Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

Overview

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz

Polymerase chain reaction (PCR) is a technique used to detect microorganisms that are present in soil, water, and atmospheric environments. By amplifying specific sections of DNA, PCR can facilitate the detection and identification of target microorganisms down to the species, strain, and serovar/pathovar level. The technique can also be utilized to characterize entire communities of microorganisms in samples.

The culturing of microorganisms in the laboratory using specialized growth media is a long-established technique and remains in use for the detection of microorganisms in environmental samples. Many microbes in the natural environment, while alive, maintain low levels of metabolic activity and/or doubling times and are thus referred to as viable but non-culturable (VBNC) organisms. The use of culture-based techniques alone cannot detect these microbes and, therefore, does not provide a thorough assessment of microbial populations in samples. The use of PCR allows for the detection of culturable microbes, VBNC organisms, and those that are no longer alive or active, as the amplification of genetic sequences does not generally require the pre-enrichment of microorganisms present in environmental samples. However, PCR cannot differentiate the aforementioned states of viability and activity. When combined with one or more culture-based techniques, the viability of certain subsets of microorganisms may still be determined.

Procedure

1. Sample Collection

Collect soil using an auger or shovel down to a determined depth. If collecting soil from the rhizosphere (the narrow region of soil surrounding and influenced by plant roots and their associated microbes), only collect directly from around the plant roots by hitting the soil off into a collection barrel.
Collect water sample by dipping a sterile plastic bottle into the water while holding the end of the dipping stick.

2. Nucleic Acids Extraction

Results

In Figure 1, the DNA ladder (lane 1) provides a reference for the size and approximate concentration for bands of the PCR products. The negative control (lane 2) does not contain any genetic material, while the positive control (lane 3) is amplified from templates known to contain the target DNA to indicate size and location of target bands. Samples 4, 6, 8, and 9 exhibit similar band pattern as the positive control, therefore indicating that these samples contain the target genetic material. It can be i

Application and Summary

PCR can be employed to quickly determine the presence or absence of pathogens in the environment. For example, primers specific to the brain-eating amoeba, Naegleria fowleri, will amplify DNA and produce strong bands on a gel if the organism is present in a sample. If a single organism is not the main interest, but rather genes associated with toxin production from a variety of organisms, PCR can also be used to determine the presence or absence of these specific genetic materials.