应用实时荧光定量PCR方法定量检测双歧杆菌对Caco-2细胞的黏附

【目的】采用实时荧光定量PCR的方法定量分析黏附于Caco-2细胞的双歧杆菌，并建立一种快速有效分离黏附于细胞的细菌的方法。【方法】采用Triton X-100溶液处理黏附于Caco-2细胞上的菌体，确定获得最佳分离效果的处理时间；建立实时荧光定量PCR定量检测双歧杆菌的方法，获得标准曲线，进行特异性、灵敏度、重复性评价；应用建立的方法分析11株双歧杆菌对Caco-2细胞的黏附能力。【结果】Triton X-100处理黏附于Caco-2细胞的双歧杆菌的最佳作用时间为10 min。实时荧光定量PCR定量检测双歧杆菌的方法重复性好、特异性强、灵敏度高；起始模板浓度范围在104?108 CFU/mL之间具有良好的线形关系，相关系数>99%，在该浓度范围线性方程为：y=?3.345 2x+37.637 0。应用建立的方法定量分析双歧杆菌的黏附能力，与直接镜检法相比差异不显著(P>0.05)，检测时间由48 h缩短至4 h。【结论】Triton X-100分离处理结合实时荧光定量PCR方法是一种快速、有效的检测双歧杆菌对Caco-2细胞黏附能力的方法。

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

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.