Development of a Multiplex PCR Technique for Detection and Epidemiological Typing of Salmonella in Human Clinical Samples

Abstract

We have developed a multiplex PCR assay for Salmonella detection and epidemiological typing. Six sets of primers were designed to detect the major Salmonella serotypes and phage types in Spain. An internal amplification control was designed in order to detect PCR inhibition. The different amplification profiles obtained allowed us to detect Salmonella bacteria and to distinguish the clinically prevalent Salmonella enterica serotypes Enteritidis, Typhimurium and subspecies I serotype 4,5,12:i:؊. Using this method, we could detect a specific band for DT104 and U302 phage types in Salmonella serotype Typhimurium. Salmonella enterica serotype Hadar and other C2 serogroup strains showed two specific band profiles. In the validation stage, the assay was reproducible for all serotypes studied, apart from some C2 serogroup strains. When the technique was applied to clinical stool specimens, the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of 93%, specificity of 100%, and efficiency of 98%. Also, a low PCR inhibition rate (8%) was obtained. The overall agreement of the multiplex PCR with conventional culture-based techniques was 95% for Salmonella typing using Cohen's kappa index.

Application of PCR-based serogrouping of selected Salmonella serotypes in Malaysia

“…One way to avoid this serological cross-reactivity is to use DNA-based methods for serotyping, such as PCR, which does not involve antigens or antibodies. Various PCR tests have been developed for species identification of Salmonella enterica [1,4,6,7,22,[24][25][26][27].…”

“…These molecular methods, such as multiplex PCR, are highly sensitive, very specific, fast and reproducible. Such approaches have previously been reported by others [1,4,6,7,14,19,22,[24][25][26][27]29]. In Salmonella, the genes responsible for biosynthesis of the O antigens are normally grouped together on the chromosome in a gene cluster called rfb [6].…”

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

“…PCR amplification of the sefA gene, encoding SEF14 fimbrial antigen, was used for preliminary confirmation of the identity of Salmonella isolates (Doran et al, 1996). A chromosomally located sdfI gene reported to be unique to the serovar S. Enteritidis was targeted for the serotype-specific identification of S. Enteritidis (Agron et al, 2001;Alvarez et al, 2004;Clavijo et al, 2006;Trafny et al, 2006). A 60 kb virulence-plasmid-associated spvB gene was used as a marker for determination of the presence or absence of a typical virulence plasmid (pSEV) among S. Enteritidis isolates (Cho et al, 2007;Rotger & Casadesú s, 1999;Rychlík et al, 2006Rychlík et al, , 2008.…”

Comparison of the Detection Limits of the Culture and PCR Methods for the Detection of Clostridium difficile, Clostridium perfringens, Campylobacter jejuni, and Yersinia enterocolitica in Human Stool

“…Most laboratories are unable to diagnose the anaerobic, microaerophilic, and fastidious bacteria responsible for human gastrointestinal disorders using conventional microbiological methods. Recently, more rapid DNA-based methods for direct identification and even subtyping of these pathogens in stool specimens have been developed (14)(15)(16)(17)(18)(19)(20)(21). Polymerase chain reaction (PCR) is a powerful technique for the detection of the target DNA in various clinical specimens, including fecal samples.…”