in the presence of technical noise.

Ring chromosome 10: report on two patients and review of the literature.

PubMed

Guilherme, Roberta Santos; Kim, Chong Ae; Alonso, Luis Garcia; Honjo, Rachel S; Meloni, Vera Ayres; Christofolini, Denise Maria; Kulikowski, Leslie Domenici; Melaragno, Maria Isabel

2013-02-01

Ring chromosome 10--r(10)--is a rare disorder, with 14 cases reported in the literature, but only two with breakpoint determination by high-resolution techniques. We report here on two patients presenting a ring chromosome 10, studied by G-banding, fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and SNP-array techniques, in order to investigate ring instability and determine breakpoints. Patient 1 showed a r(10)(p15.3q26.2) with a 7.9 Mb deletion in 10q26.2-q26.2, while patient 2 showed a r(10)(p15.3q26.13) with a 1.0 Mb deletion in 10p15.3 and a 8.8 Mb deletion in 10q26.13-q26.3, both unstable. While patient 1 presented with clinical features usually found in patients with r(10) and terminal 10q deletion, patient 2 presented characteristics so far not described in other patients with r(10), such as Dandy-Walker variant, osteopenia, semi-flexed legs, and dermal pigmentation regions. Our data and the data from literature show that there are no specific clinical findings to define a r(10) syndrome.

Development of a femtosecond micromachining workstation by use of spectral interferometry.

PubMed

Bera, Sudipta; Sabbah, A J; Durfee, Charles G; Squier, Jeff A

2005-02-15

A workstation that permits real-time measurement of ablation depth while micromachining with femtosecond laser pulses is demonstrated. This method incorporates the unamplified pulse train that is available in a chirped-pulse amplification system as the probe in an arrangement that uses spectral interferometry to measure the ablation depth while cutting with the amplified pulse in thin metal films.

Colorimetric Detection of Ehrlichia Canis via Nucleic Acid Hybridization in Gold Nano-Colloids

PubMed Central

Muangchuen, Ajima; Chaumpluk, Piyasak; Suriyasomboon, Annop; Ekgasit, Sanong

2014-01-01

Canine monocytic ehrlichiosis (CME) is a major thick-bone disease of dog caused by Ehrlichia canis. Detection of this causal agent outside the laboratory using conventional methods is not effective enough. Thus an assay for E. canis detection based on the p30 outer membrane protein gene was developed. It was based on the p30 gene amplification using loop-mediated isothermal DNA amplification (LAMP). The primer set specific to six areas within the target gene were designed and tested for their sensitivity and specificity. Detection of DNA signals was based on modulation of gold nanoparticles' surface properties and performing DNA/DNA hybridization using an oligonucleotide probe. Presence of target DNA affected the gold colloid nanoparticles in terms of particle aggregation with a plasmonic color change of the gold colloids from ruby red to purple, visible by the naked eye. All the assay steps were completed within 90 min including DNA extraction without relying on standard laboratory facilities. This method was very specific to target bacteria. Its sensitivity with probe hybridization was sufficient to detect 50 copies of target DNA. This method should provide an alternative choice for point of care control and management of the disease. PMID:25111239

Laser System for Photoelectron and X-Ray Production in the PLEIADES Compton Light Source

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gibson, D J; Barty, C J; Betts, S M

2005-04-21

The PLEIADES (Picosecond Laser-Electron Interaction for the Dynamic Evaluation of Structures) facility provides tunable short x-ray pulses with energies of 30-140 keV and pulse durations of 0.3-5 ps by scattering an intense, ultrashort laser pulse off a 35-75 MeV electron beam. Synchronization of the laser and electron beam is obtained by using a photoinjector gun, and using the same laser system to generate the electrons and the scattering laser. The Ti Ti:Sapphire, chirped pulse amplification based 500 mJ, 50 fs, 810 nm scattering laser and the similar 300 {micro}J, 5 ps, 266 nm photoinjector laser systems are detailed. Additionally, anmore » optical parametric chirped pulse amplification (OPCPA) system is studied as a replacement for part of the scattering laser front end. Such a change would significantly simplify the set-up the laser system by removing the need for active switching optics, as well as increase the pre-pulse contrast ratio which will be important when part of the scattering laser is used as a pump beam in pump-probe diffraction experiments using the ultrashort tunable x-rays generated as the probe.« less

A highly sensitive SPRi biosensing strategy for simultaneous detection of multiplex miRNAs based on strand displacement amplification and AuNP signal enhancement.

PubMed

Wei, Xiaotong; Duan, Xiaolei; Zhou, Xiaoyan; Wu, Jiangling; Xu, Hongbing; Min, Xun; Ding, Shijia

2018-06-07

Herein, a dual channel surface plasmon resonance imaging (SPRi) biosensor has been developed for the simultaneous and highly sensitive detection of multiplex miRNAs based on strand displacement amplification (SDA) and DNA-functionalized AuNP signal enhancement. In the presence of target miRNAs (miR-21 or miR-192), the miRNAs could specifically hybridize with the corresponding hairpin probes (H) and initiate the SDA, resulting in massive triggers. Subsequently, the two parts of the released triggers could hybridize with capture probes (CP) and DNA-functionalized AuNPs, assembling DNA sandwiches with great mass on the chip surface. A significantly amplified SPR signal readout was achieved. This established biosensing method was capable of simultaneously detecting multiplex miRNAs with a limit of detection down to 0.15 pM for miR-21 and 0.22 pM for miR-192. This method exhibited good specificity and acceptable reproducibility. Moreover, the developed method was applied to the determination of target miRNAs in a complex matrix. Thus, this developed SPRi biosensing method may present a potential alternative tool for miRNA detection in biomedical research and clinical diagnosis.

Colorimetric detection of Ehrlichia canis via nucleic acid hybridization in gold nano-colloids.

PubMed

Muangchuen, Ajima; Chaumpluk, Piyasak; Suriyasomboon, Annop; Ekgasit, Sanong

2014-08-08

Canine monocytic ehrlichiosis (CME) is a major thick-bone disease of dog caused by Ehrlichia canis. Detection of this causal agent outside the laboratory using conventional methods is not effective enough. Thus an assay for E. canis detection based on the p30 outer membrane protein gene was developed. It was based on the p30 gene amplification using loop-mediated isothermal DNA amplification (LAMP). The primer set specific to six areas within the target gene were designed and tested for their sensitivity and specificity. Detection of DNA signals was based on modulation of gold nanoparticles' surface properties and performing DNA/DNA hybridization using an oligonucleotide probe. Presence of target DNA affected the gold colloid nanoparticles in terms of particle aggregation with a plasmonic color change of the gold colloids from ruby red to purple, visible by the naked eye. All the assay steps were completed within 90 min including DNA extraction without relying on standard laboratory facilities. This method was very specific to target bacteria. Its sensitivity with probe hybridization was sufficient to detect 50 copies of target DNA. This method should provide an alternative choice for point of care control and management of the disease.

Diffractometric Detection of Proteins using Microbead-based Rolling Circle Amplification

PubMed Central

Lee, Joonhyung; Icoz, Kutay; Roberts, Ana; Ellington, Andrew D.; Savran, Cagri A.

2010-01-01

We present a robust, sensitive, fluorescent or radio label-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method. An aptamer-based sandwich assay was performed on microcontact-printed streptavidin arranged in 15-μm-wide alternating lines, and could specifically capture and detect platelet-derived growth factor B-chain (PDGF-BB). An aptamer served as a template for the ligation of a padlock probe and the circularized probe could in turn be used as a template for RCA. The concatameric RCA product hybridized to biotinylated oligonuclotides which then captured streptavidin-labeled magnetic beads. In consequence, the signal from the captured PDGF-BB was amplified via the concatameric RCA product, and the diffraction gratings on the printed areas produced varying intensities of diffraction modes. The detected diffraction intensity and the density of the microbeads on the surface varied as a function of PDGF-BB concentration. Our results demonstrate a robust biosensing platform that is easy to construct and use, and devoid of fluorescence microscopy. The self-assembled bead patterns allow both a visual analysis of the molecular binding events under an ordinary bright-field microscope and serve as a diffraction grating biosensor. PMID:19947589

Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping.

PubMed

Nie, Bei; Yang, Min; Fu, Weiling; Liang, Zhiqing

2015-07-07

The surface invasive cleavage assay, because of its innate accuracy and ability for self-signal amplification, provides a potential route for the mapping of hundreds of thousands of human SNP sites. However, its performance on a high density DNA array has not yet been established, due to the unusual "hairpin" probe design on the microarray and the lack of chemical stability of commercially available substrates. Here we present an applicable method to implement a nanocrystalline diamond thin film as an alternative substrate for fabricating an addressable DNA array using maskless light-directed photochemistry, producing the most chemically stable and biocompatible system for genetic analysis and enzymatic reactions. The surface invasive cleavage reaction, followed by degenerated primer ligation and post-rolling circle amplification is consecutively performed on the addressable diamond DNA array, accurately mapping SNP sites from PCR-amplified human genomic target DNA. Furthermore, a specially-designed DNA array containing dual probes in the same pixel is fabricated by following a reverse light-directed DNA synthesis protocol. This essentially enables us to decipher thousands of SNP alleles in a single-pot reaction by the simple addition of enzyme, target and reaction buffers.

Male specific genes from dioecious white campion identified by fluorescent differential display.

PubMed

Scutt, Charles P; Jenkins, Tom; Furuya, Masaki; Gilmartin, Philip M

2002-05-01

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.

Hybridization of Environmental Microbial Community Nucleic Acids by GeoChip.

PubMed

Van Nostrand, Joy D; Yin, Huaqin; Wu, Liyou; Yuan, Tong; Zhou, Jizhong

2016-01-01

Functional gene arrays, like the GeoChip, allow for the study of tens of thousands of genes in a single assay. The GeoChip array (5.0) contains probes for genes involved in geochemical cycling (N, C, S, and P), metal homeostasis, stress response, organic contaminant degradation, antibiotic resistance, secondary metabolism, and virulence factors as well as genes specific for fungi, protists, and viruses. Here, we briefly describe GeoChip design strategies (gene selection and probe design) and discuss minimum quantity and quality requirements for nucleic acids. We then provide detailed protocols for amplification, labeling, and hybridization of samples to the GeoChip.

Quenching of unincorporated amplification signal reporters in reverse-transcription loop-mediated isothermal amplification enabling bright, single-step, closed-tube, and multiplexed detection of RNA viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ball, Cameron S.; Light, Yooli K.; Koh, Chung -Yan

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of themore » reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). As a result, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.« less

Quenching of unincorporated amplification signal reporters in reverse-transcription loop-mediated isothermal amplification enabling bright, single-step, closed-tube, and multiplexed detection of RNA viruses

DOE PAGES

Ball, Cameron S.; Light, Yooli K.; Koh, Chung -Yan; ...

2016-03-16

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of themore » reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read “quasar”), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). As a result, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.« less

Prognostic significance of ESR1 gene amplification, mRNA/protein expression and functional profiles in high-risk early breast cancer: a translational study of the Hellenic Cooperative Oncology Group (HeCOG).

PubMed

Pentheroudakis, George; Kotoula, Vassiliki; Eleftheraki, Anastasia G; Tsolaki, Eleftheria; Wirtz, Ralph M; Kalogeras, Konstantine T; Batistatou, Anna; Bobos, Mattheos; Dimopoulos, Meletios A; Timotheadou, Eleni; Gogas, Helen; Christodoulou, Christos; Papadopoulou, Kyriaki; Efstratiou, Ioannis; Scopa, Chrisoula D; Papaspyrou, Irene; Vlachodimitropoulos, Dimitrios; Linardou, Helena; Samantas, Epaminontas; Pectasides, Dimitrios; Pavlidis, Nicholas; Fountzilas, George

2013-01-01

Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer. Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC) and ESR1 gene copy number by dual fluorescent in situ hybridization probes. In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%), gene gain in 551 (54.6%) and amplification in 42 cases (4.2%), with only 30 tumors (3%) harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01). ESR1 clusters were observed in 9.5% (57 gain, 38 amplification) of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression) in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification) fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p = 0.003). Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066) and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029). ESR1 gene deletion and amplification do not constitute per se prognostic markers, instead they can be classified to distinct prognostic groups according to their protein-mediated functional status.

Simultaneous detection of genetically modified organisms by multiplex ligation-dependent genome amplification and capillary gel electrophoresis with laser-induced fluorescence.

PubMed

García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

2010-07-01

In this work, an innovative method useful to simultaneously analyze multiple genetically modified organisms is described. The developed method consists in the combination of multiplex ligation-dependent genome dependent amplification (MLGA) with CGE and LIF detection using bare-fused silica capillaries. The MLGA process is based on oligonucleotide constructs, formed by a universal sequence (vector) and long specific oligonucleotides (selectors) that facilitate the circularization of specific DNA target regions. Subsequently, the circularized target sequences are simultaneously amplified with the same couple of primers and analyzed by CGE-LIF using a bare-fused silica capillary and a run electrolyte containing 2-hydroxyethyl cellulose acting as both sieving matrix and dynamic capillary coating. CGE-LIF is shown to be very useful and informative for optimizing MLGA parameters such as annealing temperature, number of ligation cycles, and selector probes concentration. We demonstrate the specificity of the method in detecting the presence of transgenic DNA in certified reference and raw commercial samples. The method developed is sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 1% of GA21, 1% of MON863, and 1% of MON810 in maize samples with signal-to-noise ratios for the corresponding DNA peaks of 15, 12, and 26, respectively. These results demonstrate, to our knowledge for the first time, the great possibilities of MLGA techniques for genetically modified organisms analysis.

Ultraviolet Channeling Dynamics in Gaseous Media for X -- Ray Production

NASA Astrophysics Data System (ADS)

McCorkindale, John Charters

The development of a coherent high brightness / short duration X -- ray source has been of considerable interest to the scientific community as well as various industries since the invention of the technology. Possible applications include X -- ray lithography, biological micro-imaging and the probing of molecular and atomic dynamics. One such source under investigation involves the interaction of a high pulsed power KrF UV laser with a noble gas target (krypton or xenon), producing a photon energy from 1 -- 5 keV. Amplification in this regime requires materials with very special properties found in spatially organized hollow atom clusters. One of the driving forces behind X -- ray production is the UV laser. Theoretical analysis shows that above a critical laser power, the formation of a stable plasma channel in the gaseous medium will occur which can act as a guide for the X-ray pulse and co-propagating UV beam. These plasma channels are visualized with a triple pinhole camera, axial and transverse von Hamos spectrometers and a Thomson scattering setup. In order to understand observed channel morphologies, full characterization of the drive laser was achieved using a Transient Grating -- Frequency Resolved Optical Gating (TG-FROG) technique which gives a full temporal representation of the electric field and associated phase of the ultrashort pulse. Insights gleaned from the TG -- FROG data as well as analysis of photodiode diagnostics placed along the UV laser amplification chain provide explanations for the plasma channel morphology and X -- ray output.

Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine.

PubMed

Bi, Sai; Yue, Shuzhen; Zhang, Shusheng

2017-07-17

Developing powerful, simple and low-cost DNA amplification techniques is of great significance to bioanalysis and biomedical research. Thus far, many signal amplification strategies have been developed, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and DNA strand displacement amplification (SDA). In particular, hybridization chain reaction (HCR), a type of toehold-mediated strand displacement (TMSD) reaction, has attracted great interest because of its enzyme-free nature, isothermal conditions, simple protocols, and excellent amplification efficiency. In a typical HCR, an analyte initiates the cross-opening of two DNA hairpins, yielding nicked double helices that are analogous to alternating copolymers. As an efficient amplification platform, HCR has been utilized for the sensitive detection of a wide variety of analytes, including nucleic acids, proteins, small molecules, and cells. In recent years, more complicated sets of monomers have been designed to develop nonlinear HCR, such as branched HCR and even dendritic systems, achieving quadratic and exponential growth mechanisms. In addition, HCR has attracted enormous attention in the fields of bioimaging and biomedicine, including applications in fluorescence in situ hybridization (FISH) imaging, live cell imaging, and targeted drug delivery. In this review, we introduce the fundamentals of HCR and examine the visualization and analysis techniques for HCR products in detail. The most recent HCR developments in biosensing, bioimaging, and biomedicine are subsequently discussed with selected examples. Finally, the review provides insight into the challenges and future perspectives of HCR.

