DEVELOPMENT OF RT PCR ASSAY FOR QUANTITATIVE DETECTION OF ENTERIC CALICIVIRUSES

肠道杯状病毒定量检测 RT PCR 检测方法的开发

• 批准号: 8173022
• 负责人: KAROL SESTAK
• 金额: $ 6.18万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173022
• 关键词: Biological; Biological Assay; Blood Group Antigens; Calicivirus; Computer Retrieval of Information on Scientific Projects Database; Detection; Development; Enteral; Epidemiology; Fluorescence; Fluorescent Dyes; Funding; Future; Gastroenteritis; Genetic Variation; Genomics; Grant; Housing; Human; Infection Control; Institution; Label; Macaca mulatta; Modeling; Norovirus; Nucleotides; Oligonucleotide Probes; Plasmids; RNA; RNA-Directed RNA Polymerase; Reaction; Reporter; Reporting; Research; Research Personnel; Resources; Sampling; Source; Specificity; System; United States National Institutes of Health; Virus; antigen binding; base; design; nonhuman primate; positional cloning; prototype; tissue culture; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recently we reported the discovery of a cultivable calicivirus (Tulane virus; TV) isolated from a rhesus macaque. Based on preliminary results that show close similarities between TVs and noroviruses (NVs) in respect to their genetic diversity, epidemiology and histo-blood group antigen (HBGA) binding, plus the availability of in-house reverse genetics system, we are pursuing the development of a TV-based non-human primate surrogate model for human NV gastroenteritis. To support this effort in this study, we developed a qRT-PCR with the objective to enumerate TV RNA load in biological samples through the course of controlled infections of rhesus macaques. Based on alignments of 15 TV isolates, primers were designed to amplify a conserved 176 nucleotide fragment of the RNA dependent RNA polymerase. A 23 nucleotides long oligonucleotide probe specific for the prototype (M33) TV was designed within the amplicon and labeled with reporter fluorescent dye and fluorescence quencher. This TAQ-MAN based assay demonstrated a high level of specificity to the prototype TV against 3 other TV isolates with 47% to 82% nucleotide homology to the prototype strain in the target region (plasmid based assays). The assay allowed optimal detection in a 5-log range and a detection limit of 10 TV genomic copies per reaction. TV genomic RNA extracted from fecal samples spiked with tissue cultured prototype TV was also successfully detected. The assay described here allows for the highly specific and sensitive quantitation of the prototype TV strain in biological samples and will serve as an important tool for our future studies.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 最近，我们报道了从恒河猴中分离出的可栽培蜡膜病毒（Tulane病毒; TV）的发现。基于初步结果，在其遗传多样性，流行病学和组织血管群抗原（HBGA）抗原（HBGA）结合方面，电视和诺病毒（NVS）之间有着密切相似的结果，加上内部反向遗传系统的可用性，我们正在追求基于基于非人类的Primate Sodogate Model for HumanV gastroentisitiation的开发。为了支持这项研究的这项工作，我们开发了一个QRT-PCR，目的是通过恒河猕猴的受控感染过程来列举生物样品中的电视RNA负载。基于15个电视分离株的比对，设计引物来扩增RNA依赖性RNA聚合酶的176个核苷酸片段。在扩增子内设计了针对原型（M33）电视特异性的23个核苷酸长寡核苷酸探针，并用报告基因荧光染料和荧光猝灭剂标记。这种基于TAQ-MAN的测定法证明了针对其他3个电视的原型电视具有很高的特异性，其针对目标区域中原型菌株的47％至82％的核苷酸同源性（基于质粒的测定）。该测定允许在5-log范围内的最佳检测和每个反应的10台电视基因组拷贝的检测极限。还成功地检测到了从组织培养的原型电视尖峰的粪便样品中提取的电视基因组RNA。 此处描述的分析允许对生物样品中原型电视菌株进行高度特异性和敏感的定量，并将作为我们未来研究的重要工具。