Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Iran.
Infect Genet Evol. 2010 Dec;10(8):1247-51. doi: 10.1016/j.meegid.2010.08.008. Epub 2010 Aug 20.
Using oprL sequences, a TaqMan real time PCR was developed and used for quantitative detection of Pseudomonas aeruginosa from 99 broncoalveolar lavage and 11 sputum specimens collected from patients with health care associated pneumonia. All specimens were cultured on appropriate media to isolate bacteria. Twenty five specimens were positive by both methods. Polymicrobial infections were found in 13 specimens. Amplification of oprL in serial dilutions ranged from 10(9)CFU/ml to 10(2)CFU/ml. Standard curve of duplicated every dilution had slope 3.25±0.1 and R(2)>0.99 with SD 0.1. Our real time PCR assay showed high sensitivity (100%) and specificity (98.85%). This technique could detect and enumerate 100 bacteria directly from clinical specimens and showed that the threshold is 10(3)CFU/ml in cases with clinical symptoms. Our method can be used for quantitative detection of P. aeruginosa from BAL and sputum specimens in 1h and 10min.
利用 oprL 序列，我们开发了一种 TaqMan 实时 PCR 方法，并用于定量检测与医疗保健相关肺炎患者的 99 份支气管肺泡灌洗液和 11 份痰液标本中的铜绿假单胞菌。所有标本均在适当的培养基上培养以分离细菌。两种方法均阳性的标本有 25 份。13 份标本为混合感染。oprL 的扩增范围从 10(9)CFU/ml 到 10(2)CFU/ml。每个稀释度的标准曲线斜率为 3.25±0.1，R(2)>0.99，标准偏差为 0.1。我们的实时 PCR 检测方法显示出很高的灵敏度（100%）和特异性（98.85%）。该技术可直接从临床标本中检测和计数 100 个细菌，且结果显示在有临床症状的情况下，阈值为 10(3)CFU/ml。我们的方法可用于在 1 小时 10 分钟内定量检测 BAL 和痰液标本中的铜绿假单胞菌。