Single-molecule dilution and multiple displacement amplification for molecular haplotyping.

PubMed

Paul, Philip; Apgar, Josh

2005-04-01

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

Whole genome amplification of DNA extracted from FFPE tissues.

PubMed

Bosso, Mira; Al-Mulla, Fahd

2011-01-01

Whole genome amplification systems were developed to meet the increasing research demands on DNA resources and to avoid DNA shortage. The technology enables amplification of nanogram amounts of DNA into microgram quantities and is increasingly used in the amplification of DNA from multiple origins such as blood, fresh frozen tissue, formalin-fixed paraffin-embedded tissues, saliva, buccal swabs, bacteria, and plant and animal sources. This chapter focuses on the use of GenomePlex(®) tissue Whole Genome Amplification Kit, to amplify DNA directly from archived tissue. In addition, this chapter documents our unique experience with the utilization of GenomePlex(®) amplified DNA using several molecular techniques including metaphase Comparative Genomic Hybridization, array Comparative Genomic Hybridization, and real-time quantitative polymerase chain reaction assays. GenomePlex(®) is a registered trademark of Rubicon Genomics Incorporation.

Hetero-enzyme-based two-round signal amplification strategy for trace detection of aflatoxin B1 using an electrochemical aptasensor.

PubMed

Zheng, Wanli; Teng, Jun; Cheng, Lin; Ye, Yingwang; Pan, Daodong; Wu, Jingjing; Xue, Feng; Liu, Guodong; Chen, Wei

2016-06-15

An electrochemical aptasensor for trace detection of aflatoxin B1 (AFB1) was developed by using an aptamer as the recognition unit while adopting the telomerase and EXO III based two-round signal amplification strategy as the signal enhancement units. The telomerase amplification was used to elongate the ssDNA probes on the surface of gold nanoparticles, by which the signal response range of the signal-off model electrochemical aptasensor could be correspondingly enlarged. Then, the EXO III amplification was used to hydrolyze the 3'-end of the dsDNA after the recognition of target AFB1, which caused the release of bounded AFB1 into the sensing system, where it participated in the next recognition-sensing cycle. With this two-round signal amplified electrochemical aptasensor, target AFB1 was successfully measured at trace concentrations with excellent detection limit of 0.6*10(-4)ppt and satisfied specificity due to the excellent affinity of the aptamer against AFB1. Based on this designed two-round signal amplification strategy, both the sensing range and detection limit were greatly improved. This proposed ultrasensitive electrochemical aptasensor method was also validated by comparison with the classic instrumental methods. Importantly, this hetero-enzyme based two-round signal amplified electrochemical aptasensor offers a great promising protocol for ultrasensitive detection of AFB1 and other mycotoxins by replacing the core recognition sequence of the aptamer. Copyright © 2016 Elsevier B.V. All rights reserved.

Carbon nanotube enhanced label-free detection of microRNAs based on hairpin probe triggered solid-phase rolling-circle amplification

NASA Astrophysics Data System (ADS)

Tian, Qianqian; Wang, Ying; Deng, Ruijie; Lin, Lei; Liu, Yang; Li, Jinghong

2014-12-01

The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development.The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development. Electronic supplementary information (ESI) available: Preparation of the chemically modified multi-walled carbon nanotubes (CNTs), characterization of the CNTs and modified CNTs, preparation of the circular probe, gel electrophoresis of the RCA products, and DNA probes as noted in the text. See DOI: 10.1039/c4nr05243a

An exonuclease III and graphene oxide-aided assay for DNA detection.

PubMed

Peng, Lu; Zhu, Zhi; Chen, Yan; Han, Da; Tan, Weihong

2012-05-15

We have developed a novel DNA assay based on exonuclease III (ExoIII)-induced target recycling and the fluorescence quenching ability of graphene oxide (GO). This assay consists of a linear DNA probe labeled with a fluorophore in the middle. Introduction of target sequence induces the exonuclease III catalyzed probe digestion and generation of single nucleotides. After each cycle of digestion, the target is recycled to realize the amplification. Finally, graphene oxide is added to quench the remaining probes and the signal from the resulting fluorophore labeled single nucleotides is detected. With this approach, a sub-picomolar detection limit can be achieved within 40 min at 37°C. The method was successfully applied to multicolor DNA detection and the analysis of telomerase activity in extracts from cancer cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

PubMed Central

2014-01-01

Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability. PMID:24712299

Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification.

PubMed

Ghedira, Rim; Papazova, Nina; Vuylsteke, Marnik; Ruttink, Tom; Taverniers, Isabel; De Loose, Marc

2009-10-28

GMO quantification, based on real-time PCR, relies on the amplification of an event-specific transgene assay and a species-specific reference assay. The uniformity of the nucleotide sequences targeted by both assays across various transgenic varieties is an important prerequisite for correct quantification. Single nucleotide polymorphisms (SNPs) frequently occur in the maize genome and might lead to nucleotide variation in regions used to design primers and probes for reference assays. Further, they may affect the annealing of the primer to the template and reduce the efficiency of DNA amplification. We assessed the effect of a minor DNA template modification, such as a single base pair mismatch in the primer attachment site, on real-time PCR quantification. A model system was used based on the introduction of artificial mismatches between the forward primer and the DNA template in the reference assay targeting the maize starch synthase (SSIIb) gene. The results show that the presence of a mismatch between the primer and the DNA template causes partial to complete failure of the amplification of the initial DNA template depending on the type and location of the nucleotide mismatch. With this study, we show that the presence of a primer/template mismatch affects the estimated total DNA quantity to a varying degree.

Target-triggering multiple-cycle signal amplification strategy for ultrasensitive detection of DNA based on QCM and SPR.

PubMed

Song, Weiling; Yin, Wenshuo; Sun, Wenbo; Guo, Xiaoyan; He, Peng; Yang, Xiaoyan; Zhang, Xiaoru

2018-04-24

Detection of ultralow concentrations of nucleic acid sequences is a central challenge in the early diagnosis of genetic diseases. Herein, we developed a target-triggering cascade multiple cycle amplification for ultrasensitive DNA detection using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). It was based on the exonuclease Ⅲ (Exo Ⅲ)-assisted signal amplification and the hybridization chain reaction (HCR). The streptavidin-coated Au-NPs (Au-NPs-SA) were assembled on the HCR products as recognition element. Upon sensing of target DNA, the duplex DNA probe triggered the Exo Ⅲ cleavage process, accompanied by generating a new secondary target DNA and releasing target DNA. The released target DNA and the secondary target DNA were recycled. Simultaneously, numerous single strands were liberated and acted as the trigger of HCR to generate further signal amplification, resulting in the immobilization of abundant Au-NPs-SA on the gold substrate. The QCM sensor results were found to be comparable to that achieved using a SPR sensor platform. This method exhibited a high sensitivity toward target DNA with a detection limit of 0.70 fM. The high sensitivity and specificity make this method a great potential for detecting DNA with trace amounts in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier Inc. All rights reserved.

Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.

PubMed

del Río, Jonathan Sabaté; Yehia Adly, Nouran; Acero-Sánchez, Josep Lluis; Henry, Olivier Y F; O'Sullivan, Ciara K

2014-04-15

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers. © 2013 Elsevier B.V. All rights reserved.

Carbon nanotubes as in vivo bacterial probes.

PubMed

Bardhan, Neelkanth M; Ghosh, Debadyuti; Belcher, Angela M

2014-09-17

With the rise in antibiotic-resistant infections, non-invasive sensing of infectious diseases is increasingly important. Optical imaging, although safer and simpler, is less developed than other modalities such as radioimaging, due to low availability of target-specific molecular probes. Here we report carbon nanotubes (SWNTs) as bacterial probes for fluorescence imaging of pathogenic infections. We demonstrate that SWNTs functionalized using M13 bacteriophage (M13-SWNT) can distinguish between F'-positive and F'-negative bacterial strains. Moreover, through one-step modification, we attach an anti-bacterial antibody on M13-SWNT, making it easily tunable for sensing specific F'-negative bacteria. We illustrate detection of Staphylococcus aureus intramuscular infections, with ~3.4 × enhancement in fluorescence intensity over background. SWNT imaging presents lower signal spread ~0.08 × and higher signal amplification ~1.4 × , compared with conventional dyes. We show the probe offers greater ~5.7 × enhancement in imaging of S. aureus infective endocarditis. These biologically functionalized, aqueous-dispersed, actively targeted, modularly tunable SWNT probes offer new avenues for exploration of deeply buried infections.

