Rapid and Specific Detection ofSalmonellaspp. in Animal Feed Samples by PCR after Culture Enrichment

Abstract

A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting 1 CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain.Salmonellosis is currently a zoonotic disease of considerable magnitude (15). Animal feed is a recognized source of Salmonella enterica for farm livestock and might also act as an indirect cause of infection of people consuming foods of animal origin. Salmonellae can survive for several years in feed (14), and low concentrations have been found to be infective in, for instance, chickens (18). Transmission of salmonellae from feed to animals such as cattle and chickens is well documented (18,22). In several countries, salmonellae have been isolated from animal feed ingredients as well as the final product, of both vegetable and animal origin, with a prevalence of between 0 and 6% (17, 30). The levels of salmonellae in feed are low; D'Aoust and Sewell (14) quantified salmonellae in several kinds of feed ingredients and compound feeds and found the levels to be less than 1 bacterium per g of feed.The established culture-based methods used to detect salmonellae in animal feed are laborious, time-consuming, and often not specific enough. The standard methods used today for analyzing salmonellae involve preenrichment in buffered peptone water (BPW), selective enrichment in RappaportVassiliadis soy broth, plating on selective agar, and subsequent identification by biochemical tests, for example, NMKL-71 (7). The whole procedure takes at least 3 days to complete. Several alternative analysis strategies have been proposed, and PCR in partic...

Quantification of Campylobacter spp. in Chicken Rinse Samples by Using Flotation prior to Real-Time PCR

“…Still, no PCR inhibition was observed, which can be explained by the use of the alternative DNA polymerase Tth. Previous studies have already shown that when using this enzyme in real-time PCR, a lower sensitivity towards PCR inhibitors was observed (11,13).…”

Molecular Technologies for Salmonella Detection

“…Food and culture media both contain components that can inhibit PCR (Rosen et al, 1992;Andersen & Omiecinski, 1992;Atmar et al, 1993;Demeke & Adams, 1992;Lofstrom et al, 2004) (for a review, see Wilson, 1997). PCR inhibitors originating from the food samples include humic acid from soil (Tsai & Olson, 1992a;Tsai & Olson, 1992b), proteins and aminoglycans from animal samples such as hemoglobin, lactoferrin and heparin (AlSoud & Radstrom, 2001), polysaccharides from plant material (Demeke & Adams, 1992;Monteiro et al, 1997), melanin from hair and skin (Eckhart et al, 2000), etc.…”

Toward Standardization of Diagnostic PCR Testing of Fecal Samples: Lessons from the Detection of Salmonellae in Pigs

“…(vi) The aforementioned issues should be considered in light of the newly standardized procedures for PCR testing, which recommend the inclusion of internal amplification control, the processing of positive and negative controls (3), a consensus on the determination of the cutoff level in real-time PCR (5), and the use of statistical calculations for determining detection probability (24).…”