title: Validation of the Applied Biosystems MicroSEQ real-time PCR system for detection of E. coli O157:H7 in food.
abstract: Modern molecular methods offer the advantages of simplicity and short time-to-results compared to traditional culture methods. We describe the validation of a new Real-Time PCR method to detect E. coli O157:H7 in five food matrixes. The complete system consists of the MicroSEQ E. coli O157:H7 Detection Kit, sample preparation (two sample preparation methods, the PrepSEQ Nucleic Acid Extraction Kit and the PrepSEQ Rapid Spin Sample Preparation Kit, were validated), the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. The test method was compared to the U.S. Department of Agriculture Microbiology Laboratory Guidebook 5.04 reference method for detecting E. coli O157:H7 in 25 g and 375 g ground beef and beef trim, and to the ISO 16654 reference method for detecting E. coli O157:H7 in 25 g spinach, orange juice, and apple juice. The MicroSEQ E. coli O157:H7 Detection Kit showed equivalent detection compared to the corresponding reference method based on Mantel-Haenszel Chi-square statistics for all matrixes tested. An independent validation confirmed these findings on ground beef. The MicroSEQ kit detected all 51 E. coli O157:H7 strains tested and showed good discrimination against an exclusivity panel of 30 strains.
authors.affiliation: Life Technologies, 850 Lincoln Centre Dr, Foster City, CA 94404, USA. Lily.Wong@lifetech.com
authors.forename: Lily Y
authors.initials: LY
authors.lastname: Wong
authors.forename: Yanxiang
authors.initials: Y
authors.lastname: Cao
authors.forename: Priya
authors.initials: P
authors.lastname: Balachandran
authors.forename: Patrick
authors.initials: P
authors.lastname: Zoder
authors.forename: Manohar R
authors.initials: MR
authors.lastname: Furtado
authors.forename: Olga V
authors.initials: OV
authors.lastname: Petrauskene
authors.forename: Robert S
authors.initials: RS
authors.lastname: Tebbs
mesh_terms: D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005516:Food Microbiology / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012984:Software