Paul, J. H.. University of South Florida, firstname.lastname@example.org
Ewert, M. H.. University of South Florida, email@example.com
Wawrik, B. University of South Florida,
Stokes, R. University of South Florida,
NOVEL RNA TECHNOLOGY FOR MICROBIAL DETECTION IN AQUATIC ENVIRONMENTS
Nucleic acid-based sequence amplification (NASBA) is a method of nucleic acid amplification which primarily amplifies RNA targets and yields RNA. Unlike PCR, no Taq?polymerase is involved and the process occurs at 41o for 90 minutes. An enzyme cocktail comprised of reverse transcriptase, RNase H, and T7 RNA polymerase are employed to produce an antisense RNA message product. We have been investigating a novel RNA extraction technique which uses guanidinium isothiocyanate and silica particles ("Boom technology") to extract mRNA and then amplify the message by NASBA. We have used this technology to detect gene expression of the Ribulose bisphosphate carboxylase large subunit gene (rbcL) in phytoplankton and the heat shock 70 gene (hsp70) in pathogenic protozoans. NASBA amplification products have been visualized on gels and by linking to an electrochemiluminescence (ECL) assay developed by Organon Teknika. As few as 10 protozooal oocysts could be detected by NASBA and ECL detection. Potential application of this technology to environmental samples will be discussed.
Day: Tuesday, Feb. 2
Location: Sweeney Center