Direct detection of Salmonella cells in the air of livestock stables by real-time PCR

Abstract

A SYBR® Green real-time quantitative polymerase chain reaction (qPCR) assay for specific detection and quantification of airborne Salmonella cells in livestock housings is presented. A set of specific primers was tested and validated for specific detection and quantification of Salmonella-specific invA genes of DNA extracted from bioaerosol samples. Application of the method to poultry house bioaerosol samples showed concentrations ranging from 2.2 × 101 to 3 × 10 6 Salmonella targets m-3 of air. Salmonella were also detected by a cultivation-based approach in some samples, but concentrations were two to three magnitudes lower than the concentrations detected by molecular biological results. Specificity of results was demonstrated by cloning analyses of PCR products, which were exclusively assigned to the genus Salmonella. However, by molecular methods, microorganisms are detected independently of their viability status, leading to an overestimation of concentration. Hence, the survival rate of Salmonella cells was measured on filter surfaces during filtration samplings where 82% of the cells died within 20 min of filtration. The results clearly show the specificity and practicability of the established qPCR assay for analysis and quantification of salmonellae in bioaerosols. The results demonstrate airborne Salmonella workplace concentrations in poultry production of up to 3.3% of 4′,6-Diamidino-2-phenylindole-counted total cell numbers.