Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients.
Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients.
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DOI：
10.1186/1471-2180-9-244
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发表时间：
2009-11-29
期刊：
影响因子：
4.2
通讯作者：
Vaneechoutte M
中科院分区：
文献类型：
--
作者：
Deschaght P;De Baere T;Van Simaey L;Van Daele S;De Baets F;De Vos D;Pirnay JP;Vaneechoutte M
铜绿假单胞菌是囊性纤维化（CF）患者肺功能下降的主要病原体。早期积极的抗生素治疗已被证明是有效的，在防止慢性殖民。因此，早期检测是重要的，灵敏的检测方法是必要的。在这项研究中，我们使用了一系列稀释的铜绿假单胞菌阳性paclitata，稀释在铜绿假单胞菌阴性paclitata池中，所有这些paclitata都来自CF患者-以尽可能接近地模拟发送到常规实验室的paclitata-以比较三种培养技术与两种常规PCR形式和四种实时PCR形式的灵敏度，每种PCR形式都靶向铜绿假单胞菌oprL基因。此外，我们比较了五种DNA提取方案。在我们手中，所有三种培养方法和bioMérieux easyMAG Nuclisens方案Generic 2.0.1，之前进行蛋白酶K预处理，然后进行3种带有探针的实时PCR形式中的任何一种都是最灵敏的，并且能够检测高达50 cfu/ml的铜绿假单胞菌，即每个PCR混合物理论上最少一个细胞，当考虑到本研究中用于DNA提取的样品、DNA洗脱和PCR混合物中DNA提取物的体积时。在本研究中，微生物培养和优化的DNA提取和实时PCR检测痰液中铜绿假单胞菌的灵敏度没有差异。结果还表明，DNA提取方案和PCR格式的优化的重要性。
Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF) patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization. Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P. aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P. aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols. In our hands, all three culture methods and the bioMérieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P. aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture. In this study, no difference in sensitivity could be found for the detection of P. aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.
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DOI：
10.1186/cc702
发表时间：
2000
期刊：
Critical care (London, England)
影响因子：
--
作者：
Pirnay JP;De Vos D;Duinslaeger L;Reper P;Vandenvelde C;Cornelis P;Vanderkelen A
通讯作者：
Vanderkelen A
影响因子：
24.3
作者：
Schelstraete, P.;Van daele, S.;De Baets, F.
通讯作者：
De Baets, F.
影响因子：
9.4
作者：
Spilker, T;Coenye, T;LiPuma, JJ
通讯作者：
LiPuma, JJ
影响因子：
2.2
作者：
Dauphin, Leslie A.;Moser, Benjamin D.;Bowen, Michael D.
通讯作者：
Bowen, Michael D.
影响因子：
5.1
作者：
KEREM, E;COREY, M;LEVISON, H
通讯作者：
LEVISON, H