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An Integrated Cell Culture-PCR Assay for the Detection of Viable Coxiella Burnetii Nine Mile Phase II RSA 439 in Fluid Dairy Products
Title
An Integrated Cell Culture-PCR Assay for the Detection of Viable Coxiella Burnetii Nine Mile Phase II RSA 439 in Fluid Dairy Products
Creator
Kukreja, Ankush
Date
2011-12, 2011-12
Description
Coxiella burnetii, an obligate intracellular bacterium, has been used as the reference organism for defining milk pasteurization conditions....
Show moreCoxiella burnetii, an obligate intracellular bacterium, has been used as the reference organism for defining milk pasteurization conditions. With renewed interest in minimizing processing times and temperatures, and in ensuring product safety, development of an Integrated Cell Culture-PCR (ICC-PCR) assay may be useful for evaluating C. burnetii inactivation in fluid dairy products. The purpose of this research was to develop an ICC-PCR assay to determine viability and detection limit of C. burnetii and characterize inhibiting effects contributed by various milk formulations. Coxiella burnetii was inoculated on Vero cell culture with PBS and whole milk, incubated for 48 hours to allow infection, and then incubated for 11 days to allow propagation. The propagated C. burnetii mix was subjected to freeze-thaw followed by DNA extraction with Autogen Blood & Tissue DNA Extraction Kit using Quickgene Mini80. Extracted DNA was amplified using TaqMan-MGB based qPCR targeting published primers for the IS1111a transposase gene to verify C. burnetii growth/infectivity. For detection limit determination, serial dilutions of C. burnetii in RPMI were mixed separately in whole milk, cream, chocolate milk and eggnog. The mix was overlaid on sub-confluent Vero cell monolayers, subjected to freeze-thaw followed by DNA extraction using Autogen Blood & Tissue DNA Extraction Kit and PCR. Uninoculated wells were evaluated for dairy sample background signal, and inhibition was evaluated by comparison to purified DNA. Duplicate trials using 6 replicates per sample were performed.
M.S. in Food Safety & Technology, December 2011
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