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Laboratory analyses of cyanobacteria and water chemistry
Published in Ingrid Chorus, Martin Welker, Toxic Cyanobacteria in Water, 2021
Judit Padisák, Ingrid Chorus, Martin Welker, Blahoslav Maršálek, Rainer Kurmayer
In contrast, quantitative PCR allows the determination of actual gene copy numbers (that can be used to approximate cell equivalents) and thus, by choosing the right targets for PCRs, the proportion of toxigenic genotypes present in a sample. In quantitative PCR (qPCR), amplification of the target gene is followed in real time via the detection of a fluorescent signal generated from DNA strand-intercalating dyes at each PCR cycle (e.g., SYBR Green). qPCR is based on the principle that the target DNA sequence is doubled in each cycle and that the dynamic increase of the recorded amplification reflects the amount of target sequence originally present. Theoretically, the more target sequence (or target genotypes) can be found in a specific sample, the earlier the amplification curve exceeds a predefined fluorescence threshold. The PCR cycle when this threshold is crossed is called a threshold cycle, Ct, or quantification cycle, Cq(-value). The use of fluorescent dyes makes qPCR most sensitive with calibration curves showing a wide dynamic range (up to seven orders of magnitude). Detailed information on the application of (q)PCR in the analysis of toxin genes has been compiled through the EU-initiative CyanoCOST (Rantala-Ylinen et al., 2017).
Methods of Identifying Microbiological Hazards in Indoor Environments
Published in Rafał L. Górny, Microbiological Corrosion of Buildings, 2020
The identification technique based on classical PCR and sequencing, although enabling species identification, does not allow determination of the number of microorganisms in the test material. A quantitative PCR technique (qPCR, Real-Time PCR), because of the use of fluorescently marked oligonucleotides (called molecular probes) and adequate detection systems, allows monitoring of the increase in the number of product copies during the reaction. The advantage of this modification is both the possibility of detecting a specific DNA sequence in the test material, and also of quantifying it (although it is possible only if, in parallel with the carried-out reaction, we analyse samples with a known number of copies of the tested sequence, which allows plotting of the curve). Their adequate selection is crucial for quantitative analysis and may pose a challenge both in technical and scientific terms for laboratories studying the microbiological quality of the environment [Hospodsky et al. 2010; Nadkarni et al. 2002]. In recent years, the qPCR technique has been increasingly used in aerobiology to estimate the total count of bacteria, fungi or archaeons. Its use in bioaerosol studies in various environments showed that the number of microorganisms determined on the basis of these techniques was 100–1000 times higher than that determined by culture techniques [Blais-Lecours et al. 2015].
Quantitative PCR Approaches for Predicting Anaerobic Hydrocarbon Biodegradation
Published in Kenneth Wunch, Marko Stipaničev, Max Frenzel, Microbial Bioinformatics in the Oil and Gas Industry, 2021
Courtney R. A. Toth, Gurpreet Kharey, Lisa M. Gieg
Quantitative PCR is a well-established molecular method that allows for the simultaneous detection and quantification of target DNA. In conventional PCR, the amplified DNA product, also known as an amplicon, is detected in an end-point analysis. In qPCR, the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle. Although the concept is relatively simple, there are specific issues in qPCR that developers and users of this technology should bear in mind.
Microbiology in Water-Miscible Metalworking Fluids
Published in Tribology Transactions, 2020
Frederick J. Passman, Peter Küenzi
Quantitative PCR (qPCR), also known as real-time PCR, is a DNA amplification technique using fluorescent reporter dyes that bind to DNA. Whereas traditional PCR measures the accumulation of the product only at the end of all amplification cycles, qPCR quantifies the amplification as it occurs by measuring the increase in fluorescent signal that is dependent and directly proportional to the amount of double-stranded DNA produced during each cycle. qPCR can conveniently be used for enumeration and specification, if the appropriate sequence information of the microorganism in question is available.
Environmental sampling for disease surveillance: Recent advances and recommendations for best practice
Published in Journal of the Air & Waste Management Association, 2023
Joshua L. Santarpia, Elizabeth Klug, Ashley Ravnholdt, Sean M. Kinahan
Polymerase chain reaction (PCR and various types):a technique that can copy specific DNA segments millions of times using special enzymes (polymerases). Quantitative PCR (qPCR) is a method to quantify the initial amount of DNA using fluorescent molecules. Reverse transcriptase (RT) PCR is a method to first convert RNA to DNA, using an enzyme, and then amplify it using the PCR process.