Validation on milk and sprouts of EN ISO 16654:2001 - Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O157

Rosangela Tozzoli, Antonella Maugliani, Valeria Michelacci, Fabio Minelli, Alfredo Caprioli, Stefano Morabito

Research output: Contribution to journalArticlepeer-review


In 2006, the European Committee for standardisation (CEN)/Technical Committee 275 - Food analysis - Horizontal methods/Working Group 6 - Microbiology of the food chain (TC275/WG6), launched the project of validating the method ISO 16654:2001 for the detection of Escherichia coli O157 in foodstuff by the evaluation of its performance, in terms of sensitivity and specificity, through collaborative studies. Previously, a validation study had been conducted to assess the performance of the Method No 164 developed by the Nordic Committee for Food Analysis (NMKL), which aims at detecting E. coli O157 in food as well, and is based on a procedure equivalent to that of the ISO 16654:2001 standard. Therefore, CEN established that the validation data obtained for the NMKL Method 164 could be exploited for the ISO 16654:2001 validation project, integrated with new data obtained through two additional interlaboratory studies on milk and sprouts, run in the framework of the CEN mandate No. M381. The ISO 16654:2001 validation project was led by the European Union Reference Laboratory for Escherichia coli including VTEC (EURL-VTEC), which organized the collaborative validation study on milk in 2012 with 15 participating laboratories and that on sprouts in 2014, with 14 participating laboratories. In both studies, a total of 24 samples were tested by each laboratory. Test materials were spiked with different concentration of E. coli O157 and the 24 samples corresponded to eight replicates of three levels of contamination: zero, low and high spiking level. The results submitted by the participating laboratories were analyzed to evaluate the sensitivity and specificity of the ISO 16654:2001 method when applied to milk and sprouts. The performance characteristics calculated on the data of the collaborative validation studies run under the CEN mandate No. M381 returned sensitivity and specificity of 100% and 94.4%, respectively for the milk study. As for sprouts matrix, the sensitivity resulted in 75.9% in the low level of contamination samples and 96.4% in samples spiked with high level of E. coli O157 and specificity was calculated as 99.1%.

Original languageEnglish
Pages (from-to)53-57
Number of pages5
JournalInternational Journal of Food Microbiology
Publication statusPublished - Jan 2 2019


  • Animals
  • Escherichia coli O157/isolation & purification
  • European Union
  • Food Microbiology/methods
  • Humans
  • Milk/microbiology


Dive into the research topics of 'Validation on milk and sprouts of EN ISO 16654:2001 - Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli O157'. Together they form a unique fingerprint.

Cite this