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

Real-time imaging of gene expression in living cells has the potential to significantly impact clinical and laboratory studies of cancer, including cancer diagnosis and analysis. Molecular beacons (MBs) provide a simple and promising tool for the detection of target mRNA as tumor markers due to their high signal-to-background ratio, and their improved specificity in detecting point mutations. However, the harsh intracellular environment does limit the sensitivity of MB-based gene detection. Specifically, MBs bound to target mRNAs cannot be distinguished from those degraded by nucleases, or opened due to non-specific interactions. To overcome this difficulty, we have developed a novel dual FRET molecular beacons approach in which a pair of molecular beacons, one with a donor fluorophore and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of false-positive events. We have performed systematic in-solution and cellular studies of dual FRET molecular beacon and demonstrated that this new approach allows for real-time imaging of gene expression in living cells.

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Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

Applied and Environmental Microbiology ◽

10.1128/aem.02597-05 ◽

2006 ◽

Vol 72 (9) ◽

pp. 6424-6428 ◽

Cited By ~ 38

Author(s):

Aneta J. Gubala ◽

David F. Proll

Keyword(s):

Vibrio Cholerae ◽

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Molecular Beacon ◽

Environmental Water ◽

Molecular Beacons ◽

Pcr Assay ◽

Specificity And Sensitivity ◽

Pcr Assays

ABSTRACT A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

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A molecular beacon based multiplex real-time PCR assay to subspeciate Mycobacterium abscessus and determine macrolide susceptibility

Journal of Clinical Microbiology ◽

10.1128/jcm.00455-21 ◽

2021 ◽

Author(s):

Salvatore A.E. Marras ◽

Liang Chen ◽

Elena Shashkina ◽

Rebecca M. Davidson ◽

Michael Strong ◽

...

Keyword(s):

Real Time ◽

Molecular Beacon ◽

Accurate Method ◽

Mycobacterium Abscessus ◽

Multiplex Assay ◽

Macrolide Resistance ◽

Molecular Beacons ◽

Diagnostic Tools ◽

Significant Treatment ◽

Treatment Information

Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterial species that comprises three subspecies; M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. These predominantly environmental microorganisms have emerged as life-threatening chronic pulmonary pathogens in both immunocompetent and immunocompromised patients and their acquisition of macrolide resistance due to the erm(41) gene and mutations in the 23S rrl has dramatically impacted patient outcome. However, standard microbiology laboratories typically have limited diagnostic tools for the subspeciation of M. abscessus, and the testing for macrolide resistance is often not done. Here we describe the development of a real-time multiplex assay using molecular beacons to establish a robust, rapid and highly accurate method to both distinguish M. abscessus sub-species and to determine which strains are susceptible to macrolides. We report a bioinformatic approach to identify robust subspeciation sequence targets, the design and optimization of six molecular beacons to identify all genotypes, and the development and application of a two-tube 3-color multiplex assay that can provide clinically significant treatment information in less than 3 hours.

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Novel nucleic acid origami structures and conventional molecular beacon–based platforms: a comparison in biosensing applications

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03309-4 ◽

2021 ◽

Author(s):

Noemi Bellassai ◽

Roberta D’Agata ◽

Giuseppe Spoto

Keyword(s):

Nucleic Acid ◽

Structural Properties ◽

Molecular Beacon ◽

Dna Origami ◽

Molecular Beacons ◽

Conformational Polymorphism ◽

Functional Systems ◽

Similarities And Differences ◽

Nucleic Acid Structures ◽

Nucleic Acid Sequences

AbstractNucleic acid nanotechnology designs and develops synthetic nucleic acid strands to fabricate nanosized functional systems. Structural properties and the conformational polymorphism of nucleic acid sequences are inherent characteristics that make nucleic acid nanostructures attractive systems in biosensing. This review critically discusses recent advances in biosensing derived from molecular beacon and DNA origami structures. Molecular beacons belong to a conventional class of nucleic acid structures used in biosensing, whereas DNA origami nanostructures are fabricated by fully exploiting possibilities offered by nucleic acid nanotechnology. We present nucleic acid scaffolds divided into conventional hairpin molecular beacons and DNA origami, and discuss some relevant examples by focusing on peculiar aspects exploited in biosensing applications. We also critically evaluate analytical uses of the synthetic nucleic acid structures in biosensing to point out similarities and differences between traditional hairpin nucleic acid sequences and DNA origami. Graphical abstract

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Rapid and Sensitive Detection of Apple stem pitting virus in Apple Trees Through RNA Amplification and Probing with Fluorescent Molecular Beacons

Phytopathology ◽

10.1094/phyto.2001.91.11.1085 ◽

2001 ◽

Vol 91 (11) ◽

pp. 1085-1091 ◽

Cited By ~ 21

Author(s):

M. M. Klerks ◽

G. Leone ◽

J. L. Lindner ◽

C. D. Schoen ◽

J. F. J. M. van den Heuvel

Keyword(s):

