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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
Author(s) -
Saha Ratul,
Bestervelt Lorelle L.,
Donofrio Robert S.
Publication year - 2012
Publication title -
journal of food science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.772
H-Index - 150
eISSN - 1750-3841
pISSN - 0022-1147
DOI - 10.1111/j.1750-3841.2011.02547.x
Subject(s) - enumeration , taqman , pseudomonas fluorescens , biology , organism , pseudomonas , microbiology and biotechnology , real time polymerase chain reaction , bacteria , gene , biochemistry , genetics , mathematics , combinatorics
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. Practical Application: 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.