title: Development and validation of a real-time TaqMan assay for the detection and enumeration of Pseudomonas fluorescens ATCC 13525 used as a challenge organism in testing of food equipments.
abstract: Pseudomonas fluorescens ATCC 13525 is used as the challenge organism to evaluate the efficacy of the clean-in-place (CIP) process of food equipment (automatic ice-maker) as per NSF/ANSI Standard 12. Traditional culturing methodology is presently used to determine the concentration of the challenge organism, which takes 48 h to confirm the cell density. Storage of the challenge preparation in the refrigerator might alter the cell density as P. fluorescens is capable of growing at 4 °C. Also, background organism can grow on the Pseudomonas F agar (PFA) used for the recovery of P. fluorescens thus affecting the results of the test. Real-time TaqMan assay targeting the cpn60 gene was developed for the enumeration and the identification of P. fluorescens because of its specificity, accuracy, and shorter turnaround time. The TaqMan primer-probe pair developed using the Allele ID® 7.0 probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10 colony forming units (CFU)/mL. The assay was also successful in determining the concentration of the challenge preparation within 2 h. Based on these observations, TaqMan assay targeting the cpn60 gene can be efficiently used for strain level identification and enumeration of bacteria.
  
  
  CONCLUSIONS
  Pseudomonas fluorescens ATCC 13525 is used as a challenge organism in the efficacy testing of clean-in-place process of food equipments. Currently, culturing technique is used for its identification and estimation, which is not only time-consuming but also prone to error. Real-time TaqMan assay is more specific, sensitive, and accurate along with a shorter turnaround time compared to culturing techniques, thereby increasing the overall quality of the testing methodology to evaluate the clean-in-place process critical for the food industry to protect public health and safety.
authors.affiliation: Dept of Microbiology and Molecular Biology, NSF Intl, 789 N Dixboro Rd, Ann Arbor, MI 48105, USA. rsaha@nsf.org
authors.forename: Ratul
authors.initials: R
authors.lastname: Saha
authors.forename: Lorelle L
authors.initials: LL
authors.lastname: Bestervelt
authors.forename: Robert S
authors.initials: RS
authors.lastname: Donofrio
mesh_terms: D015169:Colony Count, Microbial; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis*; D005511:Food Handling / Q000379:methods; D005516:Food Microbiology / Q000379:methods*; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D012984:Software
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial