Date of Award

12-2003

Level of Access

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Microbiology

Advisor

Michael E. Vayda

Second Committee Member

Stellos M. Tavantzis

Third Committee Member

Carol H. Kim

Abstract

A molecular padlock assay was developed and assessed for detection of Listeria monocytogenes that operated in crude plant extracts. The molecular padlock assay was developed by Liu et al. (1996) and modified by Lizardi et al. (1998). We further modified and described a padlock probe that detected L. monocytogenes oligonucleotide, cDNA and genomic DNA containing a 16s rRNA sequence (GenBank Acc. No. X56 153). This technique was effective in the presence of crude potato leaf extracts in contrast to PCR, which failed to detect the presence of L. monocytogenes targets in crude leaf extracts. Sensitivity of the padlock procedure was determined to be 0.02 ng using L. monocytogenes genomic DNA target templates and 0.0025 nM L. monocytogenes 40 nt oligonucleotide target sequences in an aqueous solution. Results also showed the efficacy of molecular padlocks to detect L. monocytogenes pathogens in a 5 pg potato leaf RNA background and crude leaf extracts, although sensitivity of the assay is insufficient to dispense with a pre-enrichment step for reliable detection of L. monocytogenes. The advantage of the molecular padlock assay over other FDA approved serological and molecular-based assays, is that it may be possible to design the assay to be species or strain specific while still retaining the ability to be performed in crude plant extracts.

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