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• Title: Biological Sciences Paper Abstracts (Author Abstract)
• Author : Journal of the Alabama Academy of Science
• Release Date : January 01, 2008
• Genre: Engineering,Books,Professional & Technical,
• Pages : * pages
• Size : 204 KB

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"DETECTION OF TOTAL AND PATHOGENIC SALMONELLA IN OYSTER USING REAL-TIME PCR" Maulshree Gangwar, Department of Biology, University of Alabama at Birmingham Dr.Asim K.Bej, Department of Biology, University of Alabama at Birmingham. Birmingham, AL 35294. 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 0.274 kbp and Salmonella virulence plasmid-borne spvB gene generating 0.561 kbp 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 electropho-resis. 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[degrees]C (spvB), 83.06 to 85.48[degrees]C (invA) and 86.32 to 87.99[degrees]C (16S rRNA) was observed for all Salmonella strain. Negative controls and non-Salmonella strains showed an IAC-specific melt peak at 77.0[+or-]2.0 [degrees]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 Salmonella enterica. The sensitivity was confirmed in pure cultures as well as in oyster tissue homogenate enriched with Salmonella.

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