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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.
Methods of Identifying Microbiological Hazards in Indoor Environments
Published in Rafał L. Górny, Microbiological Corrosion of Buildings, 2020
Among the most promising techniques for the assessment of microbiological differentiation of bioaerosol is also the droplet digital PCR (ddPCR). It was based on the fractionation of the test sample into droplets (formed in a water-oil emulsion) of nanolitres in volume each and an independent amplification of the DNA matrix in each of them. In the traditional qPCR technique, a single sample allows only one measurement. In the ddPCR, a split sample enables the measurement of thousands of independent amplification events in each drop, which is used, among other things, for the analysis of copy number variants, detection of rare sequences, quantification of microbial nucleic acids, analysis of gene expression and genotyping of single nucleotide polymorphism. Compared to other systems, the ddPCR technique uses a smaller volume of samples and reagents, which reduces the total cost of testing while maintaining the sensitivity and precision inherent to digital PCR [Hindson et al. 2011; Mazaika and Homsy 2014].
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.
Developing mitochondrial DNA field-compatible tests
Published in Critical Reviews in Environmental Science and Technology, 2022
Bidhan C. Dhar, Christina E. Roche, Jay F. Levine
Droplet digital PCR is extensively being used in biomedical (Cao et al., 2017; Memon et al., 2017) and environmental research (Giraldo et al., 2018; He et al., 2019). Droplet digital PCR demonstrates more sensitivity and clarity across various applications. For instance, recently an environmental ddPCR assay was developed for fecal waste source tracking in surface wastewater and freshwater samples targeting the cytb gene of human mtDNA (HCYTB; Zhu et al., 2020). The authors compared the results with the human-correlated Bacteroides, a genus of gram-negative, obligate anaerobic bacteria. The authors assessed and compared the detection of human and animal feces in three geographic areas. The specificity of the HCYMT marker was markedly higher than that observed using Bacteroides detection.
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.