The identification and detection of a unique gene in Salmonella enterica subsp. enterica Weltevreden using PCR-based approach

Introduction: Salmonella enterica are important food-borne pathogens that cause human gastroenteritis, bacteremia and subsequent focal infections. The incidences of food-borne salmonellosis due to non-typhoidal Salmonella serotypes are increasing periodically in Malaysia. Other than the two predominant non-typhoidal Salmonella serotypes, S. Typhimurium and S. Enteritidis, an emerging serotype, S. Weltevreden, is increasingly being isolated from hospitalized-gastroenteritis patients in Malaysia. Serotyping is the conventional method for differentiation of this pathogen from the 2500 other serotypes known to exist. However, this method has a number of disadvantages including low specificity and high cost. Therefore, a molecular method for identification of S. Weltevreden in clinical isolates based on amplification of a specific DNA sequence present in its genome is proposed. Methods: A total of 234 clinical isolates were collected from the Department of Clinical Microbiology and Parasitology, HUSM from year 2010-2012. There were 49 S. Weltevreden isolates which have been confirmed by the Institute of Medical Research (IMR) using conventional serotyping method. Two sets of primers targeting a unique gene in S. Weltevreden (SWE349) and a pan- Salmonella gene (invA284) were designed using Primer3 online software, and a multiplex-PCR (mPCR) assay was developed to detect S. Weltevreden in clinical isolates. The optimized S. Weltevreden mPCR assay was evaluated on a panel of known Salmonella and non- Salmonella spp. to determine the specificity and sensitivity of the mPCR assay. Results & Discussion: The S. Weltevreden serovar specific primer, SWE349, and the internal control primer, invA284, resulted in 2 PCR products of 349bp and 284bp, respectively. The mPCR assay for S. Weltevreden targeting gene SWE349 showed 100% sensitivity (49/49) and specificity (36/36) in concordance with the conventional serotyping method in detecting only S. Weltevreden clinical strains, but not from the panel of 26 non-typhoidal Salmonella and 10 non- Salmonella isolates. Conclusion: A cost effective, sensitive and specific mPCR assay has been developed for the detection and confirmation of Salmonella Weltevreden from clinical isolates of gastroenteritis patients, using genes SWE349 and invA284.