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Quantitative Polymerase Chain Reaction for Companion Diagnostics and Precision Medicine Application
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
Joel L. Ramirez, Johannes R. Kratz
Unique to qPCR is the addition of a substance marked with a fluorophore that fluoresces when excited by a specific wavelength. This fluorophore can be non-specific and have the capability to bind to any dsDNA present, giving an estimation of the amount of DNA being replicated. Fluorophore markers can also be specific and contain a DNA sequence that is complimentary to the gene of interest (TaqMan technology), although this method is more expensive and more difficult to prepare then utilization of nonspecific fluorophores. Fluorescently labeled nucleotides can be used as well (SYBR technology). qPCR has a fourth stage that is characterized by excitation of the fluorophore and measurement of fluorescence. Computer software can then detect the amount of fluorescence and account for the current PCR cycle to estimate the amount of DNA from the gene of interest. This allows for direct quantification of products generated with each cycle of PCR in realtime, whereas conventional PCR would have to be proceeded by other separate techniques to quantify DNA.
Angiostrongylus
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
Praphathip Eamsobhana, Hoi-Sen Yong
Recently, a sensitive quantitative real-time TaqMan PCR assay based on the amplification of ribosomal internal transcribed spacer-1 (ITS-1) has been developed [134]. This real-time PCR test has been applied to detect DNA amounts equivalent to less than one larva in human CSF samples with a sensitivity of down to 10 plasmid copies per reaction [134]. The assay has also been reported to support the diagnosis of angiostrongyliasis in eosinophilic meningitis cases by detecting A. cantonensis DNA in CSF [135,136].
Molecular Methods for the Diagnosis of Fungal Infections
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Adam M. Bressler, Christine J. Morrison
One such real-time, quantitative PCR method is the TaqMan system (Applied Biosystems, Inc., Foster City, California, USA). This test is a 5′-exonuclease assay that uses hybridization of a probe labeled at one end with a fluorescent reporter dye and at the other end with a quencher dye. No signal is generated while the quencher dye remains in close proximity to the reporter dye. As DNA amplification occurs, however, the 5′ → 3′ exonuclease activity of the Taq DNA polymerase separates the quencher dye from the reporter dye and allows a signal to be generated; the signal is then detected fluorometrically in the same system.
Cytogenetic and molecular genetic methods for chromosomal translocations detection with reference to the KMT2A/MLL gene
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Nikolai Lomov, Elena Zerkalenkova, Svetlana Lebedeva, Vladimir Viushkov, Mikhail A. Rubtsov
PCR-based validation. PCR is applied to confirm the presence of a chimeric gene/transcript in a sample. Depending on whether localization of the breakpoint is known exactly or approximately, PCR with genomic DNA or cDNA is applied, respectively. (A, left) Genomic DNA PCR is performed with primers flanking a breakpoint (primer annealing sites mapped on the rearranged gene scheme); a nested PCR approach can be applied to increase the sensitivity and specificity. The breakpoint region (highlighted in orange) is a region of a rearranged gene within which a breakpoint (red asterisk) may be located. (a, right) RT-PCR utilizes cDNA synthesis followed by amplification with a pair of primers that bind to exon sequences adjacent to the breakpoint. (b) If multiple sites can be implemented in a translocation, a set of primers selected for different variants of chimeric mRNAs is simultaneously used for RT-PCR (i.e. a multiplex PCR approach). PCR products are detected either with an end-point strategy (e.g. gel-electrophoresis) or quantitative PCR strategy (e.g. qPCR with TaqMan probes). (c) In qPCR, the TaqMan assay utilizes oligonucleotide labeled with a pair of fluorescent reporter-quencher tags. During PCR amplification, the TaqMan probe is annealed to the target region and tags are removed by exonuclease activity of a DNA polymerase. Therefore, a quencher is moved away from a reporter, and a fluorescence signal is emitted and can be detected in each PCR cycle.
Smartphone technology facilitates point-of-care nucleic acid diagnosis: a beginner’s guide
Published in Critical Reviews in Clinical Laboratory Sciences, 2021
Vinoth Kumar Rajendran, Padmavathy Bakthavathsalam, Peter L. Bergquist, Anwar Sunna
Hydrolysis or TaqMan (Thermo Fisher Scientific, MA, USA) probes and intercalating dyes are used most commonly for detection by real-time PCR [35,36]. A TaqMan probe is designed with a reporter at the 5′ end and a quencher at the 3′ end of the DNA. The reporter dye emission is suppressed by the quencher dye due to the close proximity of the dyes. These probes are designed to anneal to specific sequences of the template that sit in the path of the polymerase enzyme. The 5′-exonuclease activity of polymerase cleaves the probe, thus increasing the distance between reporter and quencher dyes, and the amount of reporter fluorescent signal increases proportionally to the amount of product formed [37]. Although real-time PCR has found widespread utility for nucleic acid amplification, it requires the comparison of the unknown to a standard curve to obtain quantitative information [34].
The high prevalence of Clostridioides difficile among nursing home elders associates with a dysbiotic microbiome
Published in Gut Microbes, 2021
John P. Haran, Doyle V. Ward, Shakti K. Bhattarai, Ethan Loew, Protiva Dutta, Amanda Higgins, Beth A. McCormick, Vanni Bucci
NK9-NK11 for the repetitive domain of the tcdA gene and NK104-NK105 for the tcdB gene. This method is based on TaqMan technology and has been shown to have very good test characteristics with 100% sensitivity and 98.3% specificity.92 We used the SLAN RTPCR detection system (LG Life Science) according to the manufacturer’s instructions.92 Each sample needed to be positive for both tcdA and tcdB genes to then be categorized as positive for C. difficile. Estimation of C. difficile colonization using qPCR instead than directly from shotgun metagenomics has been used several times previously by us and others93,94 and it allows to determine with better resolution the presence of this bacterium which is usually found at very low abundance in the GI tract (<1%) and could be undetected from metagenomic sequencing.