Molecular Diagnosis of pathogenic fungi

PCR已经彻底改变了传染病的诊断。通过DNA或RNA检测和鉴定微生物可能更灵敏、更特异和更准确，与大多数生物的表型检测相比，几乎可以立即检测。挑剔的生物体，也许比我们所知道的要多得多，基本上是不可培养的，或者需要丰富的培养基和过长的培养时间。尽管有这种潜力，但临床实验室中基于PCR的真菌检测仍落后于病毒和细菌。缺乏广泛使用的分子工具来识别真菌是一种耻辱，因为与系统性真菌感染相关的高死亡率往往诊断得太晚。由于采样点的细胞数量较少，而且细胞壁较硬，使有效的细胞裂解变得复杂，因此真菌感染的检测对于基于PCR的方法来说是一个挑战。文献综述强调，在PCR可靠地诊断烟曲霉和其他真菌之前，需要进行更多的优化。我们的长期目标是开发快速，定量和灵敏的检测和鉴定真菌物种的方法，并最终从临床样本中检测和鉴定其抗真菌能力。为此，我们的具体目标是：1。优化样本处理，以提供任何临床来源的单细胞检测。2.修改目前的真菌细胞裂解方法，以在存在大量过量人类细胞的情况下，以有利于高通量处理的方式，以最低限度的技术人员技能，从单个真菌细胞中回收DNA。3.探索不同的方法来产生物种特异性引物或杂交探针，并将我们的引物与其他实验室开发的引物进行比较。4.将目前用于白色念珠菌和烟曲霉的真实的时间PCR能力扩展到其他真菌物种。5.对确诊或疑似真菌感染患者的临床样本进行新检测方法的验证。我们希望，在这里开发的真菌的协议和策略将扩展到其他不可培养的生物体。

PCR has revolutionized the diagnosis of infectious diseases. Detecting and identifying microorganisms by their DNA or RNA is potentially more sensitive, more specific and accurate, and allows nearly immediate detection compared to phenotypic tests for most organisms. Fastidious organisms, perhaps many more than we are aware of, are essentially unculturable, or require enriched culture medium and excessive incubation times. Despite this potential, PCR-based testing of fungi in the clinical lab is lagging behind virus and bacteria. Lack of widely used molecular tools for identifying fungi is a shame, given the high mortality rates associated with systemic fungal infections that are often diagnosed too late. Detection of fungal infections is a challenge for PCR-based methods, due to the low numbers of cells at sampling sites, and the hard cell wall that complicates efficient cell lysis. Reviews of the literature underscore that more optimization is needed before PCR will be reliable for diagnosis of Aspergillus fumigatus and other fungi. Our long term objective is to develop rapid, quantitative and sensitive assays for detection and identification of fungal species, and ultimately their antifungal susceptibilities, directly from clinical samples. Toward this goal, our specific aims are: 1. To optimize sample processing to provide single cell detection from any clinical source. 2. To modify current methods of fungal cell lysis to achieve DNA recovery from single fungal cells in the presence of a vast excess of human cells, in a manner conducive to high-throughput processing, with minimal technician skill. 3. To explore different methods of generating species-specific primers or hybridization probes, and to compare our primers with those developed in other laboratories. 4. To extend current real time PCR capabilities for Candida albicans and Aspergillus fumigatus to other fungal species. 5. To validate the new assays on clinical samples from patients with definite or probable fungal infection. We expect that the protocols and strategies developed here for fungi will extend to other, non-culturable organisms.