Diagnostic Real-Time PCR for Detection of Salmonella in Food

Abstract

A robust 5 nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10 3 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10 4 CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.

Rapid MPN-Qpcr Screening for Pathogens in Air, Soil, Water, and Agricultural Produce

“…The strategy proposed here might be more reliable than direct testing of environmental matrices due to the removal of inhibitors by dilution (Malorny et al 2004) and nonviable bacteria by the pre-enrichment step (Heaton and Jones 2008). This observation is corroborated by the more variable and less sensitive results obtained from non-enriched samples of water, soil, and tomato wash (Table 2).…”

“…Such steps could be immunomagnetic separation (Warren et al 2007), preamplification of the target DNA (Ishii et al 2013), or nonspecific enrichment (Edel and Kampelmacher 1973), aiming to increase the likelihood of detecting (but not directly enumerating) viable pathogens (Krämer et al 2011;Malorny et al 2004). The combination of nonspecific enrichment followed by DNA extraction and qPCR analysis coupled with most probable number (MPN) estimation was found to be a useful tool to increase the likelihood of pathogen detection when initial concentrations are low (Krämer et al 2011;Russo et al 2014;Wright et al 2007).…”

Species Identification of Food Spoilage and Pathogenic Bacteria by MALDI-TOF Mass Fingerprinting

“…As a consequence, a large number of RTi-PCR procedures are currently available for the specific detection and quantification of foodborne bacteria, such as Salmonella spp., L. monocytogenes, S. aures or Leuconostoc mesenteroides (Hein et al, 2001;Malorny et al, 2004;Elizaquível et al, 2008).…”

Antibiotic Resistance in Salmonella: A Risk for Tropical Aquaculture

“…In addition, molecular biology techniques may be used for rapid detection of Salmonella in foods: TaqMan PCR (Kimura et al, 1999), PCR amplification of a 152-bp segment of the gene hns , real-time PCR (Malorny et al, 2004), PCR, dot blot hybridization, RAPD and ERIC-PCR (Shabarinath et al, 2007), PCR amplification of the gene invA (Upadhyay et al, 2010) and uniplex and multiplex PCR (Raj et al, 2011).…”