Coxiella burnetii, the etiologic agent of Q fever, is a Gram-negative, obligate intracellular coccobacillus, which has been used as the... Show moreCoxiella burnetii, the etiologic agent of Q fever, is a Gram-negative, obligate intracellular coccobacillus, which has been used as the reference organism for defining milk pasteurization conditions. A developed Integrated Cell Culture-PCR (ICC-PCR) assay may be helpful for evaluating C. burnetii inactivation by thermal process in whole milk products. The purpose of this research was to characterize and optimize an ICCPCR assay in whole milk and evaluate this assay by thermal process. Coxiella burnetii was diluted into whole milk from FDA stock and treated by thermal process. Samples were sealed in glass vials and placed in ice-water to obtain a consistent starting temperature. Cooled vials were heated in a shaking waterbath at 60 °C, 62 °C and 64 °C in for various times. Thermally treated samples were diluted by RPMI+1% FBS and inoculated on Vero cell culture monolayers with PBS, incubated for 48 hours to infect the Vero cells, and then incubated for another 9 days to allow propagation. The propagated C. burnetii mix was subjected to freeze-thaw followed by DNA extraction with Autogen DNA Tissue Kit by using Quickgene Mini80. Extracted DNA was amplified by using TaqMan-MGB based qPCR targeting published primers for the IS1111a transposase gene to verify C. burnetii growth. For detection limit determination, serial dilutions of C. burnetii were mixed independently in whole milk and 1% FBS+RPMI. The mix was overlaid on confluent Vero cells for 2 days and 11 days. The DNA extractions were followed using DNA tissue kit by Quickgene Mini80 and PCR. At least duplicate trials using 3 replicates per trial were completed for each time/temperature condition. M.S. in Food Safety and Technology, May 2013 Show less
