Reliability of Quantitative Real-Time PCR for Bacterial Detection in Cystic Fibrosis Airway Specimens
Reliability of Quantitative Real-Time PCR for Bacterial Detection in Cystic Fibrosis Airway Specimens
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DOI：
10.1371/journal.pone.0015101
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发表时间：
2010-11-30
期刊：
影响因子：
3.7
通讯作者：
Harris, J. Kirk
中科院分区：
文献类型：
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作者：
Zemanick, Edith T.;Wagner, Brandie D.;Harris, J. Kirk
囊性纤维化(CF)的呼吸道微生物群是复杂的；多菌感染是常见的，包括厌氧菌在内的挑剔细菌的存在使基于培养的诊断具有挑战性。实时定量聚合酶链式反应(QPCR)提供了一种不依赖于培养物的细菌定量方法，可能会提高对CF呼吸道感染的诊断；然而，qPCR应用于CF呼吸道标本的可靠性尚不清楚。我们试图确定9种特异性细菌定量聚合酶链式反应(总细菌、3种典型的CF病原菌和5种厌氧菌)应用于CF呼吸道标本的可靠性。收集了临床稳定的儿童CF患者的呼吸道和唾液标本。定量聚合酶链式反应的重复性通过三重反应来确定。进行分裂样本测量以测量DNA提取带来的变异性。将定量聚合酶链式反应的结果与标准微生物培养结果进行比较，包括铜绿假单胞菌、金黄色葡萄球菌和流感嗜血杆菌。我们采集了16例儿童CF患者的84份痰标本、47份口咽标本和27份唾液标本。定量聚合酶链式反应在97%以上的标本中检测到细菌DNA。所有定量聚合酶链式反应的重复性均为10(2)rRNA基因拷贝数/反应数，变异系数小于20%，超过99%的样品。一式两份的样品之间也有很好的一致性。厌氧菌非常普遍，其平均数量与典型的CF病原菌相似。与复合金标准相比，定量聚合酶链式反应和培养法检测肺炎支原体呼吸道标本中的铜绿假单胞菌、金黄色葡萄球菌和流感嗜血杆菌具有不同的敏感性。通过可靠地量化挑剔的呼吸道细菌，qPCR可能会提高我们对多菌CF肺部感染、肺部疾病进展的了解，并最终改进抗菌治疗。
The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities >10(2) rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.