Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR

• To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant density of two Salmonella strains in different growth conditions was determined to be 1.065-1.092g/ml. Based on these data, an optimal discontinuous flotation with three different density layers, ~1.200, 1.102 and 1.055g/ml, was designed for extracting intact Salmonella cells from pig carcass swabs. The method allowed accurate quantification from 4.4×102 to at least 2.2×107CFU Salmonella per swab sample using qPCR (without preceding DNA extraction) or selective plating on xylose lysine deoxycholate agar. Samples with 50CFU could be detected occasionally but fell outside the linear range of the standard curve. The swab samples showed a broad biological diversity; for seven samples not inoculated with Salmonella, the microbial background flora (BGF) was determined to 5.0±2.2 log CFU/ml sample withdrawn after flotation. It was determined that the proceeding PCR step was inhibited by BGF concentrations of ≥6.1×108CFU/swab sample, but not by concentrations ≤6.1×106CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture-independent quantification method considerably. © 2010 Elsevier B.V.

Subject headings

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology (hsv//eng)

Keyword

Buoyant density

Centrifugation

Food analysis

Food microbiology

Food safety

Meat

article

bacterial strain

carcass

cell density

colony forming unit

evaluation

flotation

gauze dressing

nonhuman

quantitative analysis

real time polymerase chain reaction

risk assessment

Salmonella enterica

swine

Animals

Polymerase Chain Reaction

Salmonella

Suidae

Buoyant density

Centrifugation

Meat

Food analysis

Food safety

Food

microbiology

Publication and Content Type

ref (subject category)

art (subject category)