Molecular Diagnosis of pathogenic fungi

___^__ PCR已经彻底改变了传染病的诊断。检测和鉴定微生物， 它们的DNA或RNA可能更敏感，更特异，更准确， 与大多数生物体的表型检测相比，挑剔的有机体，也许比 我们知道，基本上是不可培养的，或需要丰富的培养基和过度的孵育 次尽管有这种潜力，但临床实验室中基于PCR的真菌检测仍落后于病毒检测， 细菌鉴于真菌的高死亡率，缺乏广泛使用的分子工具来识别真菌是一种耻辱 与系统性真菌感染有关，通常诊断得太晚。真菌感染的检测是 由于采样点的细胞数量较少且细胞壁较硬，这对基于PCR的方法来说是一个挑战 使有效的细胞裂解复杂化。对文献的回顾强调需要进行更多的优化 才能可靠地诊断烟曲霉等真菌。我们的长远目标是 开发快速、定量和灵敏的检测和鉴定真菌物种的方法， 最终，它们的抗真菌能力，直接来自临床样品。为了实现这一目标，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，以实现DNA回收从单一的真菌细胞在存在的， 大量过量的人类细胞，以有利于高通量处理的方式， 技能3.探讨产生种特异性引物或杂交探针的不同方法， 将我们的引物与其他实验室开发的引物进行比较。4.为了扩展当前的真实的时间PCR 能力为白色念珠菌和烟曲霉菌的其他真菌物种。5.要验证新的 对来自明确或可能的真菌感染患者的临床样品进行分析。我们预计 这里针对真菌开发的方案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-basedtesting 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-basedmethods, due to the low numbers of cells at sampling sites, and the hard cell wall that complicates efficient cell lysis. Reviewsof 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 assaysfor 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 minimimal 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.