PCR-Based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients
PCR-Based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients
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DOI：
10.1128/jcm.42.5.2074-2079.2004
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发表时间：
2004-05-01
影响因子：
9.4
通讯作者：
LiPuma, JJ
中科院分区：
文献类型：
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作者：
Spilker, T;Coenye, T;LiPuma, JJ
铜绿假单胞菌是囊性纤维化 (CF) 患者的主要机会性细菌病原体；大约 80% 的成年 CF 患者发生肺部感染。 CF 患者的管理很大程度上取决于痰培养中铜绿假单胞菌的准确鉴定。然而，由于 CF 痰分离株表现出明显的表型变异性以及其他密切相关物种的存在，该物种的鉴定可能存在问题。为了促进物种鉴定，我们使用 16S 核糖体 DNA (rDNA) 序列数据来设计 PCR 检测，旨在提供属或物种水平的鉴定。两种检测均产生了预测大小的 DNA 片段。我们测试了 42 株培养物保藏菌株（包括 14 株铜绿假单胞菌菌株和代表 16 个其他密切相关的假单胞菌属物种的 28 株菌株），并且从 CF 患者痰中回收了先前已鉴定为属于 28 个非假单胞菌属物种的 43 株菌株。基于这 85 个菌株，两种检测的特异性和敏感性均为 100%。为了进一步评估 PCR 检测的实用性，我们测试了 66 个最近的 CF 痰分离株。结果表明，初步表型检测存在误判。几个分离株。确定了 38 个分离株的 16S rDNA 序列，并且在所有情况下都证实了 PCR 检测的结果。因此，我们设计了两种 PCR 检测方法：一种针对假单胞菌属，另一种针对铜绿假单胞菌。两种检测均显示 100% 的敏感性和特异性。
Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified. several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Both assays show 100% sensitivity and specificity.