China
Africa
Explore chapters and articles related to this topic
Future Perspectives on Nucleic Acid Testing
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Larry J. Kricka, Paolo Fortina
A molecular beacon is a probe functionalized at one end with a fluorophore and at the other with a quencher. Molecular beacons have served successfully as DNA detection reagents. A different way of exploiting this analytical strategy is using a gold surface assembled on a quartz substrate to quench a fluorescent rhodamine reporter; 5′-thiolated beacons (3’-rhodamine label) specific for Staphylococcus aureus FemA and mecR methicillin-resistance genes were immobilized onto gold. Increases in signal of up to 26-fold were observed when the beacons bound to specific target and disrupted the energy transfer to the gold.55
Methods for Genetic Testing II
Published in Peter G. Shields, Cancer Risk Assessment, 2005
Laura Gunn, Luoping Zhang, Martyn T. Smith
Another recent contribution to this spectrum of novel methods combines the process of genotypic selection with the sensitive new molecular beacon-based PCR methodologies. Digital PCR, developed by Vogelstein and Kinzler (66) is an assay designed to quantitatively measure the proportion of mutant sequences within a DNA sample. Single molecules of DNA are isolated and amplified by PCR in a multiple-well plate, resulting in completely mutant or completely wild-type products. After amplification, asymmetric PCR is used to generate a single-stranded product to which the molecular beacons could anneal. Two molecular beacons (red and green) are added, one which recognizes only wild-type sequence and one which is common to all PCR products. Using mutations in the RAS gene, the authors designed the wild-type beacon which would react better with the wild type than any mutant sequence within the target sequence. In other words, any mutations in the region complementary to the molecular beacon sequence would inhibit wild-type beacon binding. This was possible due to the fact that RAS has proximal mutational hotspots within codons 12, 13 which are within the range of the probe sequence. This assay, however, in most cases, would require molecular beacons for the expected mutational sequences, as well as wild-type beacons. Each well is then analyzed separately for the presence (or absence) of mutation by the fluorescent probes. The ratio of red/green is determined and normalized against known controls. Those wells containing mutant sequences are then analyzed by sequencing to determine the nature of the mutation. Although the authors chose to add the molecular beacons after amplification, the beacons may also be used during the amplification process and monitored in real time.
Expression Profile Analysis of Brain Aging
Published in David R. Riddle, Brain Aging, 2007
qPCR can quantitate amplicon product formation during each cycle of amplification and has eliminated many concerns that plague conventional PCR methods. Other advantages of real-time qPCR include high throughput capabilities, the ability to simultaneously multiplex reactions, enhanced sensitivity, reduced inter-assay variation, and lack of post-PCR manipulations. Various dye chemistries are currently being exploited in qPCR systems, including hydrolysis probes, molecular beacons, and double-stranded (ds) DNA binding dyes [40]. A prime example of a hydrolysis probe is the TaqMan assay. In this method, Taq polymerase enzyme cleaves a specific TaqMan probe during the extension phase of the PCR. The probe is dual-labeled with a reporter dye and a quenching dye at two separate ends; and as long as the probe is intact (in its free form), fluorescence emission of the reporter dye is absorbed by the quenching dye via fluorescence resonance energy transfer (FRET) [41, 42]. An increase in reporter fluorescence emission occurs when separation of the reporter and quencher dyes takes place during nuclease degradation in the PCR reaction [43]. This process occurs in every cycle of PCR and does not interfere with the exponential accumulation of the amplified product. Another methodology for qPCR-based detection involves the use of molecular beacons. Molecular beacons are probes that form a stem-loop structure from a single-stranded DNA molecule [44, 45]. They are particularly useful for identifying point mutations, as targets that differ by only a single nucleotide can be delineated. DNA binding dyes such as SYBR green incorporate selectively into ds DNA. SYBR green emits undetectable fluorescence levels when it is in its free form. Upon binding to ds DNA, a robust fluorescent signal is emitted [46]. An advantage of using ds DNA binding dye chemistry is that this method can be implemented to assay practically any target sequence with virtually any set of primers, making this application quite flexible and considerably less expensive than probe-based dye chemistries [47]. However, assay sensitivity can be diminished using a ds DNA binding dye system due to the increased risk of amplifying nonspecific PCR products. Careful primer set design and rigorous assay optimization can alleviate the majority of these nonspecific issues associated with ds DNA binding dyes.
