AOAC 2018 Methods

108  B ird et al . : J ournal of AOAC I nternational V ol . 102, N o . 1, 2019

Food Biological Contaminants

Evaluation of the 3M™ Molecular Detection Assay (MDA) 2 – Cronobacter for the Detection of Cronobacter species in Select Foods and Environmental Surfaces: Collaborative Study, First Action 2018.01 P atrick B ird , M. J oseph B enzinger J r ., B en B astin , E rin C rowley , J ames A gin , and D avid G oins Q Laboratories, Inc., 1400 Harrison Ave, Cincinnati, OH 45214 L isa M onteroso 1 3M Food Safety Department, 3M Center, Building 260-6B-01, St. Paul, MN 55144 Collaborators: T. Abramovic; M. Achen; E. Budge; N. Clemens; A. Cruz; J. Cuellar; C. Diaz; Y. Hong; S. Ishii; C. Liska; C. Luquin; L. Ma; K. Martin; G. Masanz; M. Matijevic; P. McKelvey; J. Miller; S. Molina; A. Mustafic; I. Podoreski; I. Ricki; B. Schindler; A. Scollon; C. Somoza; C. Tinajero; L. Thompson; S. Wheeler; A. Winslow; W. Xu

formula (2). The organism is able to survive in low-moisture products, such as powdered infant formula, for long periods of time. Current manufacturing processes for producing powdered infant formula are not effective in eliminating the bacteria completely from the product (3). The addition of probiotics to these products, although beneficial for the recipient, can make it difficult to culture the pathogen due to background flora and bacteriocins produced by the probiotic culture. The 3M ™ Molecular DetectionAssay (MDA) 2 – Cronobacter method, uses loop-mediated isothermal amplification (LAMP) for deoxyribonucleic acid (DNA) amplification of the target organism in real-time. This involves a two-step enzymatic process in which pyrophosphate molecules, produced as a byproduct of the DNA amplification, are used to generate light. This light emission is measured by the 3M Molecular Detection Instrument and signals the detection of the target organism. In the absence of amplification, no light signal is generated resulting in no detection of target organism. The 3M MDA 2 – Cronobacte r method allows for the rapid and specific detection of Cronobacter species in select food types and environmental surfaces after 18–24 h of pre-enrichment. After enrichment, samples are evaluated using the 3M MDA 2 – Cronobacter on the 3M Molecular Detection System (MDS). Presumptive positive results are reported in real-time and negative results are displayed after completion of the 60 min run. Prior to the collaborative study, the 3MMDA2 – Cronobacter method was validated according to AOAC Guidelines (4) in a harmonized AOAC Performance Tested Method SM (PTM) study. The assay was awarded PTM 101703 on October 24, 2017. The objective of these studies was to demonstrate that the 3M MDA 2 – Cronobacter method could detect Cronobacter in select food matrixes and environmental surfaces as claimed by the manufacturer. The following matrixes were evaluated: powdered infant formula with probiotics (10 and 300 g), powdered infant cereal without probiotics (10 and 300 g), lactose powder (10 g), and environmental surface sponges (stainless steel). The purpose of this collaborative study was to compare the reproducibility of the 3M MDA 2 – Cronobacter method with ISO 22964:2017 (5).

Received July 19, 2018. Accepted by AH August 10, 2018. This method was approved by the AOAC Expert Review Panel for Cronobacter as First Action. The Expert Review Panel invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and is critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. Supplemental information is available online at: http://aoac. publisher.ingentaconnect.com/content/aoac/jaoac 1 Corresponding author’s e-mail: lmonteroso@mmm.com indicating that the difference between methods was not statistically significant at the 0.05 probability level. C  ronobacter, originally defined as a species, Enterobacter sakazakii , is a motile Gram-negative bacterium that causes foodborne illness (1). The opportunistic pathogenic organism has been linked with serious illness among infants, notably following the consumption of powdered infant The 3M™ Molecular Detection Assay (MDA) 2 – Cronobacter combines the use of loop-mediated isothermal amplification to rapidly amplify nucleic acid sequences while using bioluminescence to detect the amplification. Using a paired study design, the MDA 2 – Cronobacter was compared with ISO 22964:2017 for the detection of Cronobacter species in powdered infant formula containing probiotics. Technicians from 11 laboratories from the United States, Mexico, and Croatia participated. Collaborators received test portions with three levels of contamination. Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low-inoculum level test portions produced a difference in POD values obtained from combining all valid collaborator POD data values with 95% confidence intervals of –0.01, –0.12, and 0.10,

DOI: https://doi.org/10.5740/jaoacint.18-0233