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增加的临床问题。曲霉病和念珠菌病引起大部分真菌感染，但大量其他真菌也涉及通常致命的感染。临床实验室中用于检测和诊断真菌感染的现有技术既耗时又非常不敏感。近50%的侵袭性真菌感染被当前的临床检测方案遗漏，对患者的生存造成了毁灭性的后果。在当前的应用中，Immunetics建议调整其反向线印迹（RLB）测定技术以进行真菌感染的分子诊断。RLB测定法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与种属特异性探针杂交。30-50个探针可以同时与每个扩增的临床样品杂交，Immunetics的CodaXcel印迹系统用于同时分析多达8个临床样品。我们建议使用PCR或等温环介导扩增（LAMP）技术开发一系列泛真菌扩增引物，并设计一系列物种特异性引物来检测多种曲霉属和念珠菌属以及其他临床相关真菌物种。RLB检测系统有几个优点，使其成为真菌诊断的理想选择：1）使用适当设计的扩增引物和检测探针，该测定非常灵敏（1- 2CFU/ml通过细菌RLB测定常规检测）并且不需要任何真菌培养（意味着在测定中仍然可检测到不可培养的真菌），2）该测定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）该测定非常灵活-探针可以被设计用于任何感兴趣的物种，并且用于任何特定应用的探针组易于设计和验证，4）该测定中的技术故障很少，但是由于该过程的每个步骤的内部阳性对照的存在而易于检测，从而产生非常稳健的测定，和5）该测定也可适用于检测其它感兴趣的基因，包括抗生素和抗微生物剂抗性基因。临床相关的单碱基多态性（SNP）也很容易检测。

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.