Advisory Committee Chair
Asim K Bej
Advisory Committee Members
Thane Wibbels
Chris Murdock
Document Type
Thesis
Date of Award
2008
Degree Name by School
Master of Science (MS) College of Arts and Sciences
Abstract
Salmonella is a causative agent for food and water borne gastroenteritis in humans. A multiplex PCR-based detection revealing total and potentially virulent strains of Salmonellae was developed using oligonucleotide primers. These primers targeted chromosomally located invA gene generating a 274 bp and Salmonella virulence plasmid-borne spvB gene generating 561 bp amplified DNA fragments. The invA gene product brings about the invasion of the cells on the intestinal epithelium and is used as the marker for total Salmonella detection. The occurrence and distribution of these genes in 90 strains exhibited 100% amplification of invA gene whereas only 22% of spvB gene implying their virulent nature. Real-Time PCR platforms, in contrast to conventional PCR, are rapid and obviate the need for further confirmation steps such as gel electrophoresis. The objective of the present study was to establish simple and specific methods to detect invA and spvB gene coding using SYBR-green Real-Time PCR. In each reaction, 16S rRNA gene was targeted as a competitive Internal Amplification Control (IAC) and used to identify detection of any false positives. The sensitivity of the reaction at all 3 targeted gene loci was found to be 1 pg. The analysis showed specific PCR products identified by melting curve analysis, and a reproducible melt temperature in the range of 88.0 to 89.53°C (spvB), 83.06 to 85.48°C (invA) and 86.32 to 87.99°C (16S rRNA gene) was observed for all ii i Salmonella strain. Negative controls and non-Salmonella strains showed an IAC-specific melt peak at 77.0±2.0 °C, giving evidence of the specificity of the method. The sensitivity of Real-Time PCR was found to be 1 ng that is comparable to approximately 104 cfu S.enterica. The sensitivity was confirmed in pure cultures as well as in oyster tissue homogenate enriched with Salmonella. Another aspect of this study was to study the antibiotic resistance profile of these strains to one representative each of the most commonly groups of antibiotics being currently used to treat salmonellosis. Amikacin (Aminoglycoside), Ceftiofur (Cephalosporin) and Ofloxacin (Fluoroquinolone) were completely effective against all the strains whereas 7.7% of the 90 strains tested showed resistance to Ampicillin (Beta-lactam). The resistance to beta lactams has often been attributed to the presence of the virulence plasmid and hence the spv genes. The results show that most likely the virulence genes do not play a role in developing resistance to beta-lactams as only 62.5% of the pathogenic strains were resistant to ampicillin.
Recommended Citation
Gangwar, Maulshree, "Detection Of Total And Pathogenic Salmonella In Oysters Using Real-Time Pcr With Internal Amplification Control And Study Of Their Antibiotic Resistance Profile" (2008). All ETDs from UAB. 3573.
https://digitalcommons.library.uab.edu/etd-collection/3573