Ergebnisse eines bundesweiten Ringversuchs: Standardisierung eines molekularbiologischen Verfahrens zur Charakterisierung Shigatoxin-bildender E. coli

The results of an interlaboratory comparison in Germany for the evaluation of a molecularbiological method cascade for detection, specific isolation and characterization of Shigatoxin-producing Escherichia coli (STEC) in foods are given. 11 groups took part (2 university institutes, 2 federal institutes, 6 institutes in different Federal Laender of Germany and 1 institute of an enterprise in Germany)[1]. 10 ground beef samples were sent to each group. These samples were produced from 2 basic matrices for all groups. Five samples were contaminated artificially in a laboratory. The infection doses were between 54 cfu and 308 cfu/25 g ground beef. The whole content of microorganisms per g ground beef was between 10[5] and 10[6] cfu. In addition the samples 1 to 9 (produced from basic ground beef matrix 1) for each group were contaminated with STEC naturally . This contamination could happen during the slaughtering process. The PCR Screening by using the primer pair MK1/MK 2 showed for the quotient: sum of all right positive detected samples of all groups/sum of all positive artificially contaminated samples the value 1. In addition 10 of 11 working groups detected 1 or 2 naturally contaminated samples. The PCR Screening by using the primer pairs KS 7/KS 8 and LP 43/LP 44 showed the values 0,9773 and 0,9636, respectively. 8 of 11 working groups detected 5 artificially contaminated samples by using the DNA Hybridisation technique with DIG labelled probes produced by using PCR and the primer pair MK 1/MK 2. The results of 3 participants could not be used for the evaluation. On one hand the samples spoiled during the transport and on the other hand 2 working groups made methodical mistakes. They used the 18 hrs enriched cultures instead of the 5 hrs preenriched cultures for preparing McConkey plates. All participants had to detect the following genes in - STEC isolates by using PCR: stx 1, stx 2, and eae. 7 of 11 working groups detected all factors of their isolates from all 5 artificially contaminated samples in the right manner. One participant made a mixture of marked probes. This working group produced their own marked probes later. The results were exellent but we did not use them in the evaluation of the interlaboratory comparison. 3 other working groups isolated STEC only from 4 artificially contaminated samples and 1 participant from 3 artificially contaminated samples. These groups were understaffed. So it was not possible for them to repeat the DNA Hybridisation of those samples which were positive in screening PCR but negative in DNA Hybridisation. In addition 3 working groups isolated and characterised STEC from naturally contaminated samples. On the basis of these results the methode cascade is a standard procedure in the collection of methods of 35 LMBG in Germany now. At present it is discussed whether the molecularbiological method cascade could be a standard CEN procedure, too.