RAPID COLORIMETRIC IDENTIFICATION OF ESCHERICHIA COLI SHIGA TOXINS 1 AND 2, AS WELL AS EAE AND AGGR, USING LOOP MEDIATED ISOTHERMAL AMPLIFICATION FOLLOWED BY PNA/AUNP DETECTION

<p><em>E</em><em>scherichia coli </em>are normal residents in the intestinal tracts of humans and animals. However, some <em>E. coli </em>strains produce Shiga toxins and attachment factors that make them pathogenic. Pathogenic <em>E. coli</em> strains can cause fever, diarrhea, vomiting and abdominal pain. In severe cases they cause hemorrhagic colitis or kidney failure. Current laboratory methods can detect pathogenic <em>E. coli</em> within 24-48 hours. This timeframe puts more people at risk of exposure to pathogenic <em>E. coli</em>.<em> </em>This research utilizes a DNA amplification technique called Loop-Mediated Isothermal Amplification (LAMP) to detect different strains of pathogenic <em>E. coli</em>. Common visualization methods of LAMP amplicons include using LAMP reaction buffer containing a pH indictor dye. This method allows for easy colorimetric detection of LAMP amplicons. If the target sequence was amplified, hydrogen ion release during phosphodiester bond formation will decrease the pH of the reaction. This causes the pH indicator dye to turn from pink to yellow. There are disadvantages to a visualization method that relies solely on pH changes. This is an indirect measure of DNA amplification of the target sequence. To make the LAMP assay more specific, a peptide nucleic acid (PNA) probe complementary to either the Shiga toxin 1 or 2 genes, <em>eae</em> or <em>aggR</em> was introduced, along with gold nanoparticles (AuNP). The PNA-AuNP detection system does not depend on pH change, rather an aggregation or monodispersing of gold nanoparticles that is sequence specific. When the PNA probe detects the specified DNA sequence, it produces a distinguishable color change from blue to red if the target LAMP amplicon DNA is present. This research demonstrates the ability to detect pathogenic <em>E. coli</em> species’ DNA and live cells, as well as from inoculated lettuce sampling, within 90 minutes using LAMP followed by a PNA-AuNP detection system.</p>

Name thesis:degree_name

Master of Science

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Biology

Year dc:date.available

2020

Author dc:creator

• Nelson, Rachael

Contributors dc:contributor

• Dr. Josh Sharp

• LAMP
• PNA. AuNP
• E. coli
• pathogens
• microbiology
• rapid
• detection

Repository record dc:identifier

https://commons.nmu.edu/theses/639

OAI identifier oai:identifier

oai:commons.nmu.edu:theses-1677