Huang J, DeGraves F J, Gao D, Feng P, Schlapp T, Kaltenboeck B
College of Veterinary Medicine, Auburn University, Auburn, AL, USA.
Biotechniques. 2001 Jan;30(1):150-7. doi: 10.2144/01301rr03.
Quantitative detection of intracellular bacteria of the genus Chlamydia by the standard cell culture method is cumbersome and operator dependent. As an alternative, we adapted hot-start PCR to the glass capillary quantitative PCR format of the LightCycler. The optimized PCR was consistently more efficient than commercially available pre-assembled PCRs. Detection by quantitative PCR of as few as single copies of DNA of Chlamydia spp. was accomplished by SYBR Green fluorescence of the dsDNA product and by fluorescence resonance energy transfer (FRET) hybridization probes. The PCRs were 15-fold more sensitive than the cell culture quantitative assay of C. psittaci B577 infectious stock. The number of chlamydial genomes detected by C. psittaci B577 FRET PCR correlated well with cell culture determination of inclusion forming units (IFUs) (r = 0.96, P < 0.0008). When infected tissue samples were analyzed by cell culture and PCR, the correlation coefficient between IFUs and chlamydial genomes was higher with C. psittaci B577 FRET PCR (r = 0.90, P < 0.0004) than with Chlamydia omp1 SYBR Green PCR (r = 0.85, P < 0.002).
采用标准细胞培养方法对衣原体属细胞内细菌进行定量检测既繁琐又依赖操作人员。作为一种替代方法，我们将热启动PCR应用于LightCycler的玻璃毛细管定量PCR形式。优化后的PCR始终比市售的预组装PCR更高效。通过双链DNA产物的SYBR Green荧光和荧光共振能量转移（FRET）杂交探针，实现了对衣原体属DNA单拷贝的定量PCR检测。这些PCR比鹦鹉热衣原体B577感染原种的细胞培养定量检测法灵敏15倍。通过鹦鹉热衣原体B577 FRET PCR检测到的衣原体基因组数量与包涵体形成单位（IFU）的细胞培养测定结果相关性良好（r = 0.96，P < 0.0008）。当通过细胞培养和PCR分析感染组织样本时，鹦鹉热衣原体B577 FRET PCR的IFU与衣原体基因组之间的相关系数（r = 0.90，P < 0.0004）高于衣原体omp1 SYBR Green PCR（r = 0.85，P < 0.002）。
