POLYMERASE CHAIN REACTION-SINGLE STRAND CONFIRMATION POLYMORPHISM FOR THE DETECTI

临床标本中微孢子虫的检测和分类有多种选择。光学显微镜允许检测寄生虫，但不允许物种形成。电子显微镜是物种形成的金标准，但作为检测微孢子虫的一种方法不敏感。聚合酶链式反应(PCR)是一种敏感的技术，有许多不同的方法来确认阳性结果，并确定导致感染的属和种。物种鉴定可以通过在聚合酶链式反应分析中使用物种特定的引物，或者在进行聚合酶链式反应分析后使用DNA探针或限制性内切酶来确定物种。单链确认多态(SSCP)结合聚合酶链式反应(PCR-SSCP)已被用于细菌、真菌和病毒的鉴定。我们将用我们的PCR方法检测粪便标本中的微孢子虫，并用SSCP方法确定该寄生虫的特定属和种。来自细胞培养的生物体也将被用于验证该方法。该项目的这一部分已接近完成，不久将提交出版。该项目已扩大到将SSCP应用于检测微孢子虫的基因类型，以用于流行病学研究。新公布的引物允许在用限制性内切酶消化聚合酶链式反应产物时形成物种。SSCP分析将使这项任务比限制性内切酶分析更容易和更敏感。

There are many options for the detection and speciation of microsporidia in clinical specimens. Light microscopy allows for detection of the parasites, but does not allow speciation. Electron microscopy is the gold standard for speciation, but is insensitive as a method of detection of microsporidia. The polymerase chain reaction (PCR) is a sensitive technique with many different methods for confirming a positive result and determining the genus and species causing an infection. Speciation can be achieved by using species- specific primers in the PCR assay, or by using DNA probes or restriction endonucleases to determine the species after the PCR assay is performed. Single strand confirmation polymorphism (SSCP) combined with PCR (PCR-SSCP) has been used to identify bacteria, fungi, and viruses. We will use our PCR assay for the detec-tion of microsporidia in stool specimens and apply SSCP to determine the specific genus and species of the parasite. Organisms from cell culture will also be used to validate the method. This portion of the project is near completion and will soon be submitted for publication. The project has been expanded to apply SSCP to the detection of microsporidial genotypes for use in epidemiological studies. Newly published primers allow speciation when the PCR products are digested with restriction enzymes. SSCP analysis will make this task easier and more sensitive than restriction enzyme analysis.