Standardized Procedures for Use of Nucleic Acid-Based Tools

Carmen Lebrón | Naval Facilities Engineering Command, Expeditionary Warfare Center

ER-1561

Objective

The use of molecular biological tools (MBTs) for the detection and quantification of microbial biomarkers (e.g., DNA/RNA) in environmental samples is rapidly increasing as remedial practitioners seek to improve the design, field performance, and monitoring of bioremediation.

This project focused on identifying and minimizing the causes of variability during quantitative real-time polymerase chain reaction (qPCR) enumeration of genes of interest in groundwater, with the goal of developing a foundation for the development of standardized methods for collection, preservation, transport, storage, and processing of environmental qPCR samples collected from contaminated sites.

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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.
  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.  
  6.  Determining the distribution of Dehalococcoides (Dhc) cells between aquifer solids and groundwater to better understand and interpret groundwater enumeration results.

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Results

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 to 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 with Dhc comprised the majority of cells in the presence of growth substrates and cells were mainly planktonic in the absence of growth substrates.

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Benefits

MBTs such as qPCR have the potential to accurately enumerate microbial cells, including Dhc, in groundwater samples if proper procedures and appropriate controls are integrated into the process.  The development of optimized methods, including the use of a project developed MIAC, allowed significant improvements in data quality and the ability to better detect and correct for matrix interference. Understanding the distribution of Dhc between sediment and groundwater will allow more accurate conclusions to be drawn regarding total Dhc biomass in aquifers using groundwater data. The ability to better quantify Dhc at a variety of sites with high precision, in multiple labs, will increase remediation practitioners’ confidence in remediation focused qPCR analysis and their ability to manage bioremediation projects effectively.

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Points of Contact

Principal Investigator

Ms. Carmen Lebrón

Naval Facilities Engineering Command, Expeditionary Warfare Center

Phone: 805-982-2907

Fax: 805-982-4304

Program Manager

Environmental Restoration

SERDP and ESTCP

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