Carbon nanotubes as in vivo bacterial probes

NASA Astrophysics Data System (ADS)

Bardhan, Neelkanth M.; Ghosh, Debadyuti; Belcher, Angela M.

2014-09-01

With the rise in antibiotic-resistant infections, non-invasive sensing of infectious diseases is increasingly important. Optical imaging, although safer and simpler, is less developed than other modalities such as radioimaging, due to low availability of target-specific molecular probes. Here we report carbon nanotubes (SWNTs) as bacterial probes for fluorescence imaging of pathogenic infections. We demonstrate that SWNTs functionalized using M13 bacteriophage (M13-SWNT) can distinguish between F‧-positive and F‧-negative bacterial strains. Moreover, through one-step modification, we attach an anti-bacterial antibody on M13-SWNT, making it easily tunable for sensing specific F‧-negative bacteria. We illustrate detection of Staphylococcus aureus intramuscular infections, with ~3.4 × enhancement in fluorescence intensity over background. SWNT imaging presents lower signal spread ~0.08 × and higher signal amplification ~1.4 × , compared with conventional dyes. We show the probe offers greater ~5.7 × enhancement in imaging of S. aureus infective endocarditis. These biologically functionalized, aqueous-dispersed, actively targeted, modularly tunable SWNT probes offer new avenues for exploration of deeply buried infections.

Carbon Nanotubes as in vivo Bacterial Probes

PubMed Central

Bardhan, Neelkanth M.; Ghosh, Debadyuti; Belcher, Angela M.

2014-01-01

With the rise in antibiotic-resistant infections, noninvasive sensing of infectious diseases is increasingly important. Optical imaging, while safer and simpler, is less developed than other modalities like radioimaging; due to low availability of target-specific molecular probes. Here, we report carbon nanotubes (SWNTs) as bacterial probes for fluorescence imaging of pathogenic infections. We demonstrate that SWNTs functionalized using M13 bacteriophage (M13-SWNT) can distinguish between F'-positive and F'-negative bacterial strains. Moreover, through one-step modification, we attach an anti-bacterial antibody on M13-SWNT, making it easily tunable for sensing specific F’-negative bacteria. We illustrate detection of Staphylococcus aureus intramuscular infections, with ~3.4× enhancement in fluorescence intensity over background. SWNT imaging presents lower signal spread ~0.08×, and higher signal amplification ~1.4×, compared to conventional dyes. We show the probe offers greater ~5.7× enhancement in imaging of S. aureus infective endocarditis. These biologically-functionalized, aqueous-dispersed, actively-targeted, modularly-tunable SWNT probes offer new avenues for exploration of deeply-buried infections. PMID:25230005

[Female genital surgery, G-spot amplification techniques--state of the science].

PubMed

Bachelet, J-T; Mojallal, A; Boucher, F

2014-10-01

The G-spot amplification is a process of "functional" intimate surgery consisting of a temporary physical increase of the size and sensitivity of the G-spot with a filler injected into the septum between the bladder and the vagina's anterior wall, in order to increase the frequency and importance of female orgasm during vaginal penetration. This surgical technique is based on the existence of an eponymous anatomical area described by Dr Gräfenberg in 1950, responsible upon stimulation of systematic orgasm different from the clitoral orgasm, referring to the vaginal orgasm as described by Freud in 1905. The purpose of this article is to review the scientific basis of the G-spot, whose very existence is currently a debated topic, and to discuss the role of G-spot amplification surgery. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Weak Value Amplification is Suboptimal for Estimation and Detection

NASA Astrophysics Data System (ADS)

Ferrie, Christopher; Combes, Joshua

2014-01-01

We show by using statistically rigorous arguments that the technique of weak value amplification does not perform better than standard statistical techniques for the tasks of single parameter estimation and signal detection. Specifically, we prove that postselection, a necessary ingredient for weak value amplification, decreases estimation accuracy and, moreover, arranging for anomalously large weak values is a suboptimal strategy. In doing so, we explicitly provide the optimal estimator, which in turn allows us to identify the optimal experimental arrangement to be the one in which all outcomes have equal weak values (all as small as possible) and the initial state of the meter is the maximal eigenvalue of the square of the system observable. Finally, we give precise quantitative conditions for when weak measurement (measurements without postselection or anomalously large weak values) can mitigate the effect of uncharacterized technical noise in estimation.

Precision phase estimation based on weak-value amplification

NASA Astrophysics Data System (ADS)

Qiu, Xiaodong; Xie, Linguo; Liu, Xiong; Luo, Lan; Li, Zhaoxue; Zhang, Zhiyou; Du, Jinglei

2017-02-01

In this letter, we propose a precision method for phase estimation based on the weak-value amplification (WVA) technique using a monochromatic light source. The anomalous WVA significantly suppresses the technical noise with respect to the intensity difference signal induced by the phase delay when the post-selection procedure comes into play. The phase measured precision of this method is proportional to the weak-value of a polarization operator in the experimental range. Our results compete well with the wide spectrum light phase weak measurements and outperform the standard homodyne phase detection technique.

Identification of Vibrio splendidus as a Member of the Planktonic Luminous Bacteria from the Persian Gulf and Kuwait Region with luxA Probes

PubMed Central

Nealson, K. H.; Wimpee, B.; Wimpee, C.

1993-01-01

Hybridization probes specific for the luxA genes of four groups of luminous bacteria were used to screen luminous isolates obtained from the Persian Gulf, near Al Khiran, Kuwait Nine of these isolates were identified as Vibrio harveyi, a commonly encountered planktonic isolate, while three others showed no hybridization to any of the four probes (V. harveyi, Vibrio fischeri, Photobacterium phosphoreum, or Photobacterium leiognathi) under high-stringency conditions. Polymerase chain reaction amplification was used to prepare a luxA probe against one of these isolates, K-1, and this probe was screened under high-stringency conditions against a collection of DNAs from luminous bacteria; it was found to hybridize specifically to the DNA of the species Vibrio splendidus. A probe prepared against the type strain of V. splendidus (ATCC 33369) was tested against the collection of luminous bacterial DNA preparations and against the Kuwait isolates and was found to hybridize only against the type strain and the three unidentified Kuwait isolates. Extensive taxonomic analysis by standard methods confirmed the identification of the 13 isolates. Images PMID:16349023

Colocalization recognition-activated cascade signal amplification strategy for ultrasensitive detection of transcription factors.

PubMed

Zhu, Desong; Wang, Lei; Xu, Xiaowen; Jiang, Wei

2017-03-15

Transcription factors (TFs) bind to specific double-stranded DNA (dsDNA) sequences in the regulatory regions of genes to regulate the process of gene transcription. Their expression levels sensitively reflect cell developmental situation and disease state. TFs have become potential diagnostic markers and therapeutic targets of cancers and some other diseases. Hence, high sensitive detection of TFs is of vital importance for early diagnosis of diseases and drugs development. The traditional exonucleases-assisted signal amplification methods suffered from the false positives caused by incomplete digestion of excess recognition probes. Herein, based on a new recognition way-colocalization recognition (CR)-activated dual signal amplification, an ultrasensitive fluorescent detection strategy for TFs was developed. TFs-induced the colocalization of three split recognition components resulted in noticeable increases of local effective concentrations and hybridization of three split components, which activated the subsequent cascade signal amplification including strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). This strategy eliminated the false positive influence and achieved ultra-high sensitivity towards the purified NF-κB p50 with detection limit of 2.0×10 -13 M. Moreover, NF-κB p50 can be detected in as low as 0.21ngμL -1 HeLa cell nuclear extracts. In addition, this proposed strategy could be used for the screening of NF-κB p50 activity inhibitors and potential anti-NF-κB p50 drugs. Finally, our proposed strategy offered a potential method for reliable detection of TFs in medical diagnosis and treatment research of cancers and other related diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Ultrabroadband infrared nanospectroscopic imaging

PubMed Central

Bechtel, Hans A.; Muller, Eric A.; Olmon, Robert L.; Martin, Michael C.; Raschke, Markus B.

