Application of quantitative real-time PCR for quantification of Bifidobacterium adhesion to Caco-2 cells

Abstract:

[Objective] Bifidobacterium adherent to Caco-2 cells were quantitatively analyzed by quantitative real-time PCR, and a rapid and effective method to detach the bacteria from the Caco-2 cells was established. [Methods] Firstly, the Bifidobacterium adherent to the Caco-2 cells were treated by Triton X-100 solution with the aim of separating bifidobacterial cells from the Caco-2 cells. Secondly, quantitative real-time PCR method for quantifying Bifidobacterium was determined by generating a standard curve, evaluating the specificity, sensitivity and reproducibility of the method, respectively. Finally, the adhering capacity of 11 Bifidobacterium to the Caco-2 cells was detected by the method established in this study. [Results] The optimum time for separating Bifidobacterium from Caco-2 cells by Triton X-100 was 10 min. The quantitative real-time PCR method used for determing adherent ability of Bifidobacterium demonstrated a good reproducibility, a high specificity and sensitivity. When the initial template concentration was in the range of 104?108 CFU/mL, a good linear relationship between bacterial cell counts and Ct value was expressed with the equation of y=?3.345 2x+37.637 0, whose correlation coefficient was above 99%. Compared with the gram staining microscopic examination with the detection time of 48 h, the quantitative real-time PCR method established in this study was able to obtain non-significant results of quantifying the adherent capacity of Bifidobacterium (P>0.05), however, the time for detection was greatly shortened to 4 h. [Conclusion] Triton X-100 separation processing combining with quantitative real-time PCR is a rapid, accurate and sensitive method for detecting and quantifying the adhering capacity of Bifidobacterium to the Caco-2 cells.