Molecular Detection of Invasive Fungal Infections by Reverse Line Blot Assay

描述(由申请人提供)：侵袭性真菌感染在免疫功能低下的患者中是一个日益受到临床关注的问题。曲霉病和念珠菌病是真菌感染的主要原因，但大量其他真菌也与往往致命的感染有关。临床实验室中现有的真菌感染检测和诊断技术既耗时又非常不敏感。目前的临床检测方案漏掉了近50%的侵袭性真菌感染，对患者的生存造成了毁灭性的后果。在目前的应用中，免疫学建议采用其反向线条杂交(RLB)检测技术来进行真菌感染的分子诊断。RLB分析采用的策略是使用泛特异的引物扩增真菌或细菌的DNA序列，然后将扩增的DNA与物种特异的探针杂交。每个扩增的临床样本可以同时杂交30-50个探针，免疫公司的CodaXcel杂交系统用于同时分析多达8个临床样本。我们建议利用PCR或等温环介导扩增(LAMP)技术开发一系列泛真菌扩增引物，并设计一系列针对多种曲霉菌和念珠菌以及其他临床相关真菌物种的特异性引物。RLB分析系统有几个优点使其非常适合用于真菌诊断：1)利用适当设计的扩增引物和检测探针，该分析极其灵敏(细菌RLB分析常规地检测到1-2 cfu/ml)并且不需要任何真菌培养(这意味着在该检测中仍然可以检测到不可培养的真菌)，2)该检测比依赖于培养的诊断方法快得多，在8小时内产生特定的分子物种ID，3)该检测非常灵活--可以为任何感兴趣的物种设计探针，并且用于任何特定应用的探针板易于设计和验证，4)分析中的技术故障很少见，但很容易检测到，因为过程的每一步都有内部阳性对照，从而产生了非常可靠的分析；5)该分析还可以用于检测其他感兴趣的基因，包括抗生素和抗菌素耐药基因。临床相关的单碱基多态(SNPs)也很容易被检测到。

DESCRIPTION (provided by applicant): Invasive fungal infection is a matter of increasing clinical concern in immunocompromised patients. Aspergillosis and Candidiasis cause the bulk of fungal infections, but a large number of other fungi are also implicated in often-fatal infections. The existing technologies in clinical laboratories for detection and diagnosis of fungal infection are both time consuming and very insensitive. Nearly 50% of invasive fungal infections are missed with current clinical testing protocols, with devastating consequences for patient survival. In the current application, Immunetics proposes to adapt its Reverse Line Blot (RLB) Assay technology to perform molecular diagnosis of fungal infection. The strategy employed by the RLB assay is amplification of fungal or bacterial DNA sequences using primers that are pan-specific, followed by hybridization of the amplified DNA to species- specific probes. 30-50 probes can be simultaneously hybridized to each amplified clinical sample, and Immunetics' CodaXcel blotting system is used to analyze up to 8 clinical samples simultaneously. We propose to develop a series of pan-fungal amplification primers using either PCR or isothermal loop mediated amplification (LAMP) techniques, and design a series of species-specific primers for detection of multiple Aspergillus and Candida species as well as other clinically relevant fungal species. There are several advantages to the RLB assay system that make it ideal for use in fungal diagnostics: 1) with properly designed amplification primers and detection probes, the assay is extremely sensitive (1-2 CFU/ml are routinely detected by the bacterial RLB assay) and does not require any culturing of fungi (meaning that uncultivatable fungi are still detectable in the assay), 2) the assay is much quicker than diagnostic methods dependent on culture, generating a specific molecular species ID within 8 hours, 3) the assay is extremely flexible - probes can be designed for any species of interest, and panels of probes for any specific applications are easy to design and validate, 4) technical failures in the assay are rare but are easily detectable due to the existence of internal positive controls for each step of the process, generating an extremely robust assay, and 5) the assay can also be adapted for detection of other genes of interest, including antibiotic and antimicrobial resistance genes. Clinically-relevant single base polymorphisms (SNPs) are also readily detectable.