Biological Sciences Division, Michigan Technological University, Dow 740, 1400 Townsend Drive, Houghton, MI 49931, USA.
J Ind Microbiol Biotechnol. 2010 Aug;37(8):843-8. doi: 10.1007/s10295-010-0731-8. Epub 2010 May 11.
A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer-probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer-probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 x 10(3) and 3.9 x 10(6) CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 x 10(1) to 1.4 x 10(5) CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer-probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.
建立了一种 TaqMan 定量实时聚合酶链反应 (qPCR) 检测方法，用于检测和计数污染金属加工液 (MWF) 中属于 mendocina 亚系的三种假单胞菌 (P. oleovorans、P. pseudoalcaligenes 和 P. oleovorans subsp. lubricantis)。这些微生物是主要的定植菌，也是用过的 MWF 生物降解的指示生物。由于这些微生物在 R2A 琼脂和假单胞菌分离琼脂 (PIA) 等典型生长培养基上生长不良，因此 qPCR 等分子技术是检测这些微生物的首选方法。传统的培养技术不仅低估了这些细菌的实际分布情况，而且还很耗时。从目标细菌的回旋酶 B (gyrB) 序列开发的引物-探针对对于这三种细菌具有高度的特异性和敏感性。使用全细胞和基因组 DNA 进行 qPCR 以确认检测方法的特异性和敏感性。该检测方法的敏感性为全细胞 10(1)个菌落形成单位 (CFU)/ml，基因组 DNA 为 13.7 fg。该引物-探针对成功地确定了用过的 MWF 样品中的浓度，表明浓度范围在 2.9 x 10(3)到 3.9 x 10(6) CFU/ml 之间。相比之下，对于相同的样品，在 PIA 上回收的假单胞菌属总计数范围为 <1.0 x 10(1) 到 1.4 x 10(5) CFU/ml。基于 qPCR 检测的这些结果，设计的 TaqMan 引物-探针对可用于快速（2 小时内）确定污染 MWF 中这些假单胞菌属物种的分布。
