Validation of Real-Time PCR and Enzyme-Linked Fluorescent Assay-Based Methods for Detection of Salmonella spp. in Chicken Feces Samples

Abstract

A comparative study of enzyme-linked fluorescent assay (ELFA)-based methods and real-time polymerase chain reaction (PCR)-based methods using three and two different sample preparation protocols, respectively, and the standard culture-based method EN ISO 6579:2002/ Amd1:2007, for the detection of Salmonella spp. in chicken feces, was performed on 20 artificially and 68 naturally contaminated chicken feces. Selectivity, relative specificity, relative accuracy, relative sensitivity, and relative detection level were determined. According to criteria established in the methods comparison study included in EN ISO 16140:2003 for validation of alternative microbiological methods, the ELFA-based methods (V1 and V2) as well as a real-time PCR method (PCR2) were comparable to the reference method for the detection of Salmonella in chicken feces. They provided results in 48 h and presented a high sensitivity (97% for all of them). The three methods showed a relative specificity of 94%, V1 being the method which presented the highest relative accuracy (96%). While detection level for V2 and reference method was between 3 and 13 CFU/25 g, PCR2 method was able to detect down to 3 CFU/25 g. In conclusion, both the real-time PCR and the ELFA-based assays can be used as rapid and user-friendly screening methods for detection of Salmonella spp. in chicken feces.

Development of a multiplex real-time PCR method for simultaneous detection of Salmonella enterica, Shigella flexneri and Listeria monocytogenes in processed food samples

“…[8], Salmonella spp. [23] and L. monocytogenes [24], or multipathogen detection [1,9,25]. Multipathogen detection on a single-assay platform not only reduces the cost for testing but also provides data on the presence of pathogens in a single experiment [26].…”

Incidence of Salmonella Enteritidis in chicken layer flocks in Turkey: Results by real-time polymerase chain reaction and International Organization for Standardization culture methods

“…in animal feces and in samples from the primary production stage is International Organization for Standardization (ISO) method 6579:2002/Amd 1:2007(ISO, 2007 in the countries of the European Union, and this method also involves 4 to 6 d. To shorten this detection time in the interest of layer companies, realtime PCR (rPCR) can be applied to fecal samples before culture results (Eyigor et al, 2002;Kurowski et al, 2002;Eyigor and Carli, 2003;Eriksson and Aspan, 2007;Tomás et al, 2009;Hadjinicolaou et al, 2009). However, as has been previously pointed out (Eyigor et al, 2002), the fact that rPCR and culture can complement each other and cannot replace one another should always be kept in mind.…”

Vidas UP–Enzyme-Linked Fluorescent Immunoassay Based on Recombinant Phage Protein and FluorescenceIn SituHybridization as Alternative Methods for Detection ofSalmonella entericaSerovars in Meat

“…The erroneous selection of the colonies for further confirmation tests may have been the cause of the false-negative results. Some authors have suggested that the multistaged nature of testing and subjective readings based on visual color changes may prompt mistakes in interpreting the results (Tomás et al, 2009). Ö zer and Kimiran-Erde (2013) and Vieira-Pinto (2005) had a different explanation for the higher number of positive results generated by the FISH technique in comparison with the standard method.…”