Novel Method for Probe Labeling

探针标记的新方法

• 批准号: 6736441
• 负责人: PHILLIP T MOEN
• 金额: $ 12.53万
• 依托单位: ONE CELL SYSTEMS, INC
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6736441
• 关键词: Epstein Barr virus; biotechnology; cell line; communicable disease diagnosis; flow cytometry; haptens; in situ hybridization; method development; nucleic acid probes; nucleic acid quantitation /detection; oligonucleotides; virus diseases

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.

描述（由申请人提供）：这个小企业创新研究第一阶段项目的目的是开发一种新的探针标记方法，以增加原位杂交检测的灵敏度。虽然这种探针标记方法可用于各种RNA和DNA检测形式，但该提案将集中于开发用于病毒检测的技术，其中迫切需要信号放大技术。目前用于检测病毒感染细胞的方法包括原位PCR和荧光原位杂交。然而，原位PCR易于出现几种假象，例如非特异性靶扩增和扩增子泄漏出细胞。信号放大方法，如酪胺信号放大（TSA），增加了原位杂交测定的灵敏度，然而，TSA的使用通过增加几个程序步骤而增加了测定的复杂性。通过酶促“加尾”修饰的寡核苷酸探针增加了原位杂交测定的灵敏度，然而，这种修饰使标记程序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。该建议将联合收割机结合一种新的探针标记方法和图像扫描细胞术，以开发用于检测罕见病毒感染细胞的灵敏的原位杂交方法。