Περίληψη:
Twenty well characterized isolates of methicillin-resistant
Staphylococcus aureus were used to study the optimal resolution and
interlaboratory reproducibility of pulsed-field gel electrophoresis
(PFGE) of DNA macrorestriction fragments. Five identical isolates tone
PFGE type), 5 isolates that produced related PFGE subtypes, and 10
isolates with unique PFGE patterns were analyzed blindly in 12 different
laboratories by inhouse protocols. In several laboratories a
standardized PFGE protocol with a commercial kit was applied
successfully as well. Eight of the centers correctly identified the
genetic homogeneity of the identical isolates by both the in-house and
standard protocols. Four of 12 laboratories failed to produce
interpretable data by the standardized protocol, due to technical
problems (primarily plug preparation). With the five related isolates,
five of eight participants identified the same subtype
interrelationships with both in-house and standard protocols. However,
two participants identified multiple strain types in this group or
classified some of the isolates as unrelated isolates rather than as
subtypes. The remaining laboratory failed to distinguish differences
between some of the related isolates by utilizing both the in-house and
standardized protocols. There were large differences in the relative
genome lengths of the isolates as calculated on the basis of the gel
pictures. By visual inspection, the numbers of restriction fragments and
overall banding pattern similarity in the three groups of isolates
showed interlaboratory concordance, but centralized computer analysis of
data from four laboratories yielded percent similarity values of only
85% for the group of identical isolates. The differences between the
data sets obtained with in-house and standardized protocols could be the
experimental parameters which differed with respect to the brand of
equipment used, imaging software, running time (20 to 48 h), and pulsing
conditions. In conclusion, it appears that the standardization of PFGE
depends on controlling a variety of experimental intricacies, as is the
case with other bacterial typing procedures.
Συγγραφείς:
van Belkum, A
van Leeuwen, W
Kaufmann, ME
Cookson, B and
Forey, F
Etienne, J
Goering, R
Tenover, F
Steward, C and
O'Brien, F
Grubb, W
Tassios, P
Legakis, N
Morvan, A and
El Solh, N
de Ryck, R
Struelens, M
Salmenlinna, S and
Vuopio-Varkila, J
Kooistra, M
Talens, A
Witte, W and
Verbrugh, H
