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(a)
PCR loading block should be stored at 2-5 °C.
(b)
Use aseptic techniques.
(c)
Change pipette tips between samples.
(d)
Change pipette tips for the PCR Master Mix and the DNA samples.
(e)
Use gloves and protective laboratory wear.
(f)
Do not touch any PCR equipment or supplies without wearing gloves.
(g)
In case of contamination, laboratory benches, apparatus, and pipettes can be
decontaminated with a 1/10 dilution of commercial bleach solution.
General Preparation
Sample Preparation and DNA Amplification
Performing Enrichment
(a) Fresh Spinach (25 g test portions)
A 25 g test portion is added to 225 mL of pre-warmed modified tryptic soy broth
containing casamino acids (mTSB+CAA), homogenized by stomaching for 1.5
min+10 sec, and incubated at 42 ± 1°C for 10 ± 1 hour.
(b) Raw Ground Beef (70% lean) (325 g test portions)
A 325 g test portion is added to 975 mL of pre-warmed mTSB+CAA, homogenized
by stomaching for, and incubated at 42 ± 1°C for 10 ± 1 hour.
(c) Raw Beef Trim (325 g test portions)
A 325 g test portion is added to 975 mL ofpre-warmed mTSB+CAA, homogenized
by stomaching for 2.0 min +10 sec, and incubated at 42 ± 1°C for 10 ± 1 hour.
Preparing Samples
(a) Manual mericonDNA Extraction Method
1) Gently mix all enriched samples to disperse
E. coli
organisms throughout the
sample. Transfer 1 mL of enriched sample to a sterile 2 mL screw cap tube and
centrifuge for 5 minutes at 13,000 x
g
. Discard the supernatant with a pipettor,
taking care not to disrupt the pellet. Add 200µLof Fast Lysis Buffer to the
bacterial pellet, tightly cap tube and vortex to resuspend the pellet. Place the
microcentrifuge tube into a heat block (100 ± 1°C) for 10 min ±10 sec. Remove
the sample and allow it to cool to 15-24 °C for 2 min ± 10 sec. Centrifuge the
tube at 13,000 x
g
for 5 minutes.
2) For all matrixes, dilute the extracted DNA 1:10 using sterile nuclease-free water.
OMAMAN-36 E/ AOAC PTM Report
ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only