Performance SummaryiQ-Check STEC VirX and SerO II MethodIntroduction
The iQ-Check STEC VirX and SerO II PCR Detection Kits are
tests based on gene amplification and detection by real-time
PCR after food and environmental samples are enriched inbuffered peptone water (BPW). Ready-to-use PCR reagents in
the iQ-Check STEC VirX Kit contain oligonucleotides (primersand probes) highly specific for stx1, stx2, and eae virulence
genes of Shiga toxin–producing Escherichia coli (STEC) while
the iQ-Check STEC SerO II Kit identifies the serogroup ofthe top seven STEC strains, E. coli O157:H7, E. coli O26,E. coli O45, E. coli O103, E. coli O111, E. coli O121, andE. coli O145. A synthetic DNA internal control is included inthe reaction mix. An internal control is critical in any reaction
to monitor for inhibitors and allow for the validation of any
negative result. The iQ-Check STEC VirX and SerO II method
has been rigorously tested and validated by internationallyrecognized validation agencies (Table 1).
Table 1. Validations for the iQ-Check STEC VirX and SerO II method.
Validation

Certificate NumberAOAC

PTM 121203

USDA FSIS MLG

Chapter 5C.00

Inclusivity/Exclusivity Testing
Inclusivity testing is performed to verify that the method candetect STEC virulence genes and target serogroups while
exclusivity studies test non-STEC organisms, including otherE. coli strains, to ensure there is no cross-reactivity. Exclusivity
strains were enriched in nonselective brain heart infusion
broth for 18–24 hr at 35 ± 1°C and were tested at high levels.
A target of 10–100 colony forming units (CFU) of each STEC
inclusivity strain was cultured in BPW for 8 hr at 41.5 ± 1°C
and diluted to a low level (~103) before testing. Results areshown in Table 2.
Table 2. Results of inclusivity/exclusivity testing.
Strains Tested

Positives Detected

Results

58 STEC

58/58

100% inclusivity

78 non-STEC

0/78

100% exclusivity

Limit of Detection
Limit of detection (LOD50) is an estimation of the contaminationlevel required to achieve positive detection in 50% of cases.
This is measured by inoculating food matrices with STEC strains
and carrying out the validated enrichment, extraction, anddetection protocols (Table 3).
The average LOD50 of the iQ-Check STEC VirX and SerO II
method was determined to be 0.7 (range: 0.4–1.2).
Table 3. LOD50 for the iQ-Check STEC VirX and SerO II method.
Matrix/Strain Pair

LOD50, CFU/sample size (range)

Raw beef trim (375 g)/E. coli O26

0.6 (0.4–1.1)

Raw ground beef (375 g)/E. coli O103

0.7 (0.4–1.3)

Fresh spinach (375 g)/E. coli O145

0.5 (0.3–1.0)

Cannabis flower (10 g)/E. coli O157:H7

0.8 (0.4–1.3)

Method Comparison/Matrix Studies
Matrix testing is critical to demonstrating the performance ofa method compared to the reference method with real-world
food samples. The iQ-Check STEC VirX and SerO II method
has been verified with external and internal testing on a wide
variety of foods. No significant difference was found between
the reference method and alternative method for all matrices
tested (Table 4).
Table 4. Matrices tested with iQ-Check STEC VirX and SerO II method.
CategoryMatrices Tested

Meat products

Raw ground beef, raw beef trim

Dairy products

Raw milk, raw milk cheese

Fresh produce

SpinachEnvironmental samples

MicroTally swabs

Cannabis plants and flowers

Cannabis flower

Performance Summary: iQ-Check STEC VirX and SerO II Method

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