Publication Date

2012-07-25

Availability

Open access

Embargo Period

2012-07-25

Degree Name

Master of Science (MS)

Department

Marine Biology and Fisheries (Marine)

Date of Defense

2012-05-08

First Committee Member

Michael Schmale

Second Committee Member

Jack Fell

Third Committee Member

Christopher Sinigalliano

Abstract

The monitoring of recreational waters is important to gauge risks to human health. The methods currently used are time-intensive (18-24 hours to test results) and because of this, results may be inaccurate. There are now molecular techniques capable of delivering faster results on multiple microbiological targets, including source tracking markers. However, these techniques cannot be utilized most effectively until extraction efficiencies of environmental DNA are known. Earlier studies of bacterial indicators in recreational beach waters using quantitative polymerase chain reaction (qPCR) showed variation in the DNA extraction efficiency of filtered marine water samples. Silica-based binding matrix micro-columns used in DNA extraction kits, have a limited binding capacity for DNA. The elution of DNA off the column appears to be released in a non-linear fashion, suggesting that for robust extraction a minimum amount of DNA must be bound. By using known quantities of Lactococcus lactis to mimic the characteristics of enterococci, along with a minimum amount of background DNA it is possible to determine DNA recovery for the extraction/purification process. This was accomplished by adding a known amount of Lactococcus lactis cells to samples of recreational water along with large volumes of water or known amounts of background DNA. After extraction, the Lactococcus lactis DNA was quantified using qPCR. The percentage of Lactococcus lactis remaining in the elution was then used to quantify the results from the enterococci assay. Source tracking markers for dog associated bacteroidales and gull associated catellococus were adapted into Taqman™ chemistry qPCR assays. These assays were then developed with standards and tested against South Florida water samples. These assays can help provide new information to the contributor of fecal waste to beach and coastal environments. These assays performed well in the field and should be incorporated into the testing of beach water that allows dogs, or has a large gull population.

Keywords

Extraction controls qPCR; Water quality; enterococci; Lactococci lactis; Gull-2; Dog associated bacteroidales

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