Purpose: To optimize and characterize an ICC-PCR assay for detection of viable Cbin whole milk.
Methods: In order to optimize Cb infection, confluent Vero cell monolayers were infected for 2, 24, and 48 h with Cb resuspended in whole milk. Infected cells were then supplied with fresh RPMI media and further incubated for 9 days to encourage Cb propagation. The ability of Cb in whole milk to propagate in Vero cells was evaluated by diluting Cb in either whole milk or tissue culture media followed by 48 h infection and 9-day propagation. The level of Cb propagation was determined by subtracting the Day 9 post-infection Cb ge/ml from the Day 0 post-infection level. Replicate samples were performed for each inoculation level. In all cases, infected cells were subjected to freeze-thaw followed by spin column DNA extraction and amplification using a TaqMan-MGB qPCR assay based on published primers for the CbIS1111a transposase gene.
Results: From three independent trials, the optimal infection period was determined to be 48 h with an approximate 0.5 log increase in propagated levels over cells infected for only 2 h. In addition, even with extended infection time of 48 h, no visible monolayer damage was noted. The detection limit was 13 cells/ml in whole milk, regardless of diluent. The maximum level of propagation in whole milk as determined by qPCR was 2 log ge/ml. When resuspended in RPMI + 1% FBS, Cb levels increased 2.5 – 3 log ge/ml after 9 days propagation. Background signals were negligible.
Significance: This optimized ICC-PCR assay allows detection of infectious Cb in whole milk, and may be used to study factors affecting Cb inactivation during processing.