Extraction-Free Filter-Based Template Preparation for Rapid and Sensitive PCR Detection of Pathogenic Parasitic Protozoa

摘要

Within the last several years, the protozoan parasites Cyclospora cayetanensis, Cryptosporidium parvum, and microsporidia have become recognized as important, rapidly emerging human pathogens in immunocompromised and immunocompetent individuals. Since the early 1990s, many of the reported outbreaks of enteric illness caused by these microorganisms have been attributed to food- and water-borne contamination. Many inherent obstacles affect the success of current surveillance and detection methods used to monitor and control levels of contamination by these pathogens. Unlike methods that incorporate preenrichment for easier and unambiguous identification of bacterial pathogens, similar methods for the detection of parasitic protozoa either are not currently available or cannot be performed in a timely manner. We have developed an extraction-free, filter-based protocol to prepare DNA templates for use in PCR to identify C. cayetanensis and C. parvum oocysts and microsporidia spores. This method requires only minimal preparation to partially purify and concentrate isolates prior to filter application. DNA template preparation is rapid, efficient, and reproducible. As few as 3 to 10 parasites could be detected by PCR from direct application to the filters. In studies, as few 10 to 50 Encephalitozoon intestinalis spores could be detected when seeded in a 100-μl stool sample and 10 to 30 C. cayetanensis oocysts could be detected per 100 g of fresh raspberries. This protocol can easily be adapted to detect parasites from a wide variety of food, clinical, and environmental samples and can be used in multiplex PCR applications.