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首页> 外文期刊>Journal of Clinical Microbiology >Use of Real-Time PCR with Multiple Targets To Identify Pseudomonas aeruginosa and Other Nonfermenting Gram-Negative Bacilli from Patients with Cystic Fibrosis
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Use of Real-Time PCR with Multiple Targets To Identify Pseudomonas aeruginosa and Other Nonfermenting Gram-Negative Bacilli from Patients with Cystic Fibrosis

机译：使用实时PCR和多个目标来鉴定囊性纤维化患者的铜绿假单胞菌和其他非发酵性革兰阴性杆菌

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Pseudomonas aeruginosa and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity. We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and gyrB PCR and sequencing as a “gold standard.” Of 50 isolates easily identified phenotypically as P. aeruginosa, all were positive with PCR primers for gyrB or oprI, 98% were positive with exotoxin A primers, and 90% were positive with algD primers. Of 50 P. aeruginosa isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with gyrB or oprI primers, 96% were positive with exotoxin A primers, and 92% were positive with algD primers. For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as P. aeruginosa; all 13 were positive with gyrB primers, 12 of 13 were positive with oprI primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with algD primers. A single false-positive P. aeruginosa result was seen with oprI primers. The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group. Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time. Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical P. aeruginosa and for non-P. aeruginosa gram-negative isolates.
机译：铜绿假单胞菌和其他革兰氏阴性分离株来自囊性纤维化（CF），可能由于其明显的表型多样性而难以鉴定。我们检查了来自CF呼吸道标本的200克阴性临床分离株，并使用生化测试和分子鉴定（包括16S rRNA部分测序和 gyrB）的标准化组合，通过生化测试和实时PCR与多个不同靶序列进行鉴定比较将PCR和测序作为“黄金标准”。在50个分离物中，很容易在表型上被识别为 P。铜绿假单胞菌， gyrB 或 oprI 的PCR引物均为阳性，外毒素A引物为98％， algD为90％ 入门。 50个 P。可以通过基本生化检测鉴定的铜绿假单胞菌分离物，采用 gyrB 或 oprI 引物的实时PCR阳性率为100％，外毒素为96％阳性一个引物， algD 引物为阳性。对于需要更广泛的生化评估的分离株，鉴定出13个分离株为 P。铜绿; gyrB 引物全部13例阳性， oprI 引物13例中12例，外毒素A引物13例中11例，阳性> algD 引物。单个假阳性 P。用 oprI 引物可以看到铜绿假单胞菌的结果。表现最好的商业生化测试与这一最困难的人群的分子鉴定只有60％的时间完全吻合。实时PCR的成本与商业生化测试的成本相似，但是周转时间短得多。考虑到这些CF分离株的多样性，结合两个靶序列的实时PCR似乎是鉴定非典型P的最佳选择。铜绿和非 P。铜绿菌革兰氏阴性菌。

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