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For fresh spinach, the 25 g test portions were enriched in 225 ml of pre-warmed
mTSB+CAA, homogenized by stomaching for 1.5 min ± 10 sec, and incubated for 10±1hrat
42 ± 1°C.
Following 10 hours of incubation at 42 ± 1°C, the manual and automated DNA preparation
methods were conducted as outlined in the “General Preparation”section.
Real-Time PCR
The 72 rotor disk or strip tubes containing samples were loaded into the Rotor-Gene
Q instrument after which the prompts were followed using the Rotor-Gene Q series
software to identify samples and controls. The
mericon
assayswere initiated and
results were obtained within 90 minutes.
Interpretation of Results
Determining the presence or absence of pathogen DNA is carried out based on the
amplification of the target sequence and is visualized in real time on the
amplification plot generated by the application software of the real-time PCR
instrument used. A positive result is visible as a final point on the fluorescence curve
that lies clearly above the cycle threshold. Table 4 below shows a summary of
possible outcomes analyzed by the software.
Table 4: Summary of Possible Outcomes
Amplification of Sample
Amplification of Internal
Control
Result
C
T
range <38
C
T
range 24-30
Positive
C
T
range 38.01 – 40
C
T
range 24-30
Indeterminate; repeat test
No C
T
C
T
range 24-30
Negative
No C
T
C
T
≥ 30.01 or No C
T
IC invalid, PCR inhibited;
dilute sample and repeat test
Confirmation
All samples analyzed by the
mericon
assay, regardless of presumptive result, were
confirmed by procedures outlined in the reference methods specified for each
matrix. Final confirmation was achieved by VITEK 2 GN Biochemical Identification,
AOAC OMA 2011.17.
Robustness Evaluation
Both the
mericon
E.coliO157 Screen Plus and
mericon
E.coliSTEC O-Type Pathogen
Detection Assays were evaluated for robustness by examining the performance of the
assay when small changes to the operating conditions were made. The following
parameters were evaluated: (1) enrichment incubation time, (2) manual heat lysis time, and
(3) sample and Master Mix volume ratio. Only DNA extracted with the manual extraction
method was used in these studies.
OMAMAN-36 E/ AOAC PTM Report
ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only