The Development of molecular genetic tools for detection of Salmonella pathogen

Gökduman, Kurtuluş
Although traditional microbiological methods are accepted standard for Salmonella detection, they are labor intensive and time consuming. Therefore, for food industry and public health, finding sensitive and rapid methods is required. As a rapid and reliable tool, Real-Time PCR is one of the most common methods in molecular detection and research area. The aim of the current study is to develop rapid, sensitive and quantitative Salmonella detection method using Real-Time PCR technique based on inexpensive, easy to produce, convenient and standardized plasmid based positive control for the first time. To achieve this, two plasmids were constructed as reference molecules by cloning two most commonly used Salmonella specific target regions ‘invA and ttrRSBC’ into them. Standard curves were constructed for the plasmids and reproducibility, PCR efficiency, amplification efficiency values were calculated. To illustrate the applicability of the developed method, enriched (as used commonly for Salmonella detection with Real-Time PCR) 105 to 100 CFU/ml level (estimated by standard plate counts before enrichment) S. Typhimurium ATCC 14028 cultures were tried to detect and quantify, also compared with traditional culture method. In addition, detection limits of the developed technique were determined by serial dilution of DNA extracted from 105 CFU/ml level. The results revealed much faster detection ability of the developed plasmid based Salmonella detection method (in comparison to traditional culture method, ISO 6579:2004) allowing quantitative evaluation with perfect reproducibility, sensitivity (except for lower concentrations for invA target), detection limit, PCR efficiency, amplification efficiency for both invA and ttrRSBC targets. The detection and quantification ability of the method developed by using S. Typhimurium ATCC 14028 cultures were tested also with 15 Salmonella species using milk as a representative food. The results also revealed much faster (in comparison to traditional culture method, ISO 6579:2004) quantitative detection ability of the developed method. Thus, the developed method has great potential to be used in food industry for rapid and quantitative Salmonella detection.


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Numerous foodborne infections and outbreaks are associated with Salmonella which makes it a challenge in terms of human health and economy. Therefore, reducing the prevalence of Salmonella in food and food processing areas is of great importance. Antibiotics are the substances that are commonly used in various stages of food production in order to fight against Salmonella. However, concerns related with the antibiotic use like antibiotic resistance give rise to pursuit of safer methods to eliminate Salmonel...
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A microarray was developed to simultaneously screen Escherichia coli and Salmonella enterica for multiple genetic traits. The final array included 203 60-mer oligonucleotide probes, including 117 for resistance genes, 16 for virulence genes, 25 for replicon markers, and 45 other markers. Validity of the array was tested by assessing inter-laboratory agreement among four collaborating groups using a blinded study design. Internal validation indicated that the assay was reliable (area under the receiver-opera...
Citation Formats
K. Gökduman, “The Development of molecular genetic tools for detection of Salmonella pathogen,” Ph.D. - Doctoral Program, Middle East Technical University, 2012.