2014-01-01

Characterizing and ultimately controlling the heterogeneity underlying biomolecular functions, quantum behavior of complex matter, photonic materials, or catalysis requires large-scale spectroscopic imaging with simultaneous specificity to structure, phase, and chemical composition at nanometer spatial resolution. However, as with any ultrahigh spatial resolution microscopy technique, the associated demand for an increase in both spatial and spectral bandwidth often leads to a decrease in desired sensitivity. We overcome this limitation in infrared vibrational scattering-scanning probe near-field optical microscopy using synchrotron midinfrared radiation. Tip-enhanced localized light–matter interaction is induced by low-noise, broadband, and spatially coherent synchrotron light of high spectral irradiance, and the near-field signal is sensitively detected using heterodyne interferometric amplification. We achieve sub-40-nm spatially resolved, molecular, and phonon vibrational spectroscopic imaging, with rapid spectral acquisition, spanning the full midinfrared (700–5,000 cm−1) with few cm−1 spectral resolution. We demonstrate the performance of synchrotron infrared nanospectroscopy on semiconductor, biomineral, and protein nanostructures, providing vibrational chemical imaging with subzeptomole sensitivity. PMID:24803431

Image-guided genomics of phenotypically heterogeneous populations reveals vascular signalling during symbiotic collective cancer invasion

PubMed Central

Konen, J.; Summerbell, E.; Dwivedi, B.; Galior, K.; Hou, Y.; Rusnak, L.; Chen, A.; Saltz, J.; Zhou, W.; Boise, L. H.; Vertino, P.; Cooper, L.; Salaita, K.; Kowalski, J.; Marcus, A. I.

2017-01-01

Phenotypic heterogeneity is widely observed in cancer cell populations. Here, to probe this heterogeneity, we developed an image-guided genomics technique termed spatiotemporal genomic and cellular analysis (SaGA) that allows for precise selection and amplification of living and rare cells. SaGA was used on collectively invading 3D cancer cell packs to create purified leader and follower cell lines. The leader cell cultures are phenotypically stable and highly invasive in contrast to follower cultures, which show phenotypic plasticity over time and minimally invade in a sheet-like pattern. Genomic and molecular interrogation reveals an atypical VEGF-based vasculogenesis signalling that facilitates recruitment of follower cells but not for leader cell motility itself, which instead utilizes focal adhesion kinase-fibronectin signalling. While leader cells provide an escape mechanism for followers, follower cells in turn provide leaders with increased growth and survival. These data support a symbiotic model of collective invasion where phenotypically distinct cell types cooperate to promote their escape. PMID:28497793

Cryptic intragenic deletion of the SHOX gene in a family with Léri-Weill dyschondrosteosis detected by Multiplex Ligation-Dependent Probe Amplification (MLPA).

PubMed

Funari, Mariana F A; Jorge, Alexander A L; Pinto, Emilia M; Arnhold, Ivo J P; Mendonca, Berenice B; Nishi, Mirian Y

2008-11-01

LWD is associated to SHOX haploinsufficiency, in most cases, due to gene deletion. Generally FISH and microsatellite analysis are used to identify SHOX deletion. MLPA is a new method of detecting gene copy variation, allowing simultaneous analysis of several regions. Here we describe the presence of a SHOX intragenic deletion in a family with LWD, analyzed through different methodologies. Genomic DNA of 11 subjects from one family were studied by microsatellite analysis, direct sequencing and MLPA. FISH was performed in two affected individuals. Microsatellite analysis showed that all affected members shared the same haplotype suggesting the involvement of SHOX. MLPA detected an intragenic deletion involving exons IV-VIa, which was not detected by FISH and microsatellite analysis. In conclusion, the MLPA technique was proved to be the best solution on detecting this small deletion, it has the advantage of being less laborious also allowing the analysis of several regions simultaneously.

Dispersive Readout of a Superconducting Flux Qubit Using a Microstrip SQUID Amplifier

NASA Astrophysics Data System (ADS)

Johnson, J. E.; Hoskinson, E. M.; Macklin, C.; Siddiqi, I.; Clarke, John

2011-03-01

Dispersive techniques for the readout of superconducting qubits offer the possibility of high repetition-rate, quantum non-demolition measurement by avoiding dissipation close to the qubit. To achieve dispersive readout, we couple our three-junction aluminum flux qubit inductively to a 1-2 GHz non-linear oscillator formed by a capacitively shunted DC SQUID. The frequency of this resonator is modulated by the state of the qubit via the flux-dependent inductance of the SQUID. Readout is performed by probing the resonator in the linear (weak drive) regime with a microwave tone and monitoring the phase of the reflected signal. A microstrip SQUID amplifier (MSA) is used to increase the sensitivity of the measurement over that of a HEMT (high electron mobility transistor) amplifier. We report measurements of the performance of our amplification chain. Increased fidelity and reduced measurement backaction resulting from the implementation of the MSA will also be discussed. This work was funded in part by the U.S. Government and by BBN Technologies.

Synthesis and evaluation of a photoresponsive quencher for fluorescent hybridization probes.

PubMed

Kovaliov, Marina; Wachtel, Chaim; Yavin, Eylon; Fischer, Bilha

2014-10-21

Nowadays, most nucleic acid detections using fluorescent probes rely on quenching of fluorescence by energy transfer from one fluorophore to another or to a non-fluorescent molecule (quencher). The most widely used quencher in fluorescent probes is 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL). We targeted a nucleoside-DABCYL analogue which could be incorporated anywhere in an oligonucleotide sequence and in any number, and used as a quencher in different hybridization sensitive probes. Specifically, we introduced a 5-(4-((dimethylamino)phenyl)azo)benzene)-2'-deoxy-uridine (dU(DAB)) quencher. The photoisomerization and dU(DAB)'s ability to quench fluorescein emission have been investigated. We incorporated dU(DAB) into a series of oligonucleotide (ON) probes including strand displacement probes, labeled with both fluorescein (FAM) and dU(DAB), and TaqMan probes bearing one or two dU(DAB) and a FAM fluorophore. We used these probes for the detection of a DNA target in real-time PCR (RT-PCR). All probes showed amplification of targeted DNA. A dU(DAB) modified TaqMan RT-PCR probe was more efficient as compared to a DABCYL bearing probe (93% vs. 87%, respectively). Furthermore, dU(DAB) had a stabilizing effect on the duplex, causing an increase in Tm up to 11 °C. In addition we showed the photoisomerisation of the azobenzene moiety of dU(DAB) and the dU(DAB) triply-labeled oligonucleotide upon irradiation. These findings suggest that dU(DAB) modified probes are promising probes for gene quantification in real-time PCR detection and as photoswitchable devices.

A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization.

PubMed

Behr, T; Koob, C; Schedl, M; Mehlen, A; Meier, H; Knopp, D; Frahm, E; Obst, U; Schleifer, K; Niessner, R; Ludwig, W

2000-12-01

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.

Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA

DOE PAGES

Meagher, Robert J.; Priye, Aashish; Light, Yooli K.; ...

2018-03-27

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer-dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer-dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMPmore » or RT-LAMP assays. In this study, we examine the impact of primer-dimers and hairpins on previously-published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter to predict the probability of non-specific amplification associated with LAMP primers.« less

Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meagher, Robert J.; Priye, Aashish; Light, Yooli K.

