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Lebron FINAL REPORT Standardized Procedures for Use of Nucleic Acid-Based Tools SERDP PROJECT ER-1561. Strategic Environmental Research and Development Program, Washington, DC, USA
Citation:
Lebron, C. Lebron FINAL REPORT Standardized Procedures for Use of Nucleic Acid-Based Tools SERDP PROJECT ER-1561. Strategic Environmental Research and Development Program, Washington, DC, USA. U.S. Environmental Protection Agency, Washington, DC, 2014.
Impact/Purpose:
SERDP project ER-1561, Standardized Procedures for Use of Nucleic Acid-Based Tools for Microbial Monitoring, focused on identifying and minimizing the causes of variability during qPCR enumeration of genes of interest in groundwater, especially dhc. The goal was to develop a foundation for the development of standardized methods for collection, preservation, transport, storage and processing of environmental qPCR samples collected from contaminated sites.
Description:
Technical Approach A technology review on the status of MBTs was performed at the beginning of the project to determine MBT use in other industries. The review focused project goals and activities, which included: 1) Comparing qPCR to non-PCR-based enumeration methods to validate and increase confidence in qPCR methods; 2) Comparing and assessing baseline variability within and between laboratories at the outset of the project using a multi-laboratory “round robin” approach; ER-1561 2 July 2014 3) Development and testing of a microbial internal amplification control (MIAC), for use in spike and recovery protocols, in the extraction and qPCR process to assess biomarker extraction efficiency and matrix interference; 4) Assessment and optimization of methods including sampling, biomass collection, nucleic extraction, and qPCR protocols; 5) Assessment of inter-laboratory variability after integration of the MIAC and optimized methods; and 6) Determining the distribution of Dhc cells between aquifer solids and groundwater to better understand and interpret groundwater enumeration results. Results and Discussion Significant results of the project included. 1) Obtaining agreement of qPCR methods with non-molecular methods such as plate counts and microscopy, these speak to the fundamental accuracy of qPCR. 2) The development of an effective MIAC, consisting of a modified E. coli with a chromosomal insertion of a firefly luciferase gene, for monitoring by qPCR. The MIAC can be used quantify biomass losses, qPCR inhibition and flag suspect samples/analyses. 3) Optimization of methods and protocols so that five independent labs were able to generate similar Dhc enumeration results for identical groundwater samples with maximum variability decreasing from as high as 40-fold to as little as 1.1-fold over the course of the study. 4) An improved understanding of the distribution of Dhc between aquifer solids and groundwater derived from column experiments. This study indicated that aquifer solids associated Dhc, comprised the majority of cells in the presence of growth substrates and cells were mainly planktonic in the absence of growth substrates.
URLs/Downloads:
https://www.serdp-estcp.org/Program-Areas/Environmental-Restoration/Contaminated-Groundwater/Monitoring/ER-1561/ER-1561/(modified)/06Oct2014