title: Characterization of the effects of heat stress on the DNA-intercalating dye EvaGreen for potential use with the joint biological agent identification and diagnostic system.
abstract: Although advances in real-time polymerase chain reaction (PCR) technology and equipment have facilitated field research, only a limited selection of reagents do not require cold storage. This study explored the temperature stability of the commercially available DNA-intercalating dye EvaGreen after exposure to a spectrum of temperatures for 176 days by analyzing quantification cycle (Cq) and end fluorescence levels during amplification of the invA gene of Salmonella typhimurium. To further characterize potential dye stability, the effects of small differences in dye volume were examined and dye samples were subjected to an Air Force deployment to the Middle East. Significant differences in Cq and end fluorescence were found; however, the magnitude of mean Cq differences was less than one cycle and the magnitude of mean fluorescence differences was less than that attributable to a difference of 0.25 μL of dye per 25 μL reaction. Liquid EvaGreen dye may thus be stable at temperatures as high as 65°C for up to 6 months for use in real-time PCR. These results warrant further investigation by using liquid EvaGreen dye to adapt traditional lab-based real-time PCR assays for Joint Biological Agent Identification and Diagnostic System use and testing the assays in the field.
authors.affiliation: United States Air Force Academy, 2355 Faculty Drive, USAF Academy, CO 80840.
authors.forename: Craig D
authors.initials: CD
authors.lastname: Nowadly
authors.affiliation: United States Air Force Academy, 2355 Faculty Drive, USAF Academy, CO 80840.
authors.forename: Jason W
authors.initials: JW
authors.lastname: David
authors.affiliation: United States Air Force Academy, 2355 Faculty Drive, USAF Academy, CO 80840.
authors.forename: Melanie L M
authors.initials: ML
authors.lastname: Grogger
authors.affiliation: United States Air Force Academy, 2355 Faculty Drive, USAF Academy, CO 80840.
authors.forename: Erik R
authors.initials: ER
authors.lastname: Demkowicz
authors.affiliation: Harding University College of Pharmacy, 915 East Market Street, Searcy, AR 72149.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Atchley
authors.affiliation: United States Air Force Academy, 2355 Faculty Drive, USAF Academy, CO 80840.
authors.forename: Donald V
authors.initials: DV
authors.lastname: Veverka
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis*; D004355:Drug Stability; D004356:Drug Storage; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006358:Hot Temperature; D007364:Intercalating Agents / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012486:Salmonella typhimurium / Q000235:genetics; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D007364:Intercalating Agents; C075518:invA protein, Bacteria