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer-dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer-dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMPmore » or RT-LAMP assays. In this study, we examine the impact of primer-dimers and hairpins on previously-published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter to predict the probability of non-specific amplification associated with LAMP primers.« less

Relative contributions from the ventricle and arterial tree to arterial pressure and its amplification: an experimental study

PubMed Central

Alastruey, Jordi; Chowienczyk, Phil; Rutten, Marcel C. M.; Segers, Patrick; Schaeffter, Tobias

2017-01-01

Arterial pressure is an important diagnostic parameter for cardiovascular disease. However, relative contributions of individual ventricular and arterial parameters in generating and augmenting pressure are not understood. Using a novel experimental arterial model, our aim was to characterize individual parameter contributions to arterial pressure and its amplification. A piston-driven ventricle provided programmable stroke profiles into various silicone arterial trees and a bovine aorta. Inotropy was varied in the ventricle, and arterial parameters modulated included wall thickness, taper and diameter, the presence of bifurcation, and a native aorta (bovine) versus silicone. Wave reflection at bifurcations was measured and compared with theory, varying parent-to-child tube diameter ratios, and branch angles. Intravascular pressure-tip wires and ultrasonic flow probes measured pressure and flow. Increasing ventricular inotropy independently augmented pressure amplification from 17% to 61% between the lower and higher systolic gradient stroke profiles in the silicone arterial network and from 10% to 32% in the bovine aorta. Amplification increased with presence of a bifurcation, decreasing wall thickness and vessel taper. Pulse pressure increased with increasing wall thickness (stiffness) and taper angle and decreasing diameter. Theoretical predictions of wave transmission through bifurcations werre similar to measurements (correlation: 0.91, R2 = 0.94) but underestimated wave reflection (correlation: 0.75, R2 = 0.94), indicating energy losses during mechanical wave reflection. This study offers the first comprehensive investigation of contributors to hypertensive pressure and its propagation throughout the arterial tree. Importantly, ventricular inotropy plays a crucial role in the amplification of peripheral pressure wave, which offers opportunity for noninvasive assessment of ventricular health. NEW & NOTEWORTHY The present study distinguishes contributions from cardiac and arterial parameters to elevated blood pressure and pressure amplification. Most importantly, it offers the first evidence that ventricular inotropy, an indicator of ventricular function, is an independent determinant of pressure amplification and could be measured with such established devices such as the SphygmoCor. PMID:28576835

Relative contributions from the ventricle and arterial tree to arterial pressure and its amplification: an experimental study.

PubMed

Gaddum, Nicholas; Alastruey, Jordi; Chowienczyk, Phil; Rutten, Marcel C M; Segers, Patrick; Schaeffter, Tobias

2017-09-01

Arterial pressure is an important diagnostic parameter for cardiovascular disease. However, relative contributions of individual ventricular and arterial parameters in generating and augmenting pressure are not understood. Using a novel experimental arterial model, our aim was to characterize individual parameter contributions to arterial pressure and its amplification. A piston-driven ventricle provided programmable stroke profiles into various silicone arterial trees and a bovine aorta. Inotropy was varied in the ventricle, and arterial parameters modulated included wall thickness, taper and diameter, the presence of bifurcation, and a native aorta (bovine) versus silicone. Wave reflection at bifurcations was measured and compared with theory, varying parent-to-child tube diameter ratios, and branch angles. Intravascular pressure-tip wires and ultrasonic flow probes measured pressure and flow. Increasing ventricular inotropy independently augmented pressure amplification from 17% to 61% between the lower and higher systolic gradient stroke profiles in the silicone arterial network and from 10% to 32% in the bovine aorta. Amplification increased with presence of a bifurcation, decreasing wall thickness and vessel taper. Pulse pressure increased with increasing wall thickness (stiffness) and taper angle and decreasing diameter. Theoretical predictions of wave transmission through bifurcations werre similar to measurements (correlation: 0.91, R 2 = 0.94) but underestimated wave reflection (correlation: 0.75, R 2 = 0.94), indicating energy losses during mechanical wave reflection. This study offers the first comprehensive investigation of contributors to hypertensive pressure and its propagation throughout the arterial tree. Importantly, ventricular inotropy plays a crucial role in the amplification of peripheral pressure wave, which offers opportunity for noninvasive assessment of ventricular health. NEW & NOTEWORTHY The present study distinguishes contributions from cardiac and arterial parameters to elevated blood pressure and pressure amplification. Most importantly, it offers the first evidence that ventricular inotropy, an indicator of ventricular function, is an independent determinant of pressure amplification and could be measured with such established devices such as the SphygmoCor. Copyright © 2017 the American Physiological Society.

HER2 Amplification and HER2 Mutation Are Distinct Molecular Targets in Lung Cancers.

PubMed

Li, Bob T; Ross, Dara S; Aisner, Dara L; Chaft, Jamie E; Hsu, Meier; Kako, Severine L; Kris, Mark G; Varella-Garcia, Marileila; Arcila, Maria E

2016-03-01

Human epidermal growth factor receptor 2 gene (HER2 [also known as ERBB2]) alterations have been identified as oncogenic drivers and potential therapeutic targets in lung cancers. The molecular associations of HER2 gene amplification, mutation, and HER2 protein overexpression in lung cancers have not been distinctly defined. To explore these associations, Memorial Sloan Kettering Cancer Center and the University of Colorado combined their data on HER2 alterations in lung cancers. Tumor specimens from 175 patients with lung adenocarcinomas and no prior targeted therapy were evaluated for the presence of HER2 amplification and mutation and HER2 protein overexpression. Amplification was assessed by fluorescence in situ hybridization (FISH) and defined as an HER2-to-chromosome enumeration probe 17 ratio of at least 2.0. Mutation was assessed by fragment analysis, mass spectrometry genotyping, and Sanger sequencing. Overexpression was assessed by immunohistochemical (IHC) staining. The frequencies of HER2 amplification and mutation and HER2 overexpression were calculated and their overlap examined. HER2 amplification was detected by FISH in 5 of 175 cases (3%). HER2 mutation was detected in 4 of 148 specimens (3%), including three identical 12-base pair insertions (p.A775_G776insYVMA) and a 9-base pair insertion, all in exon 20. None of the HER2-mutant cases was amplified. HER2 overexpression (2+ or 3+) on IHC staining was not detected in the 25 specimens available for testing, and negative IHC staining correlated with the negative results according to FISH. HER2 mutations are not associated with HER2 amplification, thus suggesting a distinct entity and therapeutic target. HER2-positive lung cancer may not be an adequate term, and patient cohorts for the study of HER2-targeted agents should be defined by the specific HER2 alteration present. Copyright © 2015 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

The present situation and forecasts of semiconductor elements performance within the microwave range, 1970-1985

NASA Technical Reports Server (NTRS)

Peterson, B.

1978-01-01

The present situation and possible developments over the period 1970-1985 for active semiconductor elements in the microwave range are outlined. After a short historical survey of FT techniques, the following are discussed: Generation, power amplification, amplification of small signals, frequency conversion, detection, electronic signal control and integrated microwave circuits.

High peak-power kilohertz laser system employing single-stage multi-pass amplification

DOEpatents

Shan, Bing; Wang, Chun; Chang, Zenghu

2006-05-23

The present invention describes a technique for achieving high peak power output in a laser employing single-stage, multi-pass amplification. High gain is achieved by employing a very small "seed" beam diameter in gain medium, and maintaining the small beam diameter for multiple high-gain pre-amplification passes through a pumped gain medium, then leading the beam out of the amplifier cavity, changing the beam diameter and sending it back to the amplifier cavity for additional, high-power amplification passes through the gain medium. In these power amplification passes, the beam diameter in gain medium is increased and carefully matched to the pump laser's beam diameter for high efficiency extraction of energy from the pumped gain medium. A method of "grooming" the beam by means of a far-field spatial filter in the process of changing the beam size within the single-stage amplifier is also described.

Local modulation of double optomechanically induced transparency and amplification.

PubMed

Yang, Q; Hou, B P; Lai, D G

2017-05-01

We consider the probe absorption properties in a mechanically coupled optomechanical system in which the two coupled nanomechanical oscillators are driven by the time-dependent forces, respectively. It is found that the mechanical interaction splits the transparency window for a usual single-mode optomechanical system into two parts and then leads to appearance of the double optomechanically induced transparency. The distance between the two transparency positions (the frequency for the maximal transparency) is determined by the mechanical interaction amplitude. This can be explained by using optomechanical dressed-mode picture which is analogue to the interacting dark resonances in coherent atoms. When the mechanical resonators are driven by the external forces, the transparencies in the double-transparency spectrum can be increased into amplifications or be suppressed by tuning the amplitude of the forces. Additionally, it is shown that the double transparencies or the amplifications oscillate with the initial phases of the forces with a period of 2π. These investigations will be useful for more flexible controllability of multi-channel optical communication based on the optomechanical systems.

Combination of Mass Signal Amplification and Isotope-Labeled Alkanethiols for the Multiplexed Detection of miRNAs.

