Polymerase Chain Reaction-Single Strand Confirmation Polymorphism for the Detect

临床标本中微孢子虫的检测和物种形成有多种选择。光学显微镜可以检测到寄生虫，但不能形成物种。电子显微镜是物种形成的金标准，但对微孢子虫的检测并不敏感。聚合酶链反应（PCR）是一种敏感的技术，有许多不同的方法来确认阳性结果和确定引起感染的属和种。物种形成可以通过在PCR检测中使用物种特异性引物来实现，或者在进行PCR检测后使用DNA探针或限制性内切酶来确定物种。单链确认多态性（SSCP）结合PCR-SSCP已被用于细菌、真菌和病毒的鉴定。我们将使用PCR方法检测粪便标本中的微孢子虫，并使用SSCP方法确定寄生虫的具体属和种。来自细胞培养的生物体也将用于验证该方法。该项目已扩大到将SSCP应用于流行病学研究中的微孢子虫基因型检测。新发表的引物允许在PCR产物被限制性内切酶消化时形成物种。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。我们在美国国立卫生研究院的微孢子虫病患者中发现了两种不同的双胞虫基因型，这将用于完成本研究。这个项目已经完成了。

There are many options for the detection and speciation of microsporidia in clinical specimens. Light microscopy allows detection of the parasites, but does not allow speciation. Electron microscopy is the gold standard for speciation, but is not as sensitive a method for the detection of microsporidia. Polymerase chain reaction (PCR) is a sensitive technique with many different methods for confirming a positive result and for 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 detection 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.The project has been expanded to apply SSCP to the detection of microsporidial genotypes in epidemiologic 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. We have identified two different genotypes among the Enterocytozoon bieneusi detected in patients with microsporidiosis at the NIH, which will be used to finish this study.This project has been completed.