Polymerase Chain Reaction-Single Strand Confirmation Polymorphism for the Detect

临床标本中微孢子虫的检测和形态鉴定有多种选择。光学显微镜可以检测寄生虫，但不允许物种形成。电子显微镜是形态鉴定的金标准，但不是检测微孢子虫的灵敏方法。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。可以通过在PCR测定中使用种特异性引物，或通过在进行PCR测定后使用DNA探针或限制性内切核酸酶来确定种来实现物种形成。单链构象多态性（SSCP）结合PCR（PCR-SSCP）已被用于鉴定细菌、真菌和病毒。我们将使用我们的PCR方法检测粪便标本中的微孢子虫，并应用SSCP确定寄生虫的特定属和种。本研究亦会利用细胞培养所得的微生物来验证该方法，并将SSCP技术应用于流行病学研究中微孢子虫基因型的检测。当PCR产物用限制性内切酶消化时，新公布的引物允许物种形成。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NIH微孢子虫病患者中检测到的比氏肠细胞虫中鉴定出两种不同的基因型，将用于完成本研究。

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.