Field Testing ◽

Molecular Beacons ◽

Fluorescent Detection ◽

Rna Amplification ◽

Apple Trees ◽

Apple Stem ◽

Bark Tissue ◽

Specific Amplification ◽

Apple Stem Pitting Virus ◽

Stem Pitting

Currently, detection of Apple stem pitting virus (ASPV; genus Foveavirus) in apple trees for certification purposes occurs by woody indexing. This method requires a minimum of 12 to 24 weeks in greenhouse testing to up to 2 years in field testing. In this paper, the development of a single tube AmpliDet RNA system for the rapid gel-free detection of ASPV in apple tree tissues is described. The system relies on the specific amplification of the viral RNA by nucleic acid sequence-based amplification and the simultaneous fluorescent detection of the amplification product through molecular beacons. A sensitivity of a minimum of 100 molecules of transcript RNA was obtained by the ASPV-specific AmpliDet RNA. All biologically characterized ASPV isolates from a field trial and 12 of 14 isolates from a plant virus collection were readily detected with this AmpliDet RNA system. In addition, the efficiency of this method for detecting ASPV in ‘Golden Delicious’ and ‘Gravenstein’ apple trees was compared throughout the year with mechanical inoculation onto Nicotiana occidentalis 37B, a candidate indicator for ASPV. This revealed that only AmpliDet RNA consistently detected the virus in bark tissue, irrespective of the season. Season-specific tissues such as buds, petals, and fruits, but not leaves, also were reliable sources for detection of ASPV by the AmpliDet RNA system.

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2021 Frank Stinchfield Award: A novel cemented hip hemiarthroplasty infection model with real-time in vivo imaging in rats

The Bone & Joint Journal ◽

10.1302/0301-620x.103b7.bjj-2020-2435.r1 ◽

2021 ◽

Vol 103-B (7 Supple B) ◽

pp. 9-16

Author(s):

William J. Hadden ◽

Mazen Ibrahim ◽

Mariam Taha ◽

Kerstin Ure ◽

Yun Liu ◽

...

Keyword(s):

Real Time ◽

In Vivo Imaging ◽

Biofilm Formation ◽

Joint Infection ◽

Volumetric Analysis ◽

In Vivo Model ◽

Infection Model ◽

Sprague Dawley ◽

Hip Hemiarthroplasty

Aims The aims of this study were to develop an in vivo model of periprosthetic joint infection (PJI) in cemented hip hemiarthroplasty, and to monitor infection and biofilm formation in real-time. Methods Sprague-Dawley rats underwent cemented hip hemiarthroplasty via the posterior approach with pre- and postoperative gait assessments. Infection with Staphylococcus aureus Xen36 was monitored with in vivo photoluminescent imaging in real-time. Pre- and postoperative gait analyses were performed and compared. Postmortem micro (m) CT was used to assess implant integration; field emission scanning electron microscopy (FE-SEM) was used to assess biofilm formation on prosthetic surfaces. Results All animals tolerated surgery well, with preservation of gait mechanics and weightbearing in control individuals. Postoperative in vivo imaging demonstrated predictable evolution of infection with logarithmic signal decay coinciding with abscess formation. Postmortem mCT qualitative volumetric analysis showed high contact area and both cement-bone and cement-implant interdigitation. FE-SEM revealed biofilm formation on the prosthetic head. Conclusion This study demonstrates the utility of a new, high-fidelity model of in vivo PJI using cemented hip hemiarthroplasty in rats. Inoculation with bioluminescent bacteria allows for non-invasive, real-time monitoring of infection. Cite this article: Bone Joint J 2021;103-B(7 Supple B):9–16.

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Simultaneous detection of five different DNA targets by real-time Taqman PCR using the Roche LightCycler480: Application in viral molecular diagnostics

Journal of Virological Methods ◽

10.1016/j.jviromet.2006.12.007 ◽

2007 ◽

Vol 141 (2) ◽

pp. 205-211 ◽

Cited By ~ 44

Author(s):

Richard Molenkamp ◽

Alwin van der Ham ◽

Janke Schinkel ◽

Marcel Beld

Keyword(s):

Real Time ◽

Molecular Diagnostics ◽

Simultaneous Detection ◽

Taqman Pcr

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Real-time PCR using molecular beacons for accurate detection of the Y chromosome in single human blastomeres

MHR Basic science of reproductive medicine ◽

10.1093/molehr/6.12.1155 ◽

2000 ◽

Vol 6 (12) ◽

pp. 1155-1164 ◽

Cited By ~ 35

Author(s):

K. E. Pierce

Keyword(s):

Real Time ◽

Y Chromosome ◽

Real Time Pcr ◽

Molecular Beacons ◽

Accurate Detection

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A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons

Biosensors ◽

10.3390/bios11120479 ◽

2021 ◽

Vol 11 (12) ◽

pp. 479

Author(s):

Soumi Sukla ◽

Prasenjit Mondal ◽

Subhajit Biswas ◽

Surajit Ghosh

Keyword(s):

Nucleic Acid ◽

Dengue Virus ◽

Magnetic Beads ◽

Molecular Beacon ◽

Detection System ◽

Denv Infection ◽

Molecular Beacons ◽

Nucleic Acid Amplification ◽

Complementary Sequence ◽

Reverse Transcription Pcr

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient’s serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

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