Inclusivity, exclusivity and limit of detection of commercially available real-time PCR assays for the detection of Salmonella

Abstract

The traditional cultural detection of Salmonella spp. is both time- and labour-intensive. Salmonella is often a release criterion for the food industry and time to result is therefore an important factor. Storage of finished products and raw materials can be costly and may adversely impact available shelf-life. The application of real-time PCR for the detection of Salmonella spp. in food samples enables a potential time-saving of up to four days. The advancement of real-time PCR coupled with the development of commercially available systems in different formats has made this technology accessible for laboratories in an industrial environment. Ideally these systems are reliable and rapid as well as easy to use. The current study represents a comparative evaluation of seven commercial real-time PCR systems for the detection of Salmonella. Forty-nine target and twenty-nine non-target strains were included in the study to assess inclusivity and exclusivity. The limit of detection for each of the method was determined in four different food products. All systems evaluated were able to correctly identify the 49 Salmonella strains. Nevertheless, false positive results (Citrobacter spp.) were obtained with four of the seven systems. In milk powder and bouillon powder, the limit of detection was similar for all systems, suggesting a minimal matrix effect with these samples. Conversely, for black tea and cocoa powder some systems were prone to inhibition from matrix components. Up to 100% of the samples were inhibited using the proprietary extracts but inhibition could be reduced considerably by application of a DNA clean-up kit.

Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay

“…Differences between the no-matrix and test samples for each product were 0, 0•21, 0•84, 2•17, and 2•84 cycles for skim, whole, heavy cream, eggnog and chocolate milks, respectively, indicating that the only milk products which would cause significant (>2 cycle) PCR inhibition were the eggnog and chocolate milk. This is consistent with the presence of inhibitory compounds such as egg components (Woodward & Kirwan, 1996), spices, and various stabilisers in the eggnog, and the polyphenols from the chocolate in the chocolate milk (Margot et al 2013).…”

Microbiological survey of raw and ready-to-eat leafy green vegetables marketed in Italy

“…Initially, some laboratories experienced a problem with PCR inhibition during analyses of L. monocytogenes and Campylobacter. Recently, it was demonstrated that the extraction procedures recommended in the ADIAFOOD® kits are not able to eliminate all the inhibitors from some vegetable samples (Margot et al, 2013). The cause was linked to Table 1 Summary of microbiological results (n. positive samples) obtained, for fresh leafy (A) and Ready to Eat (B) vegetables, by various laboratories, using RT-PCRs and reference culturing ISO methods, in Italy.…”

Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations

“…The detection limit was approximately 8–79 CFU/mL. Besides, Margot et al ( 2013 ) evaluated seven commercial qPCR kits for the detection of Salmonella which include BAX® system Q7 real-time PCR (DuPont Qualicon) and other commercial qPCR kit such as ADIAFOOD® Salmonella (AES chemunex), BIOTECON foodproof Salmonella Detection Kit (Biotecon Diagnostics), BioControl Assurance GDS® TM Salmonella (BioControl), Genedisc® Shiga Toxic E. colii and Salmonella spp. (Pall GeneDisc® Technologies), BioRad iQ-Check Salmonella 2 (Biorad) and MicroSeq® Salmonella spp.…”