Identification of Pseudomonas aeruginosa, Burkholderia cepacia complex, and Stenotrophomonas maltophilia in respiratory samples from cystic fibrosis patients using multiplex PCR
Identification of Pseudomonas aeruginosa, Burkholderia cepacia complex, and Stenotrophomonas maltophilia in respiratory samples from cystic fibrosis patients using multiplex PCR
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DOI：
10.1002/ppul.20016
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发表时间：
2004-06-01
影响因子：
3.1
通讯作者：
Ramos, SRTS
中科院分区：
文献类型：
--
作者：
da Silva, LVF;Tateno, AF;Ramos, SRTS
建立了鉴定铜绿假单胞菌B的多重PCR方法。cepacia complex和S.嗜麦芽窄食单胞菌直接在CF患者的痰和口咽样品中。从2000年9月至2001年4月，对106例（53例男性和53例女性）在我们的肺病诊所就诊的患者进行了研究。在选择性培养基中培养257个样品，并进行多重PCR反应，使用三对引物靶向每个物种的特定基因组序列，另外一对引物靶向一段核糖体16 S DNA，细菌通用，作为对照。铜绿假单胞菌在56%的样本中通过培养分离，B。cepacia复合体4.3%;嗜麦芽窄食单胞菌占2.7%，多重PCR检出铜绿假单胞菌占78.7%，B. cepacia复合体的比例为3.9%;嗜麦芽窄食单胞菌占3.1%。多重PCR结果通过使用文献中描述的不同种属特异性引物的PCR反应和来自一些样品的扩增子的DNA测序进行验证。与培养结果比较，多重PCR鉴定铜绿假单胞菌的敏感性和特异性分别为97.2%和45.5%，鉴定B菌的敏感性和特异性分别为45.5%和97.9%。cepaciacomplex，分别为40%和97.6%.嗜麦芽窄食症B的所有10个多重PCR阳性结果。使用文献中描述的其他物种特异性引物证实了洋葱复合体的结果，而这种方法证实了S.嗜麦芽窄食单胞菌检出率为87.5%（7/8）。对铜绿假单胞菌和B培养阴性但多重PCR阳性的样本的扩增子进行测序。洋葱复合体证实了它们的身份，而扩增子之间的微小核苷酸差异排除了PCR污染的假设。(C)2004 Wiley-Liss，Inc.
A multiplex PCR method was developed to identify P. aeruginosa, B. cepacia complex, and S. maltophilia directly in sputum and oropharyngeal samples from CF patients. One hundred and six patients (53 male, and 53 female) attending our pulmonology clinic were studied from September 2000-April 2001. Two hundred and fifty-seven samples were cultured in selective media and submitted to multiplex PCR reactions, using three primer pairs targeting specific genomic sequences of each species, with an additional primer pair targeting a stretch of ribosomal 16S DNA, universal for bacteria, to act as a control. P. aeruginosa was isolated by culture in 56% of samples, B. cepacia complex in 4.3%, and S. maltophilia in 2.7%, while multiplex PCR identified P. aeruginosa in 78.7%, B. cepacia complex in 3.9%, and S. maltophilia in 3.1 % of samples. Multiplex PCR results were verified by PCR reactions using different species-specific primers described in the literature and DNA sequencing of amplicons from a few samples. Comparing to culture results, the sensitivity and specificity values of multiplex PCR for bacterial identification were, respectively, 97.2% and 45.5% for P. aeruginosa, 45.5% and 97.9% for B. cepaciacomplex, and 40% and 97.6 for S. maltophilia. All 10 multiplex PCR-positive results for B. cepacia complex were confirmed using other species-specific primers described in the literature, while this approach confirmed results for S. maltophilia identification in 7/8 samples (87.5%). Sequencing of amplicons from samples culture-negative but multiplex PCR-positive for P. aeruginosa and B. cepacia complex confirmed their identity, while minor nucleotide differences among amplicons ruled out the hypothesis of PCR contamination. (C) 2004 Wiley-Liss, Inc.