DEVELOPMENT OF METHODS FOR EPIDEMIOLOGIC TYPING OF PNEUMOCYSTIS CARINII

卡氏肺孢子虫流行病学分型方法的开发

• 批准号: 5201189
• 负责人: C P CARTWRIGHT
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5201189
• 关键词: Pneumocystis carinii; bacterial RNA; bacterial genetics; genetic strain; genetic techniques; genome; method development; microorganism classification; molecular genetics; nucleic acid sequence; operon; polymerase chain reaction; restriction mapping; ribosomal RNA

The aim of this project was to investigate the feasibility of developing a PCR-based technique for analyzing genetic relatedness among isolates of Pneumocystis carinii from patients at the Clinical Center. Since this organism cannot be cultivated in vitro, the selective amplification of potentially variable portions of the genome directly from clinical specimens provides the only possibility for examining genetic variation. The genetic composition of P. carinii is poorly understood, and thus a limited number of targets are available for such exploration. We chose to examine intergenic spacer regions of the rRNA operon, a known source of intraspecific genetic variation in other microorganisms. The results of this study were very disappointing. Although we were able to amplify rRNA regions successfully from clinical specimens, we could find little evidence from exhaustive restriction endonuclease analysis for between- isolate variation in the sequence of the spacer region. Recently published data from laboratories employing similar approaches to us have shown that small differences in nucleotide sequence could be detected in this region by sequencing PCR products (a technique not well suited for clinical laboratories interested in typing P. carinii) and, that even when this highly sensitive method was employed, only 3-4 subtypes could be differentiated when a large number of isolates were examined. Such poor discriminatory power severely compromises the epidemiologic value of any data generated using this technique. Given these results, we have decided to discontinue this project until developments in P.carinii research make the possibility of developing a discriminatory typing system more feasible.

该项目的目的是研究开发的可行性 一种基于PCR的技术，用于分析分离株之间的遗传相关性， 临床中心病人身上的卡氏肺孢子虫。由于这 生物体不能在体外培养，选择性扩增 基因组的潜在可变部分直接来自临床 标本提供了检查遗传变异的唯一可能。 卡氏肺吸虫的遗传组成知之甚少，因此， 可供这种勘探的目标数量有限。我们选择 检查rRNA操纵子的基因间间隔区，这是一个已知的 其他微生物的种内遗传变异。的结果 这项研究非常令人失望。尽管我们能够扩增rRNA 区域成功地从临床标本，我们可以找到很少 从详尽的限制性内切酶分析的证据之间- 分离间隔区序列中的变异。最近发表 采用与我们类似方法的实验室的数据表明， 在该区域中可以检测到核苷酸序列的微小差异 通过对PCR产物进行测序（一种不太适合临床应用的技术）， 对卡氏肺孢子虫分型感兴趣的实验室），而且，即使这 采用高度敏感的方法，仅能检测3-4个亚型， 当检测大量分离株时，这么差 歧视的权力严重损害流行病学价值的任何 使用该技术生成的数据。鉴于这些结果，我们决定 在卡氏肺吸虫研究取得进展之前， 发展歧视性打字制度的可能性 可行