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Using Molecular Methods to Identify and Monitor Xenobiotic-Degrading Genes for Bioremediation
Published in Ederio Dino Bidoia, Renato Nallin Montagnolli, Biodegradation, Pollutants and Bioremediation Principles, 2021
Edward Fuller, Victor Castro-Gutiérrez, Juan Carlos Cambronero-Heinrichs, Carlos E. Rodríguez-Rodríguez
Newer technologies with distinctive advantages have emerged. One of such technologies is digital PCR (dPCR), in which the sample is partitioned in droplets or distributed in nanolitre chambers, and then a standard Taq polymerase PCR reaction is used to amplify target DNA. However, different to qPCR, data is acquired at the reaction endpoint (Taylor et al. 2017). This minimizes the influence of sample contaminants on the amplification, and leads to more accurate and reproducible results when compared with qPCR, and allows for very low target quantitation (Rkki et al. 2014, Taylor et al. 2015). dPCR is yet to be used for pesticide-degrading gene quantification. However, it has been used for the determination of alkane hydrocarbon-degrading genes (alkB1) in a pilot scale biopile field experiment at freeze-thaw temperatures (Kim et al. 2018). The authors performed nutrient amendment in one of the biopiles and found that degrading gene copy numbers were higher in the treated biopile versus the untreated one during the seasonal freezing and thawing phases. More studies using this technology for xenobiotic-degrading gene quantification will undoubtedly emerge in the coming years as it becomes increasingly available.
Advanced Topics in Molecular Biology
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
A new approach to DNA quantification, enabled by modern microfluidics, is called digital PCR (dPCR). In dPCR, a sample is diluted into many individual PCR reactions, called partitions. Some of the partitions are positive, containing the target molecule, and others are negative. During amplification, dye-labeled probes indicate the target sequence; negative partitions contain no signal. After the PCR is run, the fraction of negative reactions is used to generate a count of the number of target DNAs in the sample. This eliminates the need for standards, can indicate small changes, and works in complex mixtures. To perform dPCR, a special dPCR machine is required. These are available commercially. Specialized chips are used for each reaction.
The lavatory lens: Tracking the global movement of pathogens via aircraft wastewater
Published in Critical Reviews in Environmental Science and Technology, 2023
Aaron Bivins, Robert Morfino, Andrew Franklin, Stuart Simpson, Warish Ahmed
Target specific methods include techniques typically used for diagnostic assays including PCR-based techniques (qPCR, RT-qPCR, dPCR, etc.). These techniques have been widely used for wastewater surveillance. While they can be very sensitive and specific, they require the design of reagents specific to the target of interest a priori, which precludes their usefulness for measuring unknown targets such as an entirely novel pathogen. Additionally, many qPCR platforms only allow testing for up to six targets simultaneously in a single experimental run. Nonetheless, PCR-based techniques could be very useful for sensitive screening of aircraft wastewater for known pathogens, especially in highly parallel and multi-target formats such as TaqMan array cards (TAC), Fluidigm BioMark HD real-time PCR or microarrays (Capone et al., 2020; Wang et al., 2002). Other analytical techniques that warrant further investigation include loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA) which can return results in shorter time frames and have been implemented on lateral flow test strips in clinical and environmental settings (Bivins et al., 2022; Kolm et al., 2019; Zasada et al., 2022). More novel techniques that could become relevant include mass spectrometry for the detection of proteins relevant to specific pathogens (Lara-Jacobo et al., 2022), enzyme-linked immunosorbent assays (ELISA) to detect specific antibodies (Agan et al., 2022), or clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic assays (Broughton et al., 2020; Kaminski et al., 2021).
Assays and enumeration of bioaerosols-traditional approaches to modern practices
Published in Aerosol Science and Technology, 2020
Maria D. King, Ronald E. Lacey, Hyoungmook Pak, Andrew Fearing, Gabriela Ramos, Tatiana Baig, Brooke Smith, Alexandra Koustova
Digital PCR (dPCR) carries out a single reaction within the sample, however, it is separated into a large number of partitions (droplets) and the reaction is carried out in each partition individually, allowing a more reliable collection and sensitive, digital measurement of nucleic acid concentration. By measuring the number of droplets that are fluorescing due to amplification, dPCR provides absolute quantification while qPCR relies on the standard curve. The method has been routinely used for clonal amplification of samples for next-generation sequencing. Damit (2017) developed a droplet microfluidics-based bioaerosol detector and analyzed bioaerosols impinged directly into the droplet.