Development of molecular based serogrouping of Salmonella

Lim, Bee Kim (2009) Development of molecular based serogrouping of Salmonella. PhD thesis, University of Malaya.

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Salmonellosis remains a major public health problem in Malaysia. Differentiation of Salmonella enterica into its serogroups is important from an epidemiological point of view. The conventional serogrouping method is time-consuming and not cost effective as it requires hundreds of anti-sera and well-trained technicians. Moreover, the complexity of the system and difficulty of interlaboratories comparison of results limit the application of serotyping to reference laboratories. Polymerase chain reaction (PCR) is now considered a fairly standard procedure in most molecular laboratories. The technique is easy and fast to perform. A multiplex PCR method was developed to identify the major serogroups A, B, C1, D, E and Vi-positive strains of Salmonella enterica commonly encountered in Malaysia. Six sets of primers were selected to target defined regions of the O antigen synthesis genes (rfb genes cluster) and Vi antigen gene (viaB). Another H-typing multiplex PCR which identified flagellar antigen “a”, “b” and “d” was also optimized for confirmation of some common clinical serotypes (Salmonella ser. Paratyphi and Salmonella ser. Typhi). Both PCR techniques have an internal control to fulfill the quality assurance requirement for standard diagnostic methods. Optimization was carried out by adjusting the concentration of MgCl2, Taq and primers as well as denaturation time, annealing and extension temperature. Using the optimized conditions (2.5mM MgCl2 and 1.75U Taq), 77 bacterial cultures (Salmonella,n=67; and non-Salmonella, n=10) were used for verification and the results showed 100% concordance to the conventionally typed serogroups. DNA sequences analysis of the representative amplicons produced by each primer pairs showed high identity level ranging 97% to 99% to the GenBank sequence database. Further validation with 58 blind coded Salmonella strains including negative control strains yielded 100% accuracy and specificity. Based on this study, the multiplex PCR is a rapid and accurate method for serogrouping Salmonella isolates.

Item Type: Thesis (PhD)
Subjects: Q Science > Q Science (General)
Date Deposited: 31 Jul 2013 03:05
Last Modified: 31 Jul 2013 03:05

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