Molecular Detection of Invasive Fungal Infections by Reverse Line Blot Assay

描述（由申请人提供）：侵袭性真菌感染是免疫功能低下患者日益关注的临床问题。曲霉病和念珠菌病引起大部分真菌感染，但大量其他真菌也与通常致命的感染有关。临床实验室现有的真菌感染检测和诊断技术既耗时又不敏感。目前的临床检测方案遗漏了近50%的侵袭性真菌感染，这对患者的生存造成了毁灭性的后果。在目前的应用中，Immunetics建议采用其反向行印迹（RLB）检测技术来进行真菌感染的分子诊断。RLB检测采用的策略是使用泛特异性引物扩增真菌或细菌DNA序列，然后将扩增的DNA与物种特异性探针杂交。每个扩增的临床样品可以同时杂交30-50个探针，并且使用Immunetics的CodaXcel印迹系统同时分析多达8个临床样品。我们建议利用PCR或等温环介导扩增技术（LAMP）开发一系列泛真菌扩增引物，并设计一系列物种特异性引物，用于检测多种曲霉和念珠菌以及其他临床相关的真菌物种。RLB检测系统有几个优点，使其非常适合用于真菌诊断：1)使用适当设计的扩增引物和检测探针，该分析非常敏感（1-2 CFU/ml是细菌RLB试验的常规检测值），并且不需要任何真菌培养（这意味着在该试验中仍然可以检测到不可培养的真菌），2)该分析比依赖培养的诊断方法要快得多，在8小时内产生特定的分子物种ID。3)分析极为灵活-探针可用于任何感兴趣的物种,和小组调查为任何特定的应用程序很容易设计和验证,4)技术故障分析是罕见的,但却很容易被由于存在内部积极控制过程的每个步骤,生成一个非常健壮的分析,和5)分析还可以用于检测其他感兴趣的基因,包括抗生素和抗生素耐药性基因。临床相关的单碱基多态性（snp）也很容易检测到。

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.