AOAC-RI ERP Book MICRO.pdf

QIAGEN

mericon

STEC Workflow Collaborative Study Protocol

May 2016

DRAFT

preset threshold. Inhibited samples are retested at a 1/10 dilution. The

mericon

E. coli Screen

Plus targets the

stx1

stx2

O157

, and

eae

genes. The

mericon

E. coli STEC O-Type targets the big

six O-groups (O26, O45, O103, O111, O121, and O145) and O157:H7. It does not distinguish

between the big six O-groups.

Confirmation of presumptive results is carried out according to MLG 5.09 (for

E. coli

O157:H7 in

ground beef or beef trim) or MLG 5B.05 (for non-O157 STEC in ground beef or beef trim).

1.2

Summary of PTM Study

The

mericon

methods were validated according to the current AOAC Guidelines (2012) and

granted PTM certification for detection of

E. coli

O157 and non-O157 STECs in ground beef, beef

trim, and spinach. The

mericon

®E. coliO157 Screen PlusPathogen Detection Assay was assigned

PTM #101503 and the

mericon

®E. coliSTEC O-typePathogen Detection Assay was assigned PTM

#101504. The two methods were co-validated and compared to the FSIS MLG 5.09:

Detection,

Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and

Environmental Sponges

for raw ground beef (70% lean), or the FSIS MLG 5B.05:

Detection and

Isolation of non-O157 Shiga Toxin-Producing Escherichia coli (STEC) from Meat Products and

Carcass and Environmental Sponges

for raw beef trim, and FDA BAM Chapter 4A:

Diarrheagenic

Escherichia coli

for fresh spinach. Raw ground beef was inoculated with

E. coli

O157:H7 and raw

beef trim with

E. coli

O26 and the studies were carried out with paired 325 g test portions

enriched in modified Tryptic Soy Broth with casaminoacids (mTSB + CAA) at 42 ± 1°C. The

mericon

methods were performed at 10 ± 1 h incubation time, while the MLG methods were

performed at 15-24 h incubation time. Fresh spinach was inoculated with

E. coli

O111 and the

study design was unpaired with the

mericon

methods using 25 g test portions enriched in mTSB

+ CAA at 42 ± 1°C for 10 ± 1 h and the BAM reference method using 200 g test portions

(prepared from 25 g of the inoculated spinach mixed with 175 g of uninoculated spinach) in

modified Buffered Peptone Water with pyruvate (mBPWp) without homogenization at 37 ± 1°C

for 5 h, followed by addition of selective agents, and incubation at 42 ± 1°C for 18-24 h.

Table 1 shows a summary of the PTM study and proposed collaborative study.

Note that spinach

is not claimed in the OMA method

. The ground beef and beef trim use the same enrichment

conditions and test portion size, so only ground beef will be tested in the collaborative study.

Table 1. Comparison of PTM study and proposed Collaborative study

Matrix

mericon

Test

Portion Size

PTM Study

Collaborative Study - Proposed

Organism

Reference

Method

Organism

Reference

Method

Spinach

25 g

E. coli

O111

BAM Chapter 4A

Ground beef

325 g

E. coli

O157:H7

MLG 5.09

E. coli

O157:H7

MLG 5.09

Beef trim

325 g

E. coli

O26

MLG 5B.05

NC = Not Claimed for OMA; NA = Not applicable (not included in the collaborative study)

OMAMAN-36 B : Collaborative Study Protocol

For ERP Use Only

January 2017

AOAC Research Institute

Expert Review Panel Use Only