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3M Molecular Detection Assay Salmonella Collaborative Study
July 2012
OMA-2012-July-XXX
Either the API20E (Official Method 978.24) or the VITEK GN (Official
1
Method 2011.17) will be used as an alternative to the conventional
2
biochemical tests. The somatic (O) and flagellar (H) tests will also be
3
performed.
4
5
2.0
FDA-BAM
6
2.1
To each 25g test portion, add 225 mL lactose broth. Blend 2 min. Let stand 60±5
7
min at room temperature. Mix and adjust pH to 6.8±0.2 if necessary. Incubate 24
8
± 2h at 35°C.
9
2.2
Transfer 0.1 ml to 10 ml Rappaport-Vassiliadis (RV) broth (prepared from
10
individual ingredients) and 1 mL to 10 mL TT broth.
Pay special attention to
11
the preparation of RV and TT broths as USDA and FDA use different
12
formulations.
Incubate the RV at 42
±
0.2°C for 24
±
2 h in a circulating
13
waterbath. Incubate the TT broth at 35 ± 2.0
°
C for 24
±
2 h in a forced air
14
incubator.
15
2.3
Mix and streak 3 mm loopful (10
µ
L) RV broth onto BS, XLD and HE agars.
16
Repeat from TT broth. Incubate at 35
°
C for 24
±
2 h. Follow isolation procedure
17
according to FDA-BAM.
18
2.4
Transfer typical colonies to TSI and LIA. Incubate at 35
°
C for 24
±
2hr.
19
2.5
Confirm 1 typical colony per test portion with biochemical/serological procedures
20
prescribed by the FDA-BAM method. Either the API20E (Official Method
21
978.24) or the VITEK GN (Official Method 2011.17) will be used as an
22
alternative to the conventional biochemical tests. The somatic (O) and flagellar
23
(H) tests will also be performed.
24
25
Raw ground beef – separate test portion preparations
(See Appendix 8.6 for a flow diagram)
26
NOTE
– use stomacher bag with filter for all 3M test portions.
27
28
3.0
3M Molecular Detection Assay
Salmonella
29
3.1
To each 25g test portion, add 225 mL 3M BPW ISO. Homogenize each sample
30
for approximately two minutes and incubate 8 h at 37 ± 1°C.
31
3.2
Prepare the 3M Molecular Detection speed loader tray, chill block insert, heat
32
block insert and instrument following the 3M Molecular Detection Assay
33
Salmonella
IFU and 3M Molecular Detection Instrument manual.
34
3.3
Transfer 20 µL enriched sample into a LS tube.
35
3.4
Transfer 20 µL NC into a LS tube after all enriched samples have been
36
completed.
37
3.5
Invert 3-5 times and heat 15±1 min at 100±1°C.
38
3.6
Place tubes (without rack lid) in chill block insert for 10±1 min.
39
3.7
Replace rack lid and invert 3-5 times. Allow to sit on bench at least 5 min.
40
3.8
Transfer 20 µL sample lysate from the upper portion of fluid in the LS tube into
41
regent tube. Mix gently by pipetting up and down 5 times.
42
3.9
Transfer 20 µL of NC lysate into a reagent tube.
43
3.10
Transfer 20 µL of NC lysate into a Reagent Control tube.
44
3.11
Load capped tubes into speed loader tray and close lid.
45
3.12
Start assay.
46
DRAFT DOCUMENT
15