Development of a Standard Curve to Account for Viable Loads of Bifidobacterium animalis subsp. lactis HNO19 Using RNA by Real-Time PCR

Background: Bifidobacterium animalis subsp. lactis HN019 is widely used as a probiotic in various dairy preparations. To ensure optimal health benefits, an adequate number of probiotics should be viable in products, but the culture-based methods require longer periods to account for the strain. Objective: Develop a standard curve to account viable load of B. animalis subsp. lactis HN019 by real-time PCR. Methods: The growth curve was developed according to plate count method, and cycle threshold (C-T) curve was prepared using C-T values. These two curves were then combined to construct the standard curve. To validate the method, the strain was proliferated in whole milk, and viable loads were enumerated by plate counting and relative PCR counting followed by determining the SEM between the two counts. Results: The growth curve and C-T curve, respectively, showed the highest viable load (2.13 x 10(8) CFU/mL) and lowest C-T value (18.29) after 18 h, and during the entire growth phase (0-18 h), viable loads are inversely proportional to the CT values. The standard curve revealed the model y = 2E + 18e(-1.233x) (y = cells/mL, x = C-T value; R-2 = 0.992). In validation, the highest SEM (+/- 0.70 x 10(8)) was found between the plate count (1.15 x 10(8) cells/mL) and relative PCR count (1.29 x 10(8) cells/mL) after cultivating for 14 h. Conclusions: The method could be readily used in dairy industries to quantify viable B. animalis subsp. lactis HN019 by a shorter period. Highlights: Culture-independent enumeration of viable B. animalis subsp. lactis HN019 by real-time PCR.