MOLECULAR DIAGNOSIS OF ORTHOPAEDIC BACTERIAL INFECTIONS

描述(改编自申请人的摘要)：本研究方案 满足骨科社区对快速、准确的 和廉价的方法来确定矫形标本是否， 例如滑液，以及其他整形外科组织，例如骨和骨 骨髓都被感染了。实验室标准培养和革兰氏染色检测 尤其是检测关节中的细菌感染 由于假阴性测试的频率相对较高，令人失望 结果。申请者正在开发、测试和提炼一种分子 利用聚合酶链式反应(PCR)提供的生物技术 快速准确地检测食品中微生物DNA 整形外科标本。这种方法涉及到一种新的处理方法 整形外科样本实现细菌裂解和DNA释放，使 用聚合酶链式反应直接对DNA进行分子检测。细菌特异性聚合酶链式反应 人们正在使用引物来扩增高度保守的通用16S 细菌基因组所特有的核糖体RNA(RRNA)基因。初步研究 正在进行这项诊断技术的有效性测试 检测滑液中的感染。最初，提议的流程是 用接种了已知病毒的无菌滑液进行检测 各种骨科相关细菌的浓度。 其次，申请人建议将这一方法扩展到滑膜测试。 全膝关节置换术患者的体液标本 (TKA)手术，以及谁可能在 受影响的关节。还提出了设计使用聚合酶链式反应的策略。 用于确定传染病病原体种类的诊断学。一个 将进行统计比较，比较申请者的PCR值 具有标准诊断模式的检测方法。放置一个 假阴性导致污染关节的翻修植入物 术前微生物数据将导致感染的修订TKA， 需要额外的手术，花费巨大，就医疗费用和 病人感到不适。调查人员建议，随着 应用高灵敏的聚合酶链式反应诊断技术，快速、 准确识别受感染的样本可以极大地帮助 建立手术和/或抗生素治疗方案。因此，它是 推测这项拟议的研究可以提供一种有价值的方法 对于骨科临床医生以及基础研究人员，当 正试图毫不含糊地确定 临床骨科标本中的感染性物质。

DESCRIPTION (Adapted from the Applicant's Abstract): This research proposal addresses the need within the orthopaedic community for a rapid, accurate and inexpensive method for determining whether or not orthopaedic specimens, such as synovial fluid, and other orthopaedic tissues, such as bone and bone marrow, are infected. Standard laboratory culturing and Gram stain testing to detect bacterial infections in articular joints have been particularly disappointing due to the relatively high frequency of false negative test results. The applicants are developing, testing and refining a molecular biological technique using the polymerase chain reaction (PCR) to provide rapid and accurate determination of the presence of microorganismal DNA in an orthopaedic specimen. The methodology involves a novel way of processing orthopaedic samples to achieve bacterial lysis and release of DNA, allowing the direct molecular detection of that DNA by PCR. Bacteria-specific PCR primers are being used that amplify the highly conserved, universal 16S ribosomal RNA (rRNA) gene unique to bacterial genomes. A preliminary study is being undertaken to test the efficacy of this diagnostic technology for detecting infection in synovial fluid. Initially, the proposed process is being tested with sterile synovial fluid inoculated with known concentrations of various orthopaedically relevant bacterial species. Secondly, the applicants propose to extend this methodology to test synovial fluid specimens from patients who have undergone total knee replacement (TKA) surgery, and who may have acquired bacterial infections in the affected joints. Also proposed are strategies designed to use PCR diagnostics for the determination of the species of the infectious agent. A statistical comparison will be performing comparing the applicants' PCR detection methodology with standard diagnostic modalities. Placement of a revision implant into a contaminated joint because of false negative preoperative microbiological data will result in an infected revision TKA, requiring additional surgery at great expense, in terms of medical costs and patient discomfort. It is suggested by the investigators that, with the application of highly sensitive PCR diagnostic technology, rapid and accurate identification of infected specimens could greatly assist in the establishment of surgical and/or antibiotic treatment regimens. Thus, it is speculated that this proposed research could provide a valuable methodology for the orthopaedic clinician, as well as the basic researcher, when either is attempting to determine unambiguously the presence and level of infectious agents in a clinical orthopaedic specimen.