China
Africa
Explore chapters and articles related to this topic
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
Since its introduction and dissemination, most studies dealing with pesticide catabolic gene quantification have relied on real-time PCR, also known as quantitative PCR (qPCR) (Klein 2002). qPCR allows the monitoring of the PCR amplification and quantification of its products in real time through fluorescence measurements. This technique can provide a sensitive, robust, and fast estimation of catabolic gene copy numbers in samples taken from pesticide bioremediation settings. Despite the obvious advantages of the method, the adoption and transfer of inadequate and varied protocols amongst researchers has led to problems when trying to reproduce data (Hayden et al. 2008, Jaworski et al. 2018, Taylor et al. 2019). Therefore, the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines have been proposed to increase the uniformity and reliability of published research using this method (Bustin et al. 2009).
A framework for standardized qPCR-targets and protocols for quantifying antibiotic resistance in surface water, recycled water and wastewater
Published in Critical Reviews in Environmental Science and Technology, 2022
Ishi Keenum, Krista Liguori, Jeanette Calarco, Benjamin C. Davis, Erin Milligan, Valerie J. Harwood, Amy Pruden
Table 3 summarizes key quality assurance/quality control and reporting considerations for qPCR-based monitoring of antibiotic resistance in water environments. There are numerous specific aspects of qPCR that must be considered to ensure that the data produced are representative and comparable across studies. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., 2009) provides a helpful starting place. MIQE establishes common reporting guidelines for qPCR, including assay efficiencies, LOD, LOQ, R2 for calibration curves, PCR inhibition, and inclusion of negative controls. However, of the 117 studies assessed in this work only 69 (59%) reported any of these parameters. This is consistent with the recent findings of Borchardt et al. (2021), who recently further adapted MIQE guidelines for environmental microbiology samples, noting that only 33% of the evaluated studies implementing digital and qPCR complied with MIQE guidelines. The lack of reporting in regards to LOD and LOQ is especially problematic, as a non-detect in one sample medium may not be equivalent to a non-detect for another medium, due to factors such as differences in sample volumes, concentration factors, template volume in assays, and dilution of DNA extracts (U.S. Environmental Protection Agency 2012). In many studies, it also is not always clear if the LOD/LOQ values provided are in the units of gc/µL of DNA extract or original sample volume, with sample volume being more informative and relevant for comparison to other studies.
Effects of Bisphenol A on Foxl2 gene expression and DNA damage in adult viviparous fish Goodea atripinnis
Published in Journal of Toxicology and Environmental Health, Part A, 2020
Isabel Cervantes-Camacho, Sandra M. Guerrero-Estévez, María Fernanda López, Ernesto Alarcón-Hernández, Eugenia López-López
RT-qPCR was performed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al. 2009) (Supplementary Table S2). RT-qPCR was run using specific primers and a probe for each gene designed by Applied Biosystems™ (Thermo Fisher Scientific). All tests were entered in the NCBI probe database and conducted in quadruplicate. TaqMan® probes were labeled with the fluorophore reporter 6-carboxylfluorescein (FAM) dye attached to the 5ʹ end and a quencher non-fluorescent dye with MGB (Minor Groove Binder) attached to the 3ʹ end. The reaction was carried out in the instrument Step One™ Real-Time PCR System Thermal Cycling Block (Applied Biosystems™). Each PCR reaction contained 20x of each primer and each probe (final concentration 900 nm and 250 nM of each initiator and each probe, respectively), 10 µl TaqMan® Universal Master Mix II (Applied Biosystems™), and 5 µl cDNA sample, brought to a final reaction volume of 20 µl. The reaction volume was transferred to FG Fast optical 48-well RXN plates and covered with FG Optical adhesive film. The reaction conditions were 50°C for 2 min, 95°C for 10 min, and 40 cycles each consisting of 95°C for 15 sec followed by 60°C for 1 min.