PubMed

Kang, Hyunook; Hong, Seol-Hye; Sung, Jiha; Yeo, Woon-Seok

2017-08-04

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope-labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am-tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am-tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am-tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO 4 concentrations. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A single tube PCR assay for simultaneous amplification of HSV-1/-2, VZV, CMV, HHV-6A/-6B, and EBV DNAs in cerebrospinal fluid from patients with virus-related neurological diseases.

PubMed

Yamamoto, T; Nakamura, Y

2000-10-01

Cerebrospinal fluid (CSF) specimens from 27 patients with encephalitis, meningitis, and other neurological diseases were studied for the presence of herpes simplex virus types 1 and 2 (HSV-1/-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), human herpesviruses 6A and 6B (HHV-6A/-6B) and Epstein-Barr virus (EBV) DNA using the polymerase chain reaction (PCR) method. The DNAs were amplified using two sets of consensus primer pairs in a single tube, bringing simultaneous amplification of the herpesviruses. The PCR products were analyzed by agarose gel electrophoresis, and Southern blot hybridization with virus-type specific probes, thus allowing discrimination between the different types of herpesviruses to be made. Each virus-specific probe was highly specific for identifying the PCR product. Thirty CSF specimens from 13 patients with encephalitis and 10 specimens from 10 patients with meningitis, respectively, were examined using this method. Eight patients with encephalitis and six with meningitis were positive for different herpesviruses, including patients with coinfections (HSV-1/-2 and VZV, VZV and CMV). Among four CSF specimens from four patients with other neurological disorders, dual amplification of CMV and EBV was present. Since identification of the types of herpesviruses in this system requires a very small amount of CSF, and is completed with one PCR, it is useful for routine diagnosis of herpesvirus infections in diagnostic laboratories. The viruses responsible for central nervous system infection are easily detected with various coinfection and serial patterns of herpesviruses, by this consensus primer-based PCR method. This may give an insight into the relationship between virus-related neurological diseases (VRNDS) and herpesvirus infections.

Change in HER2 (ERBB2) gene status after taxane-based chemotherapy for breast cancer: polyploidization can lead to diagnostic pitfalls with potential impact for clinical management.

PubMed

Valent, Alexander; Penault-Llorca, Frédérique; Cayre, Anne; Kroemer, Guido

2013-01-01

The status of the HER2 (ERBB2) gene in breast cancer is not static and may change among the primary tumor, lymph node metastases, and distant metastases. This status change can be a consequence of the natural evolution of the tumor or can be induced by therapy. The HER2 gene status is, in the majority of cases, established at the moment of diagnosis. After chemotherapy, monitoring HER2 status can be a challenge because of ploidy changes induced by drugs. The cytogeneticist or the pathologist can face real difficulties in distinguishing between a true HER2 amplification and HER2 copy number increase by polyploidization. We performed a HER2 genetic examination by fluorescence in situ hybridization (FISH) of invasive breast cancers before and after taxane treatment. The majority of patients (91%) were HER2-negative both at diagnosis and after treatment. Thirty of 344 patients (9%) whose tumors were initially HER2-negative were found by FISH to have supernumerary HER2 gene copies (up to 15 copies) after neoadjuvant chemotherapy. This HER2 copy increase could not be attributed to true gene amplifications and instead reflected polyploidization events, which presumably affected all chromosomes. Indeed, when we used other FISH probes, we found other gene copy numbers to parallel those of HER2. We recommend careful checking of invasive breast carcinomas by supplementary FISH probes if the copy number of the HER2 gene is >6. This procedure allows the discrimination of specific HER2 gene amplifications and global increases in ploidy. Copyright © 2013 Elsevier Inc. All rights reserved.

Hairpin DNA-Templated Silver Nanoclusters as Novel Beacons in Strand Displacement Amplification for MicroRNA Detection.

PubMed

Zhang, Jingpu; Li, Chao; Zhi, Xiao; Ramón, Gabriel Alfranca; Liu, Yanlei; Zhang, Chunlei; Pan, Fei; Cui, Daxiang

2016-01-19

MicroRNA (miRNA) biomarkers display great potential for cancer diagnosis and prognosis. The development of rapid and specific methods for miRNA detection has become a hotspot. Herein, hairpin DNA-templated silver nanoclusters (AgNCs/HpDNA) were prepared and integrated into strand-displacement amplification (SDA) as a novel beacon for miRNA detection. The light-up platform was established based on guanine (G)-rich fluorescence enhancement that essentially converted the excitation/emission pair of AgNCs/HpDNAs from a shorter wavelength to a longer wavelength, and then achieved fluorescent enhancement at longer wavelength. On the basis of the validation of the method, the single and duplex detection were conducted in two plasma biomarkers (miR-16-5p and miR-19b-3p) for the diagnosis of gastric cancer. The probe (AgNCs/RED 16(7s)C) utilized for miR-16-5p detection adopted a better conformation with high specificity to recognize single-base mismatches by producing dramatically opposite signals (increase or decrease at 580 nm ex/640 nm em) while the probe (AgNCs/GRE 19b(5s)C) for miR-19b-3p generated dual signals (increase at 490 nm ex/570 nm em and decrease at 430 nm ex/530 nm em) with bright fluorescence in one reaction during the amplification, but unexpectedly was partially digested. This is for the first time to allow the generation of enhanced fluorescent AgNCs and the target recognition integrated into a single process, which offers great opportunity for specific miRNA detection in an easy and rapid way.

Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their gpd (glyceraldehyde-3-phosphate dehydrogenase) genes.

PubMed Central

Kreuzinger, N; Podeu, R; Gruber, F; Göbl, F; Kubicek, C P

1996-01-01

Degenerated oligonucleotide primers designed to flank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycorrhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B. edulis, A. muscaria, and L. deterrimus were cloned and sequenced. The respective nucleotide sequences of these gene fragments showed a moderate degree of similarity (72 to 76%) in the protein-encoding regions and only a low degree of similarity in the introns (56 to 66%). Introns, where present, occurred at conserved positions, but the respective positions and numbers of introns in a given taxon varied. The amplified fragment from a given taxon could be distinguished from that of others by both restriction nuclease cleavage analysis and Southern hybridization. A procedure for labeling DNA probes with fluorescein-12-dUTP by PCR was developed. These probes were used in a nonradioactive hybridization assay, with which the gene could be detected in 2 ng of chromosomal DNA of L. deterrimus on slot blots. Taxon-specific amplification was achieved by the design of specific oligonucleotide primers. The application of the gpd gene for the identification of mycorrhizal fungi under field conditions was demonstrated, with Picea abies (spruce) mycorrhizal roots harvested from a northern alpine forest area as well as from a plant-breeding nursery. The interference by inhibitory substances, which sometimes occurred in the DNA extracted from the root-fungus mixture, could be overcome by using very diluted concentrations of template DNA for a first round of PCR amplification followed by a second round with nested oligonucleotide primers. We conclude that gpd can be used to detect ectomycorrhizal fungi during symbiotic interaction. PMID:8795234

Voltammetric determination of the Escherichia coli DNA using a screen-printed carbon electrode modified with polyaniline and gold nanoparticles.

PubMed

Shoaie, Nahid; Forouzandeh, Mehdi; Omidfar, Kobra

2018-03-12

The authors describe an electrochemical assay for fast detection of Escherichia coli (E. coli). It is based on a dual signal amplification strategy and the use of a screen-printed carbon electrode (SPCE) whose surface was modified with a polyaniline (PANI) film and gold nanoparticles (AuNPs) via cyclic voltammetry (CV). In the next step, avidin was covalently immobilized on the PANI/AuNP composite on the SPCE surface. Subsequently, the biotinylated DNA capture probe was immobilized onto the PANI/AuNP/avidin-modified SPCE by biotin-avidin interaction. Then, DNA of E.coli, digoxigenin-labeled DNA detector probe and anti-digoxigenin-labeled horseradish peroxidase (HRP) were placed on the electrode. 3,3',5,5'-Tetramethylbenzidine (TMB) and H 2 O 2 solution were added and the CV electrochemical signal was generated at a potential of -0.1 V (vs. Ag/AgCl) and a scan rate 50 mV.s -1 . The assay can detect 4 × 10 6 to 4 CFU of E. coli without DNA amplification. The biosensor is highly specific over other pathogens including Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Staphylococcus haemolyticus and Pseudomonas aeruginosa. It can be concluded that this genosensor has an excellent potential for rapid and accurate diagnosis of E.coli inflicted infections. Graphical Abstract Schematic of an electrochemical E. coli genosensor based on sandwich assay on a polyaniline/gold nanoparticle-modified screen printed carbon electrode (SPCE). The biosensor can detect 4 × 10 6 to 4 CFU of E. coli without DNA amplification.

