Comparison of Real-Time PCR with Conventional PCR and Culture to Assess the Efficacy of a Live Attenuated Salmonella enterica Serovar Typhimurium Vaccine Against Salmonella enterica Serovar Enteritidis in Commercial Leghorn Chicks Vaccinated Under Field and Laboratory Conditions

Parker, W. Drew; Lungu, A. Bwalya; Berghaus, Roy D.; Sellers, Holly S.; Alvarado, I. R.; Hofacre, Charles L.

doi:10.1637/9728-956111-digest.1

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“…However, their real-time PCR method was not Salmonella Enteritidis specific and thus would have very limited use especially in egg quality programs due to its inability to specifically identify Salmonella Enteritidis. Over the years, Salmonella detection by conventional and real-time PCR methods has been evaluated against the culture method (7,12,13,17). In most of these studies, the PCR-based methods were reportedly more sensitive than the culture method.…”

Section: Discussionmentioning

confidence: 99%

“…Molecular assays such as the real-time PCR have shown increased success in detecting Salmonella spp. (3,7,9,13,17); however, these techniques are dependent on other factors such as specimen collection, transport conditions, DNA yield and quality, DNA storage, and nucleic acid assay limitations. Nucleic acid extraction method limitations are an extremely important and relevant concern in diagnostics, as these methods are paramount for the success of detecting and identifying pathogens such as Salmonella.…”

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Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

Lungu

Waltman²,

Berghaus

et al. 2012

Journal of Food Protection

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Conventional culture methods have traditionally been considered the "gold standard" for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis-specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis-specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%