Evaluation of PCR-based quantification techniques to estimate the abundance of atrazine chlorohydrolase gene atzA in rhizosphere soils.

There are many challenges in the accurate quantification of bacterial genes, such as the atrazine-degrading enzyme antA from Pseudomonas sp. strain ADP, from soil samples. We compared four quantitative methods for enumeration of atrazine-degrading bacteria in rhizosphere environments and utilized the optimal probe-based real-time polymerase chain reaction (PCR)-based method in an ongoing bioremediation experiment to monitor atzA copy number over time. We compared three quantitative PCR (qPCR) based methods--quantitative competitive PCR and two real-time qPCR methods--to traditional dilution-plate counting techniques. The optimal real-time qPCR assay was then used to monitor atzA copy number over time in the robust atrazine-degrading Pseudomonas sp. strain ADP-spiked rhizosphere environment. The use of sensitive and reliable probe-based real-time qPCRs for the enumeration of bacterial catabolic genes allows for their detection from soil samples and monitoring of potential degradative populations over time. The addition of arrazine-biodegrading bacteria into arrazine-contaminated sites to remove entrapped atrazine is a promising approach for mitigating atrazine pollution and its metabolites. The methodology contained herein will allow for optimal monitoring of atzA in rhizosphere soil with or without the addition of biodegradative Pseudomonas sp. strain ADP of bacteria.