Danijela Horvatek Tomić

European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.

The morphological characteristics of the passive flexor mechanism of birds with different digit layout

Birds possess a mechanism which allows passive flexion of the digits of the hind limbs which consists of two components: “automatic digital flexor mechanism” (ADFM) and “digital tendon-locking mechanism” (DTLM). The aim of this paper was to establish the existence and specificities of those components in examples of birds with anisodactyl (domestic chickens) and zygodactyl (parrots) digit layout using standard anatomical dissection and histological methods. Spatial distribution of the parrots’ muscles with a central role in DTLM (M. flexor digitorum longus and m. flexor hallucis longus) was the same as the chickens’. The plantar position of the fourth digit, which makes the difference between the anisodactyl and the zygodactyl digit layouts, did not cause changes in the position and function of this flexor muscles. The domestic chickens’ tendon sheath ridges, as well as the tendon tubercles of the flexor muscles were less developed while the parrots’ tubercles and ridges were clearly defined and the differences in their morphology were visible. There were no chondrogenic elements or they were barely noticeable in the parrots’ tendons. A potential cause of these distinctions could be the difference in the load which the tendons endure due to the different biomechanical composition. The parrots primarily inhabit trees where they move agilely around by grasping the branches. The Galliformes have a much larger body mass which is a consequence of their life on the ground, so the flexor muscle tendons suffer a greater load than the parrots and the cartilage tissue embedded in the tendon itself could act to distribute the weight of the body borne by the foot and the tendon.