Profile of the Alere i Influenza A & B assay: a pioneering molecular point-of-care test
Published in Expert Review of Molecular Diagnostics, 2018
Hongmei Wang, Jikui Deng, Yi-Wei Tang
The Alere i Influenza A & B assay, which is performed on the Alere i instrument, incorporates the Nicking Enzyme Amplification Reaction (NEAR) technique to detect and differentiate influenza viruses A and B [28,29]. Based on the NEAR technique, the templates are designed to target the Flu A-specific PB2 segment and the Flu B-specific PA segment. Fluorescently labeled molecular beacons are used to identify each of the specifically amplified RNA targets. As presented in Figure 1, the recognition region of the template (T2) binds to the complementary target region and is extended by the polymerase along the target and then another identical template binds to the same target, displacing the first template as it is extended (1a-3a). A different template (T1) binds to the complementary target region, is extended and creates a double-stranded nicking site, which is then cut by a Nicking Enzyme (4a-6a). Repeated nicking, polymerization, and strand displacement result in the rapid synthesis of amplification products (B). Due to the simplicity of the reaction, it is easy to replicate DNA at a constant temperature ranging from 55oC to 59oC and 20-mer to 30-mer products can be amplified 108 to 1010 -fold from genomic DNA in 2.5 to 10 min. Moreover, the method is able to amplify RNA without a separate reverse transcription step. Molecular beacons are coupled to detect the fluorescence signals that are optically filtered by a confocal lens and mirror arrangement. The results are calculated automatically and available on the user interface or can be exported for printing.
Detection of exosome miRNAs using molecular beacons for diagnosing prostate cancer
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Jinhee Lee, Min Hee Kwon, Jeong Ah Kim, Won Jong Rhee
The molecular beacon (MB) is a nano-sized oligonucleotide probe and a bi-labelled hairpin molecule with a fluorescent dye at one end and a quencher at the other end. Since the loop of the MB is designed to include a complementary sequence to target miRNA, it spontaneously hybridizes to its specific target and the hairpin-loop structure opens, resulting in fluorescence. Thus, MB can be used as an efficient probe for detecting specific exosome miRNA. Recently, we demonstrated an in situ detection method for the detection of specific miRNAs in exosomes using MBs [25,26]. This report proposed that the exosome miRNA can be detected directly using MB targeting miRNA and will open great opportunities for the diagnosis of various diseases, including cancer.
Diagnosing human cutaneous leishmaniasis using fluorescence in situ hybridization
Published in Pathogens and Global Health, 2021
Thilini Jayasena Kaluarachchi, Rajitha Wickremasinghe, Manjula Weerasekera, Surangi Yasawardene, Andrew J McBain, Bandujith Yapa, Hiromel De Silva, Chandranie Menike, Subodha Jayathilake, Anuradha Munasinghe, Renu Wickremasinghe, Shalindra Ranasinghe
Although FISH is widely used for the diagnosis of infectious diseases, studies using FISH for diagnosis of leishmaniasis are limited [7]. Application of FISH on Malaria positive blood smears had shown a sensitivity of 85.6% and a specificity of 90.6% [23]. When a species-specific digoxigenin labeled probe was used to diagnose canine CL on tissue, the sensitivity of the in situ hybridization assay was 70.6% with a specificity of 100% [8]. A German study which used the exact FISH probes that we used in our study reported positive results in 15 out of the 16 samples, with no false positives in detecting human CL [9]. Our SSS-FISH had a specificity of 96.7% and a sensitivity of 79.1%. For FFPE-FISH, we observed a higher sensitivity of 80.9% but a lower specificity of 93.4% than that reported in the German study. The lower specificity may be due to the background noise of the FISH assay. Background noise is a common drawback of FISH on FFPE tissue. Different protocols, models and optimizing steps for FISH on FFPE sections are available in literature [21,24–26]. The discussed protocol could be experimented with and improved to reduce the background noise. Optimizing fixation; adjusting incubating durations and chemical concentrations of immersion solutions and Proteinase K pretreatment; varying probe concentrations; and changing hybridization and post-hybridization washing conditions might help in achieving better results. Cryosections have been introduced superior to FFPE tissue in terms of better probe permeability [24]. Also, a specific molecular beacon could be tested with instead of the regular linear DNA probes to minimize the background noise [26].