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QIAGEN
mericon
STEC Workflow Collaborative Study Protocol
May 2016
DRAFT
3
FSIS MLG 5.09 methods. If natural contamination is found and it is high enough
to yield fractional positives, it can be used for the collaborative study. If the
level is too low, the lot can be temperature abused or alternative lots can be
screened. If no natural contamination is found, the laboratory will inoculate one
bulk amount of ground beef at a low level to achieve fractional positives (25-
75% positives) and one bulk amount at a high level designed to produce all
positive results(e.g., 2-3 fold higher inoculum than the low level). The ground
beef will be inoculated with a fresh culture of a type strain or well-characterized
strain of pathogenic
E. coli
O157. Each inoculated bulk will be kneaded to
achieve a distribution of pathogen as close to homogeneous as possible. Twenty
five-g aliquots of inoculated matrixwill be placed into individual bags containing
300 g uninoculated ground beef to create the 325 g test portions. Each bag will
be kneaded to mix, though homogeneity is not required. Once bagged and
labeled (see 4.1.2), all test portions will be chilled to 2-8°C to stabilize the
inoculum. Test portions will be shipped at 2-8°C on the day of inoculation (on a
Thursday to arrive Friday) and stored at 2-8°C at the collaborator site until
Monday. By the time analyses begin on Monday, the stabilization time will be
~96 h.
4.1.2
Test portions designated for each particular collaborator will be uniquely
randomized and blind-coded for that collaborator. For example, Collaborator 1
will receive 36 randomized test portions of ground beef comprised of 12
uninoculated test portions, 12 low level test portions, and 12 high level test
portions labeled 101-136. Likewise, Collaborator 2 will have a unique
randomization and test portions will be labeled 201-236. Thus, the collaborators
at each site can easily distinguish their test portions from the other
collaborators’ test portions at that site.
4.1.3
In addition, each collaborator will receive one uninoculated test portion of each
matrix for determination of APC on the day of initiation of analyses.
4.2
Shipment of Test Portions
4.2.1
Test portions will be packaged in leak-proof insulated containers and shipped
(according to the Dangerous Goods Regulations IATA for Infections Substances)
by overnight carrier to arrive the Friday before initiation of analysis
.
All test
portions will be packed with ice packs to maintain a temperature of <7
°
C during
transport. A temperature control sample will be included with each shipment to
verify the temperature of samples upon receipt. Upon arrival, the temperature
of the temperature control sample will be recorded and the remaining test
portions will be stored at 2-8
°
C until they are analyzed.
4.3
Analysis of Test Portions – See Appendix 8.3
OMAMAN-36 B : Collaborative Study Protocol
For ERP Use Only
January 2017
AOAC Research Institute
Expe t Review Panel Use O ly