Validation of a 20-h real-time PCR method for screening of Salmonella in poultry faecal samples

Abstract

Efficient and rapid monitoring of Salmonella in the poultry production chain is necessary to assure safe food. The objective was to validate an open-formula real-time PCR method for screening of Salmonella in poultry faeces (sock samples). The method consists of incubation in buffered peptone water for 18 ± 2 h, centrifugation of a 1-ml subsample, DNA extraction on the pellet and PCR. The total analysis time is 20 h. The validation study included comparative and collaborative trials, based on the recommendations from the Nordic organization for validation of alternative microbiological methods (NordVal). The comparative trial was performed against a reference method from the Nordic Committee on Food Analysis (NMKL187, 2007) using 132 artificially and naturally contaminated samples. The limit of detection (LOD50) was found to be 24 and 33 CFU/sample for the PCR and NMKL187 methods, respectively. The relative accuracy, relative sensitivity and relative specificity were all 100%, when including naturally contaminated samples and samples artificially contaminated with 10–100 CFU/sample. The collaborative trial included six laboratories and valid results were obtained from five of them. Apart from one of the samples that was artificially contaminated with 1–10 CFU/sample being a false negative with PCR for one of the laboratories, no false-positive or false-negative results were reported. This test supplies the growing demand for validated diagnostic PCR methods for screening of samples in the meat production chain to assure safe food.