Novel Method for Probe Labeling

描述(申请人提供)：这个小型企业创新研究第一阶段项目旨在开发一种新的探针标记方法，以提高原位杂交分析的灵敏度。虽然这种探针标记方法可以用于各种RNA和DNA分析格式，但这项提案将专注于开发用于病毒分析的技术，在病毒分析中迫切需要信号放大技术。目前检测病毒感染细胞的方法包括原位聚合酶链式反应和荧光原位杂交。然而，原位聚合酶链式反应容易产生一些伪影，如非特异性靶标扩增和扩增产物泄漏到细胞外。信号放大方法，如酪胺信号放大(TSA)，提高了原位杂交分析的灵敏度，然而，TSA的使用增加了几个程序步骤，增加了分析的复杂性。经酶“拖尾”修饰的寡核苷酸探针提高了原位杂交分析的灵敏度，但这种修饰使标记过程复杂化，并增加了生产成本。此外，由于许可问题，这些探头的使用受到限制。这项建议将把一种新的探针标记方法与图像扫描细胞术相结合，开发出检测罕见病毒感染细胞的灵敏的原位杂交方法。

DESCRIPTION (provided by applicant): This Small Business Innovation Research Phase I project aims to develop a novel probe labeling method to increase the sensitivity of in situ hybridization assays. Although this probe labeling method can be used for a variety of RNA and DNA assay formats, this proposal will focus ondeveloping the technology for use with viral assays, where there is an urgent need for signal amplification technologies. Current methods for detecting viral infected cells include in situ PCR and fluorescence in situ hybridization. In situ PCR, however, is prone to several artifacts, such as non-specific target amplification and amplicon leakage out of the cell. Signal amplification methods, such as tyramide signal amplification (TSA), have increased the sensitivity of in situ hybridization assays, however, use of TSA increases assay complexity by adding several procedural steps. Oligonucleotide probes modified by enzymatic "tailing" increase the sensitivity of in situ hybridization assays, however, this modification complicates labeling procedures and increases production costs. Furthermore, use of these probes is limited due to licensing issues. This proposal will combine a novel probe labeling method with image scanning cytometry to develop sensitive in situ hybridization assays for detecting rare viral infected cells.