ABSTRACT
Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 105 CFU/ml, while PCR displayed a limit of 5.9 x 107 CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 104 CFU/g, whereas PCR was 3.6 x 105 CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.
ABSTRACT
The present study aims to develop a system which consists of four pairs of primers that specifically detects Salmonella spp., Salmonella serovar Typhi and Salmonella serovar Paratyphi A with an internal amplification control. The system, when applied in Polymerase Chain Reaction (PCR) under specific conditions, reaction mixture and cycling temperatures produced four bands; 784 bp, 496 bp, 332 bp and 187 bp. The DNA band 784 bp is present in all Salmonella spp., while the bands of 496 bp and 332 bp are only present in S. Paratyphi A and S. Typhi, respectively. An internal amplification control as indicated by the 187 bp shows the system is working in optimum condition in all the tests. This multiplex PCR was evaluated on 241 bacterial cultures and 691 naturally contaminated samples. Overall, this multiplex PCR detection system provides a single step for simultaneous detection of DNAs of Salmonella spp., S. Typhi and S. Paratyphi A.