Real-time fluorescence ligase chain reaction for sensitive detection of single nucleotide polymorphism based on fluorescence resonance energy transfer.

PubMed

Sun, Yueying; Lu, Xiaohui; Su, Fengxia; Wang, Limei; Liu, Chenghui; Duan, Xinrui; Li, Zhengping

2015-12-15

Most of practical methods for detection of single nucleotide polymorphism (SNP) need at least two steps: amplification (usually by PCR) and detection of SNP by using the amplification products. Ligase chain reaction (LCR) can integrate the amplification and allele discrimination in one step. However, the detection of LCR products still remains a great challenge for highly sensitive and quantitative SNP detection. Herein, a simple but robust strategy for real-time fluorescence LCR has been developed for highly sensitive and quantitative SNP detection. A pair of LCR probes are firstly labeled with a fluorophore and a quencher, respectively. When the pair of LCR probes are ligated in LCR, the fluorophore will be brought close to the quencher, and thus, the fluorescence will be specifically quenched by fluorescence resonance energy transfer (FRET). The decrease of fluorescence intensity resulted from FRET can be real-time monitored in the LCR process. With the proposed real-time fluorescence LCR assay, 10 aM DNA targets or 100 pg genomic DNA can be accurately determined and as low as 0.1% mutant DNA can be detected in the presence of a large excess of wild-type DNA, indicating the high sensitivity and specificity. The real-time measuring does not require the detection step after LCR and gives a wide dynamic range for detection of DNA targets (from 10 aM to 1 pM). As LCR has been widely used for detection of SNP, DNA methylation, mRNA and microRNA, the real-time fluorescence LCR assay shows great potential for various genetic analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultrasensitive and Multiple Disease-Related MicroRNA Detection Based on Tetrahedral DNA Nanostructures and Duplex-Specific Nuclease-Assisted Signal Amplification.

PubMed

Xu, Fang; Dong, Haifeng; Cao, Yu; Lu, Huiting; Meng, Xiangdan; Dai, Wenhao; Zhang, Xueji; Al-Ghanim, Khalid Abdullah; Mahboob, Shahid

2016-12-14

A highly sensitive and multiple microRNA (miRNA) detection method by combining three-dimensional (3D) DNA tetrahedron-structured probes (TSPs) to increase the probe reactivity and accessibility with duplex-specific nuclease (DSN) for signal amplification for sensitive miRNA detection was proposed. Briefly, 3D DNA TSPs labeled with different fluorescent dyes for specific target miRNA recognition were modified on a gold nanoparticle (GNP) surface to increase the reactivity and accessibility. Upon hybridization with a specific target, the TSPs immobilized on the GNP surface hybridized with the corresponding target miRNA to form DNA-RNA heteroduplexes, and the DSN can recognize the formed DNA-RNA heteroduplexes to hydrolyze the DNA in the heteroduplexes to produce a specific fluorescent signal corresponding to a specific miRNA, while the released target miRNA strands can initiate another cycle, resulting in a significant signal amplification for sensitive miRNA detection. Different targets can produce different fluorescent signals, leading to the development of a sensitive detection for multiple miRNAs in a homogeneous solution. Under optimized conditions, the proposed assay can simultaneously detect three different miRNAs in a homogeneous solution with a logarithmic linear range spanning 5 magnitudes (10 -12 -10 -16 ) and achieving a limit of detection down to attomolar concentrations. Meanwhile, the proposed miRNA assay exhibited the capability of discriminating single bases (three bases mismatched miRNAs) and showed good eligibility in the analysis of miRNAs extracted from cell lysates and miRNAs in cell incubation media, which indicates its potential use in biomedical research and clinical analysis.

On the role of dealing with quantum coherence in amplitude amplification

NASA Astrophysics Data System (ADS)

Rastegin, Alexey E.

2018-07-01

Amplitude amplification is one of primary tools in building algorithms for quantum computers. This technique generalizes key ideas of the Grover search algorithm. Potentially useful modifications are connected with changing phases in the rotation operations and replacing the intermediate Hadamard transform with arbitrary unitary one. In addition, arbitrary initial distribution of the amplitudes may be prepared. We examine trade-off relations between measures of quantum coherence and the success probability in amplitude amplification processes. As measures of coherence, the geometric coherence and the relative entropy of coherence are considered. In terms of the relative entropy of coherence, complementarity relations with the success probability seem to be the most expository. The general relations presented are illustrated within several model scenarios of amplitude amplification processes.

Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes.

PubMed

Fujita, Hiroto; Kataoka, Yuka; Tobita, Seiji; Kuwahara, Masayasu; Sugimoto, Naoki

2016-07-19

We have developed a novel RNA detection method, termed signal amplification by ternary initiation complexes (SATIC), in which an analyte sample is simply mixed with the relevant reagents and allowed to stand for a short time under isothermal conditions (37 °C). The advantage of the technique is that there is no requirement for (i) heat annealing, (ii) thermal cycling during the reaction, (iii) a reverse transcription step, or (iv) enzymatic or mechanical fragmentation of the target RNA. SATIC involves the formation of a ternary initiation complex between the target RNA, a circular DNA template, and a DNA primer, followed by rolling circle amplification (RCA) to generate multiple copies of G-quadruplex (G4) on a long DNA strand like beads on a string. The G4s can be specifically fluorescence-stained with N(3)-hydroxyethyl thioflavin T (ThT-HE), which emits weakly with single- and double-stranded RNA/DNA but strongly with parallel G4s. An improved dual SATIC system, which involves the formation of two different ternary initiation complexes in the RCA process, exhibited a wide quantitative detection range of 1-5000 pM. Furthermore, this enabled visual observation-based RNA detection, which is more rapid and convenient than conventional isothermal methods, such as reverse transcription-loop-mediated isothermal amplification, signal mediated amplification of RNA technology, and RNA-primed rolling circle amplification. Thus, SATIC methodology may serve as an on-site and real-time measurement technique for transcriptomic biomarkers for various diseases.

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

PubMed

Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

Molecular Technique to Understand Deep Microbial Diversity

NASA Technical Reports Server (NTRS)

Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.

2012-01-01

Current sequencing-based and DNA microarray techniques to study microbial diversity are based on an initial PCR (polymerase chain reaction) amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of the minor template appears to be suppressed by the exponential amplification of the more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck has overlooked the presence of the less abundant minority population, and underestimated their role in the ecosystem maintenance. To generate PCR amplicons for subsequent diversity analysis, bacterial l6S rRNA genes are amplified by PCR using universal primers. Two distinct PCR regimes are employed in parallel: one using normal and the other using biotinlabeled universal primers. PCR products obtained with biotin-labeled primers are mixed with streptavidin-labeled magnetic beads and selectively captured in the presence of a magnetic field. Less-abundant DNA templates that fail to amplify in this first round of PCR amplification are subjected to a second round of PCR using normal universal primers. These PCR products are then subjected to downstream diversity analyses such as conventional cloning and sequencing. A second round of PCR amplified the minority population and completed the deep diversity picture of the environmental sample.

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

PubMed

Ying, Na; Ju, Chuanjing; Sun, Xiuwei; Li, Letian; Chang, Hongbiao; Song, Guangping; Li, Zhongyi; Wan, Jiayu; Dai, Enyong

2017-01-01

MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3' end modified with biotin) for a target miRNA of miR-21 and capture sequence (5' end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

Non-biased and efficient global amplification of a single-cell cDNA library

PubMed Central

Huang, Huan; Goto, Mari; Tsunoda, Hiroyuki; Sun, Lizhou; Taniguchi, Kiyomi; Matsunaga, Hiroko; Kambara, Hideki

2014-01-01

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively. PMID:24141095