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1.1.1   Background of SalmonellaSalmonella is a flagellated facultatively anaerobic Gram-negative bacillus bacteria characterized by O, H, and Vi antigens. Salmonella enterica is one of the major bacterial pathogen responsible for a large number of infections in humans and animals across the globe. Salmonella infections are generated by the ingestion of  water or food that are contaminated. The onset of fever, vomiting, abdominal cramps and bloody diarrhea, a few hours to a few days after infection are characteristic of salmonellosis. Commonly, the infection lasts for several days and most people recover without treatment needed. However, the infection under severe circumstances, can spread from the intestines to the bloodstream and then to other sites leading to death. There are over 1800 known serovars which current classification are examined to be separate species. For example, Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common food-borne pathogen and frequent cause of gastroenteritis in humans. Human-specific pathogens S. enterica serovars Typhi and Paratyphi can cause enteric fevers and the severity of the infection may vary by the resistance of each individual and their immune system.1.1.2    The hilA Gene in SalmonellaThe hilA gene encodes an OmpR/ToxR family transcriptional regulator that operates the expression of invasion genes in response to both environmental and genetic regulatory factors. Salmonella enters intestinal epithelial cells during an infection of the host. Many Salmonella typhimurium genes are encoded on a 40 kb ‘pathogenicity island’ as a requirement for bacterial entry into host cells. The recognition of hilA gene within the ‘island’ appears to encode an activator of invasion gene expression. Consequently, the hilA gene plays an important role in the regulation of Salmonella invasion during an infection. 1.1.3   Extraction of bacterial DNAExtracting DNA from Gram-positive and Gram-negative bacteria is a crucial preliminary step in species identification using techniques such as PCR, restriction digestion, pulsed-field gel electrophoresis (PFGE), and optical mapping. Isolating DNA from prokaryotes can be divided into four different steps: cell lysis, removal of RNA, removal of proteins and precipitation of DNA. A good quality DNA sample can be obtained by following these steps in DNA extraction. For the use of the polymerase chain reaction (PCR) technique, it is important to acquire good quality DNA to achieve good tests results, in which the excess of cellular structures and proteins can inhibit the amplification process. DNA extractions should provide good band standardization with sufficient quantity and quality so that interference in the electrophoresis gels migration patterns will not be caused.1.1.4   Applications of PCR and Its use as a Diagnostic Tool The Polymerase Chain Reaction (PCR) technique is used for the detection of microorganisms, diagnosing diseases, cloning and sequencing genes, carrying out quantitative and genomic studies in a rapid and sensitive manner. It possesses widespread application in different areas such as genetic analysis, medical applications, and research. Some applications of PCR include the ability to amplify RNA using RT-PCR which facilitates laboratory-based diagnostic testing of infectious agents to a greater extent. The use of PCR assay allows the identification of criminals in the field of forensic medicine. It is also used as a sensitive test for tissue typing and plays an important role in organ transplantation. Another example would be the use of diagnostic real-time PCR for the specific detection of Salmonella in foods which is increasingly being used as a rapid and reliable tool for the control of contaminated samples along the food production chain.  Real-Time PCR allows precise quantification of nucleic acids with greater reproducibility and provides a sensitive method for the accurate quantification of individual species, which could be relevant to the diagnosis of pathogens and genetic diseases. It requires a thermocycler that has an optical system which captures fluorescence and a computer software capable of capturing accurate data and performing the final analysis of the reaction.1.1.5   Difference between Polymerase Chain Reaction (PCR) and Conventional MethodsConventional culture methods have been considered as the “gold standard” for the isolation and identification of foodborne bacterial pathogens. This standardized culture method is sensitive and inexpensive, but culture methods are laborious and time-consuming, because they require at least 3 days to produce a negative result and 5-10 days for confirmed positive results. Moreover, due to environmental factors, variations in gene expression of microorganisms can occur and may affect the results of biochemical tests. On the other hand, the PCR method is a rapid procedure with high sensitivity and specificity for the prompt detection and identification of specific pathogenic bacteria in food. However, a major limitation involves the low number of pathogens occurring heterogeneously within a large volume of food and inhibition of PCR by the food matrix. Additionally when a pathogen is detected, the conventional method must still be used for confirmation. Despite these limitations, the application of PCR based assays to enrichment broths has been much more successful.1.2  AimThe aims of this study is to extract genomic DNA of Salmonella Spp from an overnight culture to be subsequently used as a positive control for the rapid detection of Salmonella Spp in contaminated raw chicken by Polymerase Chain Reactions(PCR) method and Agarose Gel electrophoresis.

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