Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli from patients with cystic fibrosis
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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DOI：
10.1128/jcm.41.9.4312-4317.2003
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发表时间：
2003-09-01
影响因子：
9.4
通讯作者：
Burns, JL
中科院分区：
文献类型：
--
作者：
Qin, X;Emerson, J;Burns, JL
铜绿假单胞菌和其他革兰氏阴性分离囊性纤维化（CF）患者可能很难确定，因为他们的显着的表型多样性。我们检查了200个来自CF呼吸道标本的革兰氏阴性临床分离株，并使用生化检测和分子鉴定的标准化组合（包括16 S rRNA部分测序和gyrB PCR和测序作为“金标准”），比较了生化检测和实时PCR与多种不同靶序列的鉴定。“在50个容易鉴定为铜绿假单胞菌的分离株中，所有gyrB或oprI的PCR引物均呈阳性，98%的外毒素A引物呈阳性，90%的algD引物呈阳性。在50株可通过基本生化检测鉴定的铜绿假单胞菌分离株中，gyrB或oprI引物的实时PCR阳性率为100%，外毒素A引物的阳性率为96%，algD引物的阳性率为92%。对于需要更广泛生化评价的分离株，13株分离株被鉴定为铜绿假单胞菌;所有13株均为gyrB引物阳性，12/13株为oprI引物阳性，11/13株为外毒素A引物阳性，10/13株为algD引物阳性。使用oprI引物观察到一个假阳性铜绿假单胞菌结果。对于这个最困难的群体，最好的商业生化检测与分子鉴定的准确率只有60%。实时PCR的成本与商业生化检测相似，但周转时间要短得多。鉴于这些CF分离株的多样性，采用两种靶序列组合的实时PCR似乎是鉴定非典型铜绿假单胞菌和非铜绿假单胞菌革兰氏阴性分离株的最佳选择。
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.