Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay.
Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay.
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使用高度多重的高分辨率熔体曲线测定法对普通革兰氏阴性病原体的形态。
DOI：
10.1038/s41598-017-18915-5
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发表时间：
2018-01-18
影响因子：
4.6
通讯作者：
Adams ER
中科院分区：
文献类型：
--
作者：
Edwards T;Sasaki S;Williams C;Hobbs G;Feasey NA;Evans K;Adams ER
鉴定导致感染的细菌种类仍然是选择抗菌治疗的重要步骤。革兰氏阴性菌是医院和社区获得性感染的重要来源，并且经常具有抗菌素耐药性。细菌的物种形成通常是通过对从临床标本中分离出来的生物体进行生化分析来进行的，这既耗时又延迟了量身定制治疗的开始。虽然已经使用了诸如PCR之类的分子方法，但它们经常面临检测和区分大范围靶标的挑战。高分辨率熔体分析是一种端点qPCR检测方法，提供比基于探针的方法更大的多路复用能力。在这里，我们报告了一种高分辨率熔体分析方法的设计，用于鉴定六种常见的革兰氏阴性病原体；大肠杆菌、肺炎克雷伯菌、氧化克雷伯菌、铜绿假单胞菌、沙门氏菌和鲍曼不动杆菌，以及通用革兰氏阴性特异性16S rRNA对照。该试验是通过收集113个临床分离的革兰氏阴性菌进行评估的。HRM法与PCR和测序参比法的一致性为98.2% (Kappa 0.96)；总灵敏度为97.1% (95% CI: 90.1 ~ 99.7%)，特异度为100% （95% CI: 91.78 ~ 100%）。
The identification of the bacterial species responsible for an infection remains an important step for the selection of antimicrobial therapy. Gram-negative bacteria are an important source of hospital and community acquired infections and frequently antimicrobial resistant. Speciation of bacteria is typically carried out by biochemical profiling of organisms isolated from clinical specimens, which is time consuming and delays the initiation of tailored treatment. Whilst molecular methods such as PCR have been used, they often struggle with the challenge of detecting and discriminating a wide range of targets. High resolution melt analysis is an end-point qPCR detection method that provides greater multiplexing capability than probe based methods. Here we report the design of a high resolution melt analysis assay for the identification of six common Gram-negative pathogens; Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella Sp, and Acinetobacter baumannii, and a generic Gram-negative specific 16S rRNA control. The assay was evaluated using a well characterised collection of 113 clinically isolated Gram-negative bacteria. The agreement between the HRM assay and the reference test of PCR and sequencing was 98.2% (Kappa 0.96); the overall sensitivity and specificity of the assay was 97.1% (95% CI: 90.1–99.7%) and 100% (95% CI: 91.78–100%) respectively.
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影响因子：
4.9
作者：
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Mc Gann P
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