Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dóm tér 10, Szeged H-6720, Hungary.
BMC Microbiol. 2013 Dec 23;13:300. doi: 10.1186/1471-2180-13-300.
Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified.The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections.
A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons.With the novel pathogen detection system, fungi, G + and G- bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube.
This modified FRET technique is specific and more rapid than the gold-standard culture-based methods. The fact that fungi, G + and G- bacteria were successfully identified in the same tube within an hour after the DNA preparation permits rapid and early evidence-based management of bloodstream infections in clinical practice.
聚合酶链反应（PCR）技术广泛用于鉴定真菌和细菌感染。尽管这些技术的临床实用性尚未完全阐明，但已有许多关于不同的、新的、实时 PCR 病原体鉴定方法的报道。本研究专注于一种新的、多重实时 PCR 病原体鉴定系统，用于快速区分常见的血流感染的细菌和真菌病原体。
引入了一种用于检测和区分真菌、革兰氏阳性（G+）和革兰氏阴性（G-）细菌的多重实时 PCR 方法。革兰氏分类使用推荐用于 LightCycler 毛细管实时 PCR 的特定荧光共振能量转移（FRET）探针进行。我们系统的新颖之处在于使用非特异性 SYBR Green 染料代替标记的锚定探针或引物来激发 FRET 探针上的受体染料。结合这一点，使用嵌入染料可以检测真菌扩增子。使用新型病原体检测系统，通过同一管中扩增子和探针的熔解温度，可以在 DNA 制备后一小时内从同一反应管中区分真菌、G+和 G-细菌。
这种改良的 FRET 技术比金标准基于培养的方法更具特异性和更快。在 DNA 制备后一小时内，成功在同一管中鉴定出真菌、G+和 G-细菌，这使得能够在临床实践中快速进行基于证据的血流感染管理。
