Development of Microarray and Multiplex Polymerase Chain Reaction Assays for Identification of Serovars and Virulence Genes in Salmonella Enterica of Human or Animal Origin

Abstract

Abstract. Salmonella enterica is an important enteric pathogen consisting of many serovars that can cause severe clinical diseases in animals and humans. Rapid identification of Salmonella isolates is especially important for epidemiologic monitoring and controlling outbreaks of disease. Although immunologic and DNA-based serovar identification methods are available for rapid identification of isolates, they are time consuming or costly or both. In the current study, 2 molecular methods for identification of Salmonella serovars were developed and validated. A 70-mer oligonucleotide spotted microarray was developed that consisted of probes that detected genes responsible for genetic variation among isolates of Salmonella that can be used for serotyping. A multiplex polymerase chain reaction (PCR) assay was also developed, which is capable of identifying 42 serovars, thus providing a valuable prediction of the pathogenicity of the isolates by detecting the presence of virulence genes sseL, invA, and spvC. The gene spvC was the best predictor of pathogenicity. In a blind study, traditional serologic methods were correlated at 93.3% with the microarraybased method and 100% with the multiplex PCR-based serovar determination.

Methodologies for Salmonella enterica subsp. enterica Subtyping: Gold Standards and Alternatives

“…A third extended version of the original multiplex assays was obtained by adding three virulence genes, enabling the detection of 12 additional serovars (total of 42 serovars) (66). In the same study, a 37-spot DNA microarray made of 70-mer oligonucleotides derived from previous work (45, 67) was evaluated in parallel and reported to perform well in assessing 56 common clinical isolates belonging to 29 serovars.…”

Quantification and characterization of Salmonella spp. isolates in sewage sludge with potential usage in agriculture

“…Peterson et al . [ 30 ] reported that up to that moment, the spvC gene had been found only in five Salmonella strains: S. Cholerasuis, S. Dublin, S. Enteritidis, and S. Typhimurium. Thus, the Salmonella Typhimurium strains mentioned in this study could represent a threat, as three virulence genes were detected even after the sewage sludge treatment.…”

“…The 40 previously selected isolates were tested in order to find the presence of s pvC , invA, and s seL virulence genes, which are in accordance to Peterson et al . [ 30 ]. Within a 25 μl reaction volume, the following quantities were added: 5 μl of DNA template, 5 μl of 5× Gotaq buffer, 0.3 μmol of each primer (Bioneer Oligo Synthesis Report - Korea), 200 μmol of dNTP (Thermo Fisher Scientific Inc.), and 1U of GO Taq Polymerase (Promega Inc.- Wisconsin -USA) (Table 4 ).…”

Ocorrência e caracterização de Salmonella não tifóide em esgotos brutos e águas superficiais da região metropolitana de São Paulo

“…Os padrões atípicos de Salmonella Enteritidis e Salmonella Typhimurium detectados no curso desta investigação podem ampliar as possibilidades de interpretação de resultados produzidos pela análise de PCR com os conjuntos de iniciadores STM e STY (SEONGHAN KIM et al, 2006). Outros pesquisadores têm se empenhado no aprimoramento deste método, avaliando-o na identificação de cepas de SNT isoladas de matrizes clínicas e fezes de animais (LEADER et al, 2009;MERTES et al, 2010;PETERSON et al, 2010) -GILLES BEAUBRUN et al, 2012;McEGAN et al, 2014;PETERSON et al, 2010;SEONGHAN KIM et al, 2006) São Paulo, entre 1996e 2003(FERNANDES et al, 2006 Lago Spring -Rio San Marcos: uso da água NE 6 coletas em 6 pontos no rio e 3 poças de água às margens (amostras de água e sedimentos).…”

“…ePETERSON et al (2010), observou-se que os padrões de PCR de sete cepas, provenientes das amostras de esgoto,…”