Quantifying Environmental Microorganisms and Viruses Using qPCR

Overview

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

Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, is a widely-used molecular technique for enumerating microorganisms in the environment. Prior to this approach, quantifying microorganisms was limited largely to classical culture-based techniques. However, the culturing of microbes from environmental samples can be particularly challenging, and it is generally held that as few as 1 to 10% of the microorganisms present within environmental samples are detectable using these techniques. The advent of qPCR in environmental microbiology research has therefore advanced the field greatly by allowing for more accurate determination of concentrations of microorganisms such as disease-causing pathogens in environmental samples. However, an important limitation of qPCR as an applied microbiological technique is that living, viable populations cannot be differentiated from inactive or non-living populations.

This video demonstrates the use of qPCR to detect pepper mild mottle virus from an environmental water sample.

Procedure

1. Sample Collection

Collect soil using an auger or shovel to a determined depth. If collecting soil from the rhizosphere, only collect soil within 7 mm around plant root, by hitting excess soil off the root and scraping desired soil into a collection barrel.
Place a sterile Nalgene bottle into dipping stick. Hold the end of the stick and collect water by submerging bottle. Place bottle into a cooler with ice.
Transfer samples to the laboratory.

2. Nucleic

Application and Summary

The ability to quantify targeted genomic segment copies using the qPCR technique is of importance in a number of scientific fields. Example applications include:

(1) Enumerating pathogens in water, soil, food, surfaces, etc.

Real-time PCR is utilized to enumerate pathogens in various environments. During outbreaks, water and soil samples can be analyzed for the pathogen of interest to find the source causing spread. The source can then be further analyzed t

References

Zhang, T., Breitbart, M., et al. RNA Viral Community in Human Feces: Prevalence of Plant Pathogenic Viruses. PLoS Biology. 4, e3 (2005).
Haramoto, E., et al. Occurrence of Pepper Mild Mottle in Drinking Water Sources in Japan. Applied Environmental Microbiology. 79, 7413-